NEUROBIOCHEMICAL AND PEPTIDOMIC APPROACHES TO ...

NEUROBIOCHEMICAL AND PEPTIDOMIC APPROACHES TO SCREEN FOR TAU KINASE INHIBITORS AND ELUCIDATE POST-TRANSLATIONAL MODIFICATIONS

FOLLOWING TRAUMATIC BRAIN INJURY

BY

HAMAD YADIKAR

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2018

To my Family and Friends

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ACKNOWLEDGMENTS

I thank Allah for all his blessings without which nothing of my work would have

been done. The body of this work would not have been completed without the patience,

guidance, scientific discussions, and encouragement from Dr. Kevin K Wang, Dr.

Richard A. Yost, Dr. Kari Basso, and Dr. Gail Fanucci. I will be forever indebted for the

experiences and the knowledge I gained as well as the extensive training obtained in

mass spectrometry. I would like to take the opportunity to give my sincere appreciation

to my research advisor, Professor Kevin Wang. He not only taught me how to be an

independent scientist, but also gave me the will to keep going on this exciting research

field. I will always admire and respect him as a research mentor; under his supervision, I

have learned many scientific skills in research, and this will be an excellent guideline for

my future career.

I would like to thank our lab research team as well, Dr. Sunny and Lab Manager

Fan Lin, they have been quite helpful to me in sample preparation for my Ph.D. project.

Wholeheartedly, I would like to thank the Yost Research Group for fostering my

scientific skills in mass spectrometry as well as solving instrumental problems I faced as

I evolved into the biochemist I am today. The weekly meeting has been very insightful

for me to solve many problems. The meetings gave me several methodological ideas to

apply to my research. I would also like to thank our undergraduate students whom I

enjoyed working with and gained experience in teaching and managing their sub-

projects, a skill I will use in my future career.

I would like to express my gratitude to my chair, Dr. Richard A. Yost, for his

interest in this work and his generous support for my research. I also would like to thank

Dr. Kari Basso, who has been truly kind and welcoming in helping me in my proteomics

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research and gave me valuable suggestions to improve my data for publication. I would

like to thank Dr. Gail Fanucci for her valuable points she made for my research project. I

express my sincere gratitude to Kuwait University Fund Doctoral Scholarship for

providing me the academic, professional, financial, and personal support through my

doctoral study at the University of Florida. I am grateful for ICBR-proteomics core at the

University of Florida for aiding me in analyzing my samples using the innovative mass

spectrometric technology, especially Dr. Jin Koh and Dr. Sixue Chen.

Lastly, I cannot end without thanking my family for their constant encouragement

and love, that I have relied on throughout my time at the UF. My parents educated me

to love science and believed in me. They were never afraid to express faith in my ability

to obtain success. The knowledge I have gained with Dr. Wang and Dr. Yost research

teams will be an invaluable asset as I move forward in my career and use my expertise

professionally to aid in bettering humanity and leaving a legacy for others to follow.

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TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 9

LIST OF FIGURES ........................................................................................................ 10

LIST OF ABBREVIATIONS ........................................................................................... 14

ABSTRACT ................................................................................................................... 19

CHAPTER

1 LITERATURE REVIEW .......................................................................................... 21

Traumatic Brain Injury (TBI) .................................................................................... 21

Chronic Traumatic Encephalopathy (CTE) ............................................................. 22 The Role of Tau Protein in Cellular Functions and Dysfunctions ............................ 24 Tau Structure .......................................................................................................... 25

Tau Post-Translational Modification ........................................................................ 27 Tau Proteolysis ....................................................................................................... 30

Tau Calpain-Mediated Proteolysis .......................................................................... 31 The Function of Tau proteolytic fragments in Disease ............................................ 32 Tau Fragments in Biofluids as Biomarkers ............................................................. 33

Serum Tau ........................................................................................................ 33

CSF Tau ........................................................................................................... 34 Plasma Tau ...................................................................................................... 34 Conclusions ...................................................................................................... 35

Peptidomics-Based Discovery of Novel Neuropeptides .......................................... 35 Current Mass Spectrometric Peptidomic Technologies .......................................... 37 The Scope of the Dissertation ................................................................................. 38

2 SCREENING OF TAU PROTEIN KINASE INHIBITORS IN A TAUOPATHY-RELEVANT CELL-BASED MODEL OF TAU HYPERPHOSPHORYLATION AND OLIGOMERIZATION ...................................................................................... 46

Introduction ............................................................................................................. 46

Materials and Methods............................................................................................ 48 Inhibitors ........................................................................................................... 48 Antibodies ......................................................................................................... 49

Cell Line ........................................................................................................... 49 Primary Cerebrocortical Neuronal Cultures ...................................................... 49 Cell Treatments ................................................................................................ 50 Cell Lysate Collection and Preparation ............................................................ 50 SDS–PAGE and Western Blots ........................................................................ 51

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Statistical Analysis ............................................................................................ 51 Ethical Statement ............................................................................................. 52

Results .................................................................................................................... 52 Okadaic Acid (OA) Induces Tau Hyperphosphorylation and Oligomerization

at Different Time Points in Mouse Neuroblastoma N2a Cells........................ 52 Testing Tau Kinase Inhibitors on OA-Induced Tau Hyperphosphorylation

and Oligomerization in N2a Cells. ................................................................. 53

Casein kinase II (CKII) inhibitor: 4, 5, 6, 7-tetrabromobenzotriazole (TBB) ...................................................................................................... 54

Calcineurin inhibitor: cyclosporine A (CsA) ................................................ 55 Calcium chelator: EGTA ............................................................................ 55 GSK3 inhibitors: LiCl, A-1070722, and AR-1014418 ................................. 56

Src/Fyn kinase inhibitor: Saracatinib .......................................................... 57

CDK5 inhibitor: Roscovitine ....................................................................... 58 Baseline and OA-Induced Tau Hyperphosphorylation and Oligomerization:

Effects of Various Kinase Inhibitors Treatment in Rat Primary Cerebrocortical Neuronal (CTX) Culture. ...................................................... 58

Pan kinase inhibitor: K252a and STS ........................................................ 61 Discussion .............................................................................................................. 62

Limitations and Future Directions ........................................................................... 67 Conclusions ............................................................................................................ 67

3 IDENTIFICATION OF LOW AND HIGH MOLECULAR WEIGHT TAU FRAGMENTS FOLLOWING IN VITRO CALPAIN DIGESTION AND CELL-BASED NEUROTOXIC CHALLENGES BY BIOCHEMICAL AND PEPTIDOMIC APPROACHES ....................................................................................................... 85

Introduction ............................................................................................................. 85 Material and Methods ............................................................................................. 88

Ethics Statement .............................................................................................. 88

Mouse Brain Collection and Samples Preparation ........................................... 89 In Vitro Calpain-1 Digestion of Purified Tau and Naïve Mouse Brain Lysate .... 89

Rat Primary Cerebrocortical Neuronal (CTX) Culture ....................................... 90

Neurotoxic Challenges ..................................................................................... 90 Preparation and Collection of Cell Lysate and Conditioned Media ................... 91 Sample Preparation and Ultrafiltration .............................................................. 91 Gel Electrophoresis and Western Blotting ........................................................ 92 Nano-LC-ESI-MS/MS ....................................................................................... 93

Thermo Orbitrap Fusion Tribrid Mass Spectrometer Analysis .......................... 94

Data Analysis ................................................................................................... 95

Statistical Analysis ............................................................................................ 96 Results and Discussion........................................................................................... 96

Immunoblot Analysis ........................................................................................ 98 Recombinant purified human tau/p-tau protein .......................................... 98 Transgenic mouse brain lysate .................................................................. 98

Primary Rat Cerebrocortical Neuronal (CTX) culture Subjected to Neurotoxic and Neurodegenerative Conditions ............................................. 99

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nLC-ESI-MS/MS Analysis of Purified Tau and Mouse Brain Lysate ............... 101 Conditioned Media of CTX Culture Analysis by nLC-ESI-MS/MS .................. 106

Phosphorylation Sites Analysis ...................................................................... 108 Conclusions .......................................................................................................... 109

4 ANALYSIS OF TEMPORAL PROFILE OF TBI-INDUCED TAU HYPERPHOSPHORYLATION AND PROTEOLYSIS IN MOUSE BRAIN BY IMMUNOBLOTTING AND MASS SPECTROMETRY........................................... 148

Introduction ........................................................................................................... 148 Cortical Impact Injury (CCI) ................................................................................... 150 Materials and Methods.......................................................................................... 152

Ethical Statement ........................................................................................... 152

Mouse Brain Samples Preparation ................................................................. 152 Ultrafiltration Method and Ultrafiltrate Processing for Mass Spectrometry ..... 153 Immunoblot Analysis ...................................................................................... 153

Reversed-Phase Nano-UPLC-MS/MS ........................................................... 154 Results and Discussion......................................................................................... 155

Immunoblot Analysis of Tau in Transgenic Mouse Brain after Experimental CCI .............................................................................................................. 157

Identification of Tau Proteolytic Fragments from Mouse Brain after CCI using nLC-nESI-MS/MS .............................................................................. 160

Conclusions .......................................................................................................... 167

5 CHARACTERIZATION OF TAU POST-TRANSLATIONAL MODIFICATION LEVELS AND TEMPORAL PROFILE IN HUMAN CEREBROSPINAL FLUID FROM SEVERE TBI AND CONTROL SUBJECTS. ............................................. 197

Introduction ........................................................................................................... 197

Materials and Methods.......................................................................................... 199 Human TBI and Control Cerebrospinal Fluid Procurement ............................ 199 Total-Tau-ELISA Kit ....................................................................................... 200

SDS-PAGE and Immunoblotting .................................................................... 200 Isolation of CSF LMW peptides ...................................................................... 200 nLC-ESI-MS/MS ............................................................................................. 200

Results and Discussion......................................................................................... 201 Conclusion ............................................................................................................ 204

6 CONCLUSIONS AND FUTURE DIRECTIONS .................................................... 222

LIST OF REFERENCES ............................................................................................. 229

BIOGRAPHICAL SKETCH .......................................................................................... 250

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LIST OF TABLES

Table page 2-1 Antibodies used in this study .............................................................................. 69

2-2 Phosphatases, kinase inhibitors, and pharmacological agents used in the study. .................................................................................................................. 70

2-3 Composite effects of tau kinase inhibitors on OA-induced tau hyperphosphorylation in N2a cells. ..................................................................... 71

2-4 Composite effects of tau kinase inhibitors on basal and OA-induced tau hyperphosphorylation in rat primary cerebrocortical neuronal cells. ................... 72

3-1 Selected human tau-441 calpain-mediated proteolytic peptides from purified protein digestion identified by nLC-MS/MS. ...................................................... 111

3-2 Selected human calpain-mediated proteolytic peptides from purified p-tau protein digestion identified by nLC-MS/MS. ...................................................... 112

3-3 Selected tau-441 calpain-mediated proteolytic peptides from mouse htau transgenic brain lysate digestion identified by nLC-MS/MS. ............................. 113

3-4 Selected rat Tau-776 peptides identified from conditioned primary CTX culture media by nLC-MS/MS. .......................................................................... 114

3-5 Global Homo sapiens database search using in vitro calpain-mediated tau-441 digested sample. ....................................................................................... 115

4-1 Identification of TBI-induced proteolytic peptides from mouse CCI cortex sample ultrafiltrate by nLC-MS/MS. .................................................................. 170

4-2 Identification of TBI-induced phosphorylated peptides from mouse CCI cortex sample ultrafiltrate by nLC-MS/MS. Adjacent amino acid are shown in brackets ............................................................................................................ 171

5-1 Brain proteins identified exclusively from human TBI-CSF ultrafiltrate samples by high resolution nLC-MS/MS. .......................................................... 205

5-2 Tau-441 proteolytic peptides identified from human TBI-CSF patients ultrafiltrate fractions by nLC-MS/MS. ................................................................ 206

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LIST OF FIGURES

Figure page

1-1 Human brain tau protein isoforms ....................................................................... 40

1-2 Phosphorylation sites on tau protein and specific epitopes for tau antibodies .... 41

1-3 Tau phosphorylation events are sequential in the development of tau pathology ............................................................................................................ 42

1-4 Schematic of tau proteolysis and its role in tauopathies ..................................... 43

1-5 TBI-relevant pathways associated with neuronal death ...................................... 44

1-6 Workflow for peptidomics platforms, which are divided into five main steps compromising different types of methods. .......................................................... 45

2-1 OA induced tau hyperphosphorylation and oligomerization at different time points in mouse neuroblastoma N2a cells .......................................................... 73

2-2 Screening of protein kinase inhibitors on OA-induced tau hyperphosphorylation and oligomerization in N2a cells (with or without cell-death linked protease inhibitors (calpain/caspase inhibitors) .............................. 74

2-3 Dose-response of TBB on OA-induced tau hyperphosphorylation and oligomerization in N2a cells ................................................................................ 76

2-4 Screening of protein kinase inhibitors on basal tau phosphorylation in rat primary cortical neuronal culture (CTX) .............................................................. 77

2-5 Screening of protein kinase inhibitors on OA-induced tau hyperphosphorylation in rat primary cortical neuronal culture ............................ 79

2-6 Effect of combining protein kinase inhibitors on OA-induced tau hyperphosphorylation and oligomerization in N2a cells. ..................................... 81

2-7 Different effects of cyclosporine A on basal and OA-induced tau hyperphosphorylation in mouse N2a vs. rat CTX culture .................................... 83

3-1 Peptidomic sample preparation workflow for the nLC-ESI-MS/MS for the analysis of purified Tau protein, conditioned cell media, and mouse brain lysate ................................................................................................................ 116

3-2 Identification of calpain-mediated tau peptides by immunoblotting and mass spectrometry. .................................................................................................... 117

3-3 Identification of calpain-mediated phosphorylated tau peptides by immunoblotting and mass spectrometry ........................................................... 119

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3-4 Identification of calpain-mediated tau peptides from transgenic htau mouse brain lysate by immunoblotting and mass spectrometry ................................... 121

3-5 Composite summary of calpain-mediated tau proteolytic peptides and phosphorylation sites. ....................................................................................... 123

3-6 View of a customized Peptigram peptide alignment map compiled from tau, p-tau, and mouse brain lysate. ......................................................................... 125

3-7 Basal and OA-induced tau proteolysis following neurotoxin challenges in rat cerebrocortical neurons .................................................................................... 126

3-8 Identification of tau neuropeptides from conditioned media following neurotoxin challenges in rat cerebrocortical neurons. ...................................... 128

3-9 Composite summary of tau proteolytic peptides and phosphorylation sites from CTX conditioned cell media ...................................................................... 130

3-10 Tau protein sequence alignments of rat and human ......................................... 131

3-11 Representative tau-441 sequences coverage of the human protein (P10636-8) detected........................................................................................................ 132

3-12 Representative tau-776 sequences coverage of the human protein (P10636-9) detected........................................................................................................ 133

3-13 Product ion spectrum of the tau peptide AEPRQEFEVMEDHAGTYG released from calpain-1 digestion ..................................................................... 134

3-14 Product ion spectrum of the tau peptide SPRHLSNVSSTGSIDMVDSPQLA released from calpain-1 digestion ..................................................................... 136

3-15 Product ion spectrum of the rat tau peptide PRHLSNVSSTGSIDMVDSPQLA released from conditioned cell media of primary cerebrocortical cultures ........ 138

3-16 Product ion spectrum of the phosphorylated tau peptide STGSIDMVDSPQLA released from calpain-1 digestion ..................................................................... 140

3-17 Human tau-441 and tau-776 isoforms amino acid sequence alignments. Human tau-441 is in the top lane while tau-776 is on the bottom lane ............. 142

3-18 Comparison between targeted database search against Human tau-441 versus Homo sapiens proteome ....................................................................... 143

4-1 Peptidomic sample preparation workflow for the nano-LC-ESI-MS/MS for the analysis after CCI treatment to htau mouse brain ............................................. 172

4-2 Characterization of tau phosphorylation, oligomerization, and fragments formation in ipsilateral and contralateral mouse cortical mouse lysate ............. 173

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4-3 Additional characterization of tau phosphorylation, oligomerization, and fragments formation in ipsilateral and contralateral mouse cortical mouse lysate after TBI ................................................................................................. 176

4-4 Assessment of cell injury using α-spectrin antibody in ipsilateral and contralateral mouse cortical mouse lysate after TBI ......................................... 178

4-5 Representative TIC-LC chromatogram for peptides isolated from ipsilateral (IC) and contralateral (CC) mouse brain cortex lysate using peptidomic workflow. .......................................................................................................... 179

4-6 Representative TIC-LC chromatogram for peptides isolated from ipsilateral (IC) mouse brain cortex lysate using peptidomic workflow ............................... 180

4-7 Tau-441 sequences coverage (P10636-8) highlighting the peptides and phosphorylation sites detected ......................................................................... 181

4-8 Tau-758 sequences coverage (P10636-1) highlighting the peptides and phosphorylation sites detected ......................................................................... 182

4-9 Top PSM TBI-induced tau peptides from mouse brain lysate ........................... 183

4-10 Schematic representation for the TBI-generated tau peptides recovered from ultrafiltrate fractions .......................................................................................... 184

4-11 Selected TBI-induced tau peptides from mouse brain lysate based on Intensity ............................................................................................................ 185

4-12 Product ion spectrum of the tau peptide EIPEGTTAEEAGIGDTPSLEDEAAGHVTQA released from TBI-induced mouse brain ...................................................................................................... 186

4-13 Product ion spectrum of the tau peptide NVSSTGSIDMVDSPQLATLADEVSASLAKQGL released from TBI-induced mouse brain ...................................................................................................... 188

4-14 Product ion spectrum of the tau peptide SPQLATLADEVSASLAKQGL released from TBI-induced mouse brain. .......................................................... 190

4-15 Product ion spectrum of the tau peptide NVSSTGSIDMVDSPQLATLADEVSASLAKQGL released from TBI-induced mouse brain ...................................................................................................... 192

4-16 Venn diagrams comparing TBI-induced tau peptides with naive and phosphorylated versus non-phosphorylated peptides ...................................... 193

4-17 Composite summary of tau proteolytic peptides from TBI mouse brain samples ............................................................................................................ 194

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4-18 View of a customized peptigram peptide alignment map from tau, p-tau, mouse brain lysate, and CCI mouse peptides .................................................. 195

5-1 The plot for the concentration of CSF total tau in control patients (n=10) and the clinically diagnosed patients with TBI (n=12) .............................................. 207

5-2 Human CSF profile of tau phosphorylation (intact and oligomeric) for TBI and control patients ................................................................................................ 208

5-3 Schematic representation for TBI-generated tau proteolytic peptides recovered from CSF ultrafiltrate fractions ......................................................... 210

5-4 Tau-441 sequences coverage (P10636-8) highlighting the peptides and phosphorylation sites detected from TBI-CSF patients. .................................... 211

5-5 Product ion spectrum of the tau peptide AEPRQEFEVMEDHAGTYGLGDRKDQGGYT released in CSF samples in TBI subjects. ..................................................................................................... 212

5-6 Product ion spectrum of the tau peptide AGTYGLGDRKDQGGYTMHQD released in CSF samples in TBI subjects. ........................................................ 214

5-7 Product ion spectrum of the tau peptide TREPKKVAVVRT released in CSF samples in TBI subjects. ................................................................................... 216

5-8 Product ion spectrum of the tau peptide KNVKSKIGSTENL released in CSF samples in TBI subjects. ................................................................................... 218

5-9 Product ion spectrum of the tau peptide MVDSPQLATLA released from TBI-induced mouse brain. ....................................................................................... 220

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LIST OF ABBREVIATIONS

3R Three Tau Domain Binding Regions

4R Four Tau Domain Binding Regions

a.a. Amino Acid

abl Abelson Murine Leukemia Protein

AD Alzheimer’s Disease

AKT Serine/Threonine-Specific Protein Kinase B

AP Alkaline phosphatase

ATP Adenosine Triphosphate

Aβ Amyloid Β

BBB Blood-Brain Barrier

BCIP 5-Bromo-4-Chloro-3-Indolyl Phosphate

BDP Breakdown Product

BEH Ethylene Bridged Hybrid

BME Beta-Mercaptoethanol

CaM Calcium-Modulated Protein

CaMKII Ca2+/Calmodulin-Dependent Protein Kinase II

cAMP 3’,5’- Cyclic Adenosine Monophosphate

CC Contralateral Cortex

CCI Control Cortical Impact

CD Circular Dichroism

CDK5 Cyclin-Dependent Kinase 5

cDNA Complementary Deoxyribonucleic Acid

CID Collision-Induced Energy

CKII Casein Kinase Ii

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CNS Central Nervous System

CsA Cyclosporine A

CSF Cerebrospinal Fluid

CT Computed Tomography

c-tau Cleaved-Tau

CTE Chronic Traumatic Encephalopathy

CTX Primary Cerebrocortical Neuron Culture

D1 Day 1 After Control Cortical Impact



DDA Data-Dependent Acquisition

Delta CN Delta Correlation

DMEM Dulbecco's Modified Eagle Medium

DMSO Dimethyl Sulfoxide

DTT Dithiothreitol

EGFP Enhanced Green Fluorescent Protein

ELISA Enzyme-Linked Immuno-Sorbent Assay

ERK Extracellular Signal-Regulated Kinase

ESI Electrospray Ionization

ESI-MS Electrospray Ionization Mass Spectrometry

ETD Electron Transfer Dissociation

eV Electron Volts

FBS Fetal Bovine Serum

FDR False Discovery Rate

FPI Fluid Percussion Injury

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FTD Frontotemporal Dementia

FTIR Fourier-Transform Infrared Spectroscopy

Fyn Proto-Oncogene Tyrosine-Protein Kinase Fyn

GFAP Glial Fibrillary Acidic Protein

GSK3 Glycogen Synthase Kinase-3

HCD High-Energy Collisional Dissociation

HMW High Molecular Weight

htau Human Tau

IC Ipsilateral Cortex

IC50 The Half Maximal Concentration

IgG Immunoglobin G

K Kilo Dalton

kDa Kilo Dalton

Ki The Inhibitory Constant

LIT Linear Ion Trap

LMW Low Molecular Weight

MALDI Matrix-Assisted Laser Desorption Ionization

MAP Microtubule-Associated Protein

MAPKs Microtubule Associated Protein Kinases

MAPT Microtubule-Associated Protein Tau

MARK Microtubule-Associated Serine/Threonine-Protein Kinase

MBD Microtubule Binding Domain

MRI Magnetic Resonance Imaging

MRM Multiple Reaction Monitoring

MTBD Microtubule Tau Binding Domain

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mTBI Mild Traumatic Brain Injury

MWCO Molecular Weight Cut-Off

N2a Mouse Neuroblastoma-2a Cells

NBT Nitro Blue Tetrazolium

NFT Neurofibrillary Tangles

nLC-ESMS/MS

Nano Liquid Chromatography-Tandem Mass Spectrometry

NMDA N-Methyl-D-Aspartate

NMDAR N-Methyl-D-Aspartate Receptor

NMR Nuclear Magnetic Resonance

OA Okadaic Acid

OptiMEM Reduced-Serum Minimal Essential Medium

p38 P38-Mitogen-Activated Protein Kinase

PAC Puromycin N-Acetyltransferase

PCS Post-Concussive Syndrome

PEP Posterior Error Probability

PHFs Pair Helical Filaments

PKA Protein Kinase A

PKB Protein Kinase B

PKC Protein Kinase C

PP1 Protein Phosphatase 1

PP2A Protein Phosphatase 2a

PP3 Protein Phosphatase 3

PSM Peptide Sequence Matches

PSP Progressive Supra Nuclear Palsy

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PVDF Polyvinylidene Difluoride

rCHI Repeated Closed Head Injury

r-mTBI Repetitive Mild Traumatic Brain Injuries

RP-UPLC Reverse Phase-Ultrahigh Pressure Liquid Chromatography

SBDP αII-Spectrin Breakdown Product

SDS-PAGE Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Ser Serine

ST Straight Filament

STS Staurosporine

Tau-BDP Tau-Breakdown Product

TBB 4,5,6,7-Tetrabromo-2-Azabenzimidazole

TBDP Tau-Breakdown Product

TBI Traumatic Brain Injury

TBST Tris-Buffered Saline-Tween

TDP 43 TAR-DNA-Binding Protein 43

Thr Threonine

TIC Total Ion Current

UCH-L1 Ubiquitin Carboxyl-Terminal Esterase L1.

UPLC Ultra-High-Pressure Liquid Chromatography

XCorr X Correlation

Z-DCB Z-Asp-2,6-Dichlorobenzoyloxymethyl Ketone

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

NEUROBIOCHEMICAL AND PEPTIDOMIC APPROACHES TO SCREEN FOR TAU KINASE INHIBITORS AND ELUCIDATE POST-TRANSLATIONAL MODIFICATIONS

FOLLOWING TRAUMATIC BRAIN INJURY

By

Hamad Yadikar

December 2018

Chair: Richard A. Yost Major: Chemistry

Traumatic brain injury (TBI) is a multifaceted injury that generates an extensive

range of medical complications. TBI, Alzheimer disease (AD) and chronic traumatic

encephalopathy (CTE) are all examples of tauopathies that involves abnormal

hyperphosphorylation and aggregation of microtubule-associated tau proteins. Previous

studies have shown that several proteolytic signaling cascades become activated within

the cell following brain injury (e.g., calpain and caspase pathways), resulting in

truncation of tau, and generating varying lengths of fragments (MW~400-45,000 Da).

Tau hyperphosphorylation and proteolysis plays a fundamental role in causing

neurodegenerative damage as in AD, TBI, and CTE. Inhibiting a specific pathway that is

involved in tau hyperphosphorylation, proteolysis and oligomerization can mitigate

tauopathies-associated neurotoxicity and improve functional outcomes for therapeutic

approaches. We have approached this study using immunological (antibody-based

detection), and peptidomic platforms (liquid chromatography tandem mass

spectrometry) to investigate tau kinases, proteolytic peptides, and pathological

phosphorylation sites derived from in vitro, cell-based, animal TBI models and human

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cerebrospinal fluid TBI samples. From our immunoblotting analysis, we have identified

novel tau kinase inhibitors (TBB and Saracatinib), hyperphosphorylation of tau sites

(e.g., pSer202, pSer396, and pSer404), and high molecular weight proteolytic fragments

following neurotoxic conditions and animal models of TBI.

As for peptidomic analysis, we subjected the samples to ultrafiltration (10 kDa

molecular weight cut-off value), and the filtrates were analyzed using nLC-nESI-MS/MS.

Using our peptidomic approach, we were able to identify novel calpain tau cleavage

sites including G19↓L20, Q49↓T50, A89↓A90, Q124↓E125, and Thr720↓Ser721 following in vitro

digestion. We also able to identify low molecular weight calpain-mediated tau peptides

including N-terminal peptide AEPRQEFEVMEDHAGTYG and C-terminal peptides

SPRHLSNVSSTGSIDMVDSPQLATLADEVS, and STGSIDMVDSPQLA in an in vitro

and in vivo animal TBI models. As for human cerebrospinal fluid samples, we were able

to observe an increase in tau phosphorylation with TBI subjects. We identified the N-

terminal peptide AEPRQEFEVMEDHAGTYGLGDRKDQGGYT that was detected in a

truncated form in the in vitro analysis of tau. Monitoring a subset of these targets

generated from TBI and the accompanied bio-fluid might provide biomarker utilities and

applied as a potential “theranostic” tool in augmenting the clinical trials for new TBI drug

development.

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CHAPTER 1 LITERATURE REVIEW

Traumatic Brain Injury (TBI)

Traumatic brain injury (TBI) results from changes in brain function caused by

external mechanical or physical force applied to the head, leading to brain tissue

injury.(1) Examples of TBI causes include blast waves, acceleration/deceleration forces,

or penetration by a projectile such as a bullet or vehicle accidents.(2) TBI is the most

usual cause of trauma-related death and disabilities in young adults, associated mostly

with male individuals.(3)

More than 300,000 of the deployed troops in the military service have

experienced TBI from wars and military training from 2000 to 2014(1). Many military

veterans have experienced short and long-term post-TBI or post-concussive

symptoms(2, 4-6). Around half of the estimated 1.9 million Americans who were

exposed to TBI each year suffer at least certain short-term disabilities (1, 2, 7).

Moreover, TBI has been a primary cause of mortalities and disabilities around the

world with the incidence rate of ~ 10 million people(1, 2). TBI can be categorized into

direct brain injuries (in which an object breaks through the skull and dura) and closed-

head injuries (in which the brain skull and dura remains undamaged)(1). TBI can also

be classified based on the severity of the damage into mild, moderate and severe,

depending on several medical conditions such as length of injury, the severity of

consciousness, the occurrence of amnesia, neurological pathologies and medical

reports of brain imaging (e.g., CT or MRI)(7, 8).

Mild TBI (m-TBI) and concussions are terms used to define the least severe

class of TBI, and they embody 80-90% of incidents (8, 9). Symptoms of mild TBI have a

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wide range of variability. Physical symptoms include nausea, vomiting, dizziness,

headache, while cognitive symptoms include poor concentration, memory

complications, behavioral changes and loss of consciousness(10). More than 70% of

mild TBI patients can recover in 7-10 days(8). About 20% may develop long-term

neurobehavioral, psychiatric or substance abuse complications that may enhance their

vulnerability to TBI and extend the healing process(11-13). Cases, where symptoms

continue for more than three months, are diagnosed with Post-Concussive Syndrome

(PCS) (10, 14). The symptoms of PCS are unpredictable and heterogeneous, which

creates problems in diagnosing the condition. Moreover, reports estimated that 10-20%

of TBI patients develop concussion(15).

Chronic Traumatic Encephalopathy (CTE)

TBI is one of the top recognized risk factors for chronic traumatic encephalopathy

(CTE) and Alzheimer’s disease (AD). CTE is a class of neurodegenerative tauopathy,

found in individuals who experienced repetitive mild traumatic brain injuries (r-mTBI) .

Tauopathy is a class of neurodegenerative disorders linked with hyperphosphorylated

tau and insoluble aggregation leading to the formation of neurofibrillary tangles

(NFT)(16). Tauopathies also occur in Alzheimer disease, frontotemporal dementia

(FTD), progressive supranuclear palsy (PSP), cerebrocortical degeneration, Pick’s

disease, and argyrophilic grain disease(16-18).

CTE is mostly associates with athletes who took part in contact sports including

American football, ice hockey, wrestling, rugby, boxing, and soccer. Other risk factors

are the military association, local violence, and multiple head accidents(19-22).

Symptoms of CTE categorize into four phases, which mostly show 8-10 years after

exposure to r-mTBI(23-25). The first phase of symptoms consists of attention deficit

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hyperactivity disorder, behavioral changes, mental confusion, dizziness, headaches,

and disorientation. The second phase of symptoms includes memory loss, social

behavior unpredictability, and weak judgment. Third and fourth phases of CTE

symptoms include dementia, decreased mental abilities, movement disorder, deafness,

dizziness, depression, ocular abnormalities, Parkinsonism, and suicidality(19, 23-26).

CTE has a unique neuropathological appearance compared to other tauopathies,

such as AD(23, 27). CTE and AD do share a similar ratio of tau isoforms and

phosphorylation state(24). The four phases of CTE symptoms have been linked with tau

pathology in brain tissue, starting in severity from focal perivascular epicenters of

neurofibrillary tangles (NFT) in the front region of the cortex to severe tauopathy

affecting several parts of the brain. On a molecular level, the neuropathology includes

neuronal loss, microtubule-associated tau protein accumulation, TAR-DNA-binding

protein 43 (TDP-43) deposit formation, alternation in white matter, and other defects.

One major hallmark of tauopathies is the presence of hyperphosphorylated tau protein

that is vulnerable to aggregation, leading to the formation of NFT deposits (16). Up to

the present time, CTE pathology has been recognized exclusively in patients with a

history of TBI(26).

Despite the progress in characterizing CTE neuropathology, CTE is still

unknown. The main reason behind this ambiguity is due to the heterogeneity of CTE

clinical features and neuropathological presentation(26). Animal studies are being

conducted to understand CTE and r-mTBI; however, several studies have not been

successful in elucidating tau pathology seen in CTE(16, 25).

24

Comprehensive diagnosis standards for CTE need to be established in the

medical and scientific fields(28). There is still on-going research to develop diagnostic

methodologies to CTE in living people and to have a better understanding of the

possible consequences of the exposure to brain injury. Thus far, all medical evidence

associated with CTE have been collected from post-mortem autopsy of people that

have been exposed to head trauma and consist of macroscopic (gross anatomical) and

microscopic (cellular and molecular) neuropathologies (28, 29).

The Gross anatomical abnormalities from the autopsy of brains are consistent

with the primary physical manifestations of CTE(23, 30, 31). The physical signs consist

of overall shrinkage in the mass of the brain, atrophy of the frontal, temporal cortices

and medial temporal lobe, enlargement of the ventricles, anterior cavum septi pellucidi,

posterior fenestrations, pallor of the substantia nigra, and locus ceruleus (23, 25, 30).

Moreover, as CTE advances, atrophy of the hippocampus entorhinal cortex and

amygdala may be developed (23). The abnormalities linked with CTE during post-

mortem examination consist of multiple histological features that reflect extracellular and

intercellular biological mechanisms of neurodegeneration(25).

The Role of Tau Protein in Cellular Functions and Dysfunctions

Microtubule-associated protein tau (MAPT) is encoded by a single gene on

chromosome 17q21.31, crossing 16 exons. Exon 2, 3, and 10 of the MAPT gene are

alternatively spliced to generate six different tau isoforms in the human brain tissue(32,

33). Tau protein is found predominantly in neurons relative to non-neuronal cells(34,

35). While tau is mainly found in the cytoskeletal axons of neurons, it can also localize

to the nucleus, plasma membranes, and post-synaptic clefts depending on cell

requirements(5, 17, 25, 36-38).

25

Tau primary function is to bind the microtubule and assist its stabilization and

depolymerization (39). Tau modulates microtubule stability by interacting with tubulin

and promotes its assembly(39). Tau has two significant ways of controlling microtubule

stability: phosphorylation and isoforms. In a normal healthy brain, other microtubule-

associated proteins (MAPs) might be able to compensate for tau function because tau

knockout mice studies did not show significant defects in brain morphology(39). Tau has

several other roles including axonal transport, regulation of actin and neurite

outgrowth(27, 32, 36, 37, 39-41). Moreover, tau can bind to heat shock proteins and be

involved in proteasomal degradation(27).

Tau Structure

Tau consists of four functional domains including N-terminal domains (0N, 1N, or

2N inserts), the proline-rich domain, the microtubule-binding domain (MBD) with

conservative and repetitive sequences, and the C-terminal domain (Figure 1-1)(34).

Three isoforms consist of three binding regions (3R), and the other three isoforms

comprise four binding regions (4R)(39). Tau isoforms are expressed differentially during

development with the fetal brain has the shortest isoform of tau (0N3R), while all six tau

isoforms are expressed by tau genes in the adult human brain (0N2R, 1N3R, 2N3R,

0N4R)(35, 42).

Tau is a hydrophilic protein with a net basic charge(43). The carboxy-terminus of

tau protein has the binding domains and contain most of the positively charged amino

acid residues(43). The positively charged Lys residues of the binding domain allow tau

to bind the negatively charged microtubule and maintain it in the stabilized form(41).

This ionic property might cause tau to dissociate from the microtubule as it becomes

hyperphosphorylated by kinases(42). Most tau mutations found in tauopathies decrease

26

tau affinity to bind microtubule and occur in MBD(41). However, it is not proven that

these mutations cause disease, or directly associated with tauopathies(44). On the

other hand, the N-terminal domain consists of acidic amino acid residues that interact

with non-microtubular proteins(39). The isoforms with the four binding domains have

improved stabilization of microtubule compared to those with the three binding

domains(45).

Tau in solution behaves as an intrinsically disordered protein(42). A variety of

probing techniques used to include circular dichroism (CD), Fourier transform infrared

spectroscopy (FTIR), X-ray diffraction, fluorescence, and other analytical methods

suggest that this protein behave as a dynamic coil, lacking a well-characterized

secondary and tertiary structure(42). The NMR data show that 343 out of 441 amino

acids of human tau lack any ordered structure(46). Six small stretches of tau sequence

show the ability to form β-strands, while three segments of tau display poly-proline

helices. Two tau segments of 10 amino acids (114-123 and 428-437) in the N-terminal

and C-terminal domains can form α-helix structures(35, 42, 47, 48).

Tau has a highly dynamic secondary and tertiary structure, compromising many

conformations under normal and pathological conditions and a complex network of

contact with a wide range of transient proteins (35, 42, 49). Tau proteins can also

assemble into dimers, oligomers and form large polymers(42). Cysteine residues

located at the microtubule-binding domain (MTBD) of the tau protein assist the

processes of dimerization and oligomerization(50). However, polyanionic compounds

such as heparin can accelerate the formation of tau aggregates, and not necessarily

need cysteine-cysteine bridges(35, 42, 51, 52). Tau dimerization and oligomerization

27

are suggested to be toxic; yet, there is some evidence suggesting that tau trimers are

the species inducing the toxicity(42). Tau monomers can self-assemble in an anti-

parallel arrangement, through MTBDs. Tau fragments that have MTBDs can assemble

into a fibrillary polymeric structure. Small peptide fragment of only six amino acid

residues having the MTBD can form paired helical filaments (PHF)(35, 42).

Tau Post-Translational Modification

Tau undergoes a variety of post-translational modifications occurring at many

sites along the protein, with most studies focusing on tau phosphorylation(41). Human

tau protein consists of 79 possible phosphorylation sites (serine, tyrosine, and

threonine) on the longest isoform, mostly located in the proline-rich region and the C-

terminal domain(35, 37, 39, 53). At least 30 of these tau sites are phosphorylated by

kinases including: glycogen synthase kinase 3β (GSK3 β), cyclin-dependent kinase 5

(CDK5) casein kinase II (CKII), the mitogen-activated protein kinases (MAPKs),

extracellular signal-regulated kinase (ERK), p38, Jun N-terminal kinase (JNK),

Ca2+/calmodulin-dependent protein kinase II (CaMKII), microtubule-affinity regulating

kinase (MARK), protein kinase A (PKA), protein kinase C (PKC), and tyrosine kinases

including Src kinase family (Src, Fyn and c-abl) (Figure 1-2) (35-37, 53-56).

Brain development regulates tau phosphorylation such as that fetal tau is more

phosphorylated in the embryonic central nervous system (CNS) compared to adult

tau(57, 58). The level of tau phosphorylation lessens in all six isoforms with age

because of the activation of phosphatases(37, 43, 57-60). Phosphatases participate in

controlling tau dephosphorylation on specific amino acid residues, affecting its ability to

associate with the microtubules(17, 35, 39). Reduction of tau phosphatases activity may

lead to tau hyperphosphorylation instead of increased kinase activity.(61)

28

Protein phosphatase 2A (PP2A) is responsible for approximately 70% of tau

dephosphorylation in the human brain(62). PP2A activity is reduced in AD patients

compared to control subjects(62, 63). It is considered normal to have 2-3 phosphate

molecules per one tau molecule. However, when phosphate molecules exceed 7-8 on

one tau molecule, tau might be regarded as hyperphosphorylated(64). Tau toxicity is

more likely to be reflected on specific phosphorylation sites rather than the number of

residues that are phosphorylated(62, 63, 65, 66).

As stated above, tau hyperphosphorylation is one major hallmark of tauopathies

such as CTE and AD. The process of tau hyperphosphorylation alone can cause the

protein aggregation, insoluble PHF formation, and bundling of straight filaments (SF)

into NFT(64). NFT play a pivotal role in initiating several neurodegenerative metabolic

disorders(67). Studies have shown that TBI is associated with a hyperphosphorylated

tau(16, 17, 23, 25). The exact mechanism of tangle development is not fully yet

understood, and it remains controversial as to whether these tangles play a primary role

in the disease development or more of a secondary role(27).

There have been unbiased strategies to study the effect of human kinases ability

to phosphorylate tau at multiple sites linked with AD(68). Approximately 45

phosphorylation sites on tau have been linked with AD pathology (59, 69). The reported

phosphorylation sites are not always universal, but several research groups reported

Ser195, Ser202, Thr205, Thr231, Ser235, Ser396 and Ser404 as possible tau pathological

phosphorylation sites (Figure 1-2) (58, 69, 70). Many of the diseased phosphorylation

sites reported leads to attenuation of tau-microtubule assembly, promoting aggregation

process. Furthermore, dissociated tau has increased vulnerability to proteolytic

29

cleavage by calpain or caspase producing peptide fragments that are susceptible to

aggregation(71).

Several antibodies-based approaches have been conducted to associate specific

phosphorylation site(s) on tau protein with the state of disease. Filamentous tau is

observed immunohistochemically by antibodies that recognize phosphorylated tau

residues. Moreover, tau phospho-antibodies can be utilized for early detection of

pathological tau, before accumulating into the toxic NFT deposits. Antibodies raised

against a specific phosphorylated residue of tau are a precious tool to be utilized in

laboratory and pre-clinical research. It is essential to validate the specificity of these

antibodies using other analytical techniques such as immunoassays for robust and

reliable detection(16, 17, 27, 30, 39, 41).

Other tau post-translational modifications (PTMs) include acetylation,

glycosylation, deamidation, sumoylation, ubiquitination, polyamination, oxidation,

nitration, isomerization, and truncation(27). Several tau PTMs have been suggested to

be associated with the accumulation of pathological tau. For instance, tau oxidation

causes cross-linkages, aggregation and isomerization of the phosphorylated site Thr231

to cis isoform leading to decreased tau dephosphorylation, thereby reducing

microtubule binding and increasing susceptibility to aggregation(27). A study had shown

that introducing human tau oligomers into mouse brain caused diminished memory

consolidation(72). On the other hand, injecting monomeric tau or filamentous tau did not

induce neuronal loss or cognitive damage despite the progressive elevation of NFT

formation(73).

30

These tau pathologies have been linked with cases of single severe TBI, in

patients that have died from sustained concussions and in young military veterans with

a history of exposure to improvised explosive devices(4, 5, 27, 38). Tau pathology

research interest has been increasing, especially in animal models of single and r-mTBI,

and in the development of tau biomarkers for TBI and CTE(74).

Tau Proteolysis

TBI is linked with complex metabolic, biochemical and cellular dysfunctionalities

including ischemia, diffusion hypoxia, mitochondrial dysfunction, increased energy

demands, metabolites imbalances (from excitotoxicity), axonal and dendritic injuries,

neuroinflammation and neuroapoptosis (4, 27). Glutamate receptors subunits genes

were observed to be changed, which is critical because increased concentrations of

glutamate intracellularly can lead to increased production of Na2+ and Ca2+ in the

cytosol which activates protein calpain, resulting proteolysis of cytoskeletal proteins

including tau (Figure 1-4 and Figure 1-5)(75).

Tau can be proteolyzed by several proteases producing fragments of varied

sizes in vitro and in vivo. Tau proteolysis has newly generated research interest

because proteolytic tau fragments ability to contribute to aggregation process(43, 71,

76). Proteolytic fragments of tau have been detected in patients cerebrospinal fluid

(CSF) and plasma with various tauopathies, making them beneficial as biomarkers for

early disease detection(70, 71, 77, 78). Tau proteolytic fragments are classified by their

cleavage site in full-length tau, biological function, the protease involved, their use as a

biomarker and the associated disease. Many proteases cleave tau, a few fragments of

tau have been found with unknown protease(79).

31

Tau Calpain-Mediated Proteolysis

The calpain family consists of 14 different cysteine proteases that are controlled

by calcium binding to a specific site conserved in all family members(80). Calpastatin

serves as an endogenous inhibitor and the substrate for calpains, which can allow tau to

avoid proteolysis by inhibiting calpain. Calpain-1 and -2 are the two most abundant

isoforms, which vary in their sensitivity to calcium(71, 81, 82). Tau is a well-established

substrate of the calcium-dependent cysteine protease, calpain(71, 83). Proteolytic

cleavage of tau by calpain-1 can lead to the production of truncated tau break down

products (tau-BDP-35K and tau-BDP-17K) which are susceptible to aggregation(71,

83). Calpain can also degrade other cytoskeletal proteins such as microtubule-

associated protein 2 (MAP2) and αII-spectrin(4, 82).

Furthermore, calpain-mediated αII-spectrin cleavage products are used to detect

post-TBI axonal injury and neurodegeneration following TBI(84, 85). Likewise,

cytochrome c release from the mitochondria leads to activation of the proteases calpain

and caspase-3, which initiate apoptosis(76, 81, 86, 87).

In Alzheimer disease, increased calpain activity and decreased levels of

endogenous calpain inhibitor, calpastatin, have been reported(81, 86, 88). The tau

fragment 17K associated with an elevation in calpain activity in different tauopathies,

including AD, FTD, and CTE (71, 83). The 17K tau fragment has a decreased

microtubule binding ability and accumulates in the perikarya(79). Calpain-mediated 17K

tau fragment form by increased levels of calcium induced by glutamate and thapsigargin

in vitro and is well known to be neurotoxic(71, 81, 82). For instance, Amyloid β (Aβ) can

activate calpain in cultured hippocampal neurons, producing the 17K tau fragment and

cause neurodegeneration properties, which are delayed by inhibition of calpain

32

activation(81, 89). Surprisingly, other studies have shown that the 17K tau does not

cause toxicity in vitro and is expressed equally in the brain of AD and normal healthy

control samples(89-92).

Additionally, calpain is an upstream activator of extracellular-regulated kinase

(ERK), which can phosphorylate tau protein(79). Increase in ERK expression leads to

early accumulation of tau in neurons and glia in various tauopathies(93-95). Although

the function of calpain-generated 17K tau fragment in neurodegeneration is

controversial, increase in calpain activity and 17K tau breakdown products are

recognized in human tauopathies. TBI can initiate the production of calpain-mediated

17K tau fragment by increasing calcium leak from dysfunctional organelles leading to

calpain activation and tau cleavage. The susceptibility of tau to calpain and caspase-3

under neurotoxic and neurodegenerative environments has been examined(71). We

have initially reported three novel calpain cleavage sites in rat tau (4-repeat isoform) as

Ser130/Lys131, Gly157/Ala158, and Arg380/Glu381. We built fragment-specific antibodies to

target tau break down products (TBDP)-35K and TBDP-45K(71). We showed that tau

was degraded in injured rat cortex following TBI in vivo to BDPs of 45-42K, 35K. 25K

and 15K(71). Tau-BDP-35K-specific antibody confirmed these products in the injured

cortex. Intravenous administration of SNJ-1945 (calpain-specific inhibitor) inhibited the

production of tau-BDP-35K (71).

The Function of Tau proteolytic fragments in Disease

Tau fragments have been shown to grow and aggregate depending on their

amino acid sequence(35, 72). The protease-resistant PHFs and straight filaments (ST)

core of NFT are made up of tau fragments. Tau neurotoxic peptides usually have the

third and fourth repeat regions; however, the first and the second repeats might also be

33

part of the peptide depending on the ratio of 3R:4R of tau in the core. The composition

of the proteolytic fragments differs between different tauopathies. Tau fragments can

participate in the process of formation of non-filamentous tau aggregates that cause

multiple cellular defects and disruption of axonal transport, mitochondria, Golgi

apparatus, and synaptic proteins. The definitive relevance of these tau fragments to the

disease is still to be determined as well as how tau fragments and full-length tau work

synergistically to initiate several tauopathy-related diseases(79).

Tau Fragments in Biofluids as Biomarkers

A biomarker has specific characteristics that are used as a reporter to measure

standard biological processes, pathological processes, or response to therapeutical

intervention. Neurodegeneration following TBI results in tau to be dissociated from the

microtubule, elevating the levels of tau and tau-BDP in bio-fluids. Research interests are

increasing in analyzing bio-fluids such as cerebrospinal fluid, plasma, or serum tau as a

biomarker for TBI and CTE.

Serum Tau

Increased tau serum levels were observed in experimental animal TBI and

human TBI(96, 97). Serum tau escape mechanism includes direct release via blood-

brain barrier (BBB) opening, ventricular wall damage, release via the glymphatic

system, or more complex intracellular transport via macrophages or phagocytosis

microglia. In human TBI, elevation of tau serum levels might be associated with injury

severity and have been reported to reach the highest levels in two days after the

injury(98). A study has shown that cleaved-tau (c-tau) is elevated in serum after severe

TBI, however, more prospective studies are needed to establish a definite

conclusion(99).

34

CSF Tau

Due to blood biomarkers performing poorly in the detection of acute mTBI,

researchers are starting to look to the example of AD research(97). CSF has a

biomarker diagnostic potential in tauopathies of AD(70). In severe TBI, increased levels

of CSF total tau and phosphorylated tau continue to rise beyond two days and reach the

maximum levels between 5 and 15 days after the injury(70, 71, 77, 78). Moreover, CSF

sample studies following severe TBI have shown that c-tau isoforms levels increase

post-injury, suggesting that proteolytic enzymes play a critical role in neuropathological

processes contributing to mortality and morbidity(76, 83, 92, 100).

Furthermore, tau concentration in CSF increases during the acute phase of

severe head trauma, which is beneficial in determining the stage of prognosis. Ultra-

sensitive analytical methods, such as Quanterix digital Simoa assay platform, allow

single molecule detection and facilitate the analysis of plasma tau levels(101-103).

Plasma Tau

A study has shown that comparing phosphorylated plasma tau and

phosphorylated tau/total tau ratio offer an efficient and consistent way as a diagnostic

and prognostic biomarker compared to total-tau alone revealing elevations among

patient with chronic TBI(101). The control plasma samples were not collected in the

study and were bought commercially. Commercial samples might have limited

demographic and health status of data donor. The reliance on commercial samples

might affect the comparison between control and patients with TBI. It is essential to

validate the data using other types of samples such as blood collected within the same

timeframe after TBI to confirm the findings.

35

Conclusions

Collectively, severe TBI, concussions, sub-concussive hits, and repetitive mild

head injuries are adequate to cause permanent dissociations of tau protein from the

microtubules leading to accumulation of peripheral tau protein(21, 27). Neuroprotective

approaches have been hypothesized centered on the phosphorylation of tau in

pathological diseases(104). Since CTE has only been reported in patients with a

background of repetitive (sometimes single) TBI, it is probable that early start of

neuroprotective treatments might efficiently prevent the potential acute, subacute and

initial chronic secondary damages, thus decreasing the chances of tauopathy

development. Although a neuroprotective strategy can reduce the advancement of brain

damage in CTE, there is more than one single mechanism involved in cellular

dysfunctions that lead to tau neurotoxicity. Therefore, combinatorial multi-mechanistic

neuroprotective strategies are essential to prevent and interrupt CTE(27).

Peptidomics-Based Discovery of Novel Neuropeptides

Peptidomics have recently been employed as a tool for studying human body

fluids; of particular interests are the blood plasma, serum, urine, saliva, and

cerebrospinal fluid(105). Natural endogenous peptides have potent biochemical

functions in respiratory, cardiovascular, endocrine, inflammatory, nervous systems.

Most neurons have biologically active peptides together with conventional

neurotransmitters. In neurodegenerative disorders, biologically active neuropeptides are

implicated in the neuropathology(106, 107).

Calpain-mediated tau peptides are secreted into extracellular space and undergo

various post-translational modifications, e.g., glycosylation, C-terminal amidation,

acetylation, sulfation, and phosphorylation(79). Several peptidomics studies

36

characterizing CSF and blood plasma supplied alternative strategies for biomarker

diagnosis, which showed increased sensitivity to endogenous peptide alterations not

evidenced by standard proteomics approaches(107, 108). Valuable information is lost

when the sample of proteins are digested artificially with trypsin in proteomics

quantification on the measurement of tryptic peptides. Therefore, from an analytical

standpoint, studying endogenous neuropeptides decrease sources of inconsistencies,

less expensive and decrease sample preparation time, which are critical factors for

building the basis for clinical biomarker research and routine work.

Natural proteolytic peptides can be separated easily by ultrafiltration from the

high molecular weight proteins, which make up the most of CSF protein pool (e.g.,

albumin and immunoglobulins). This ultrafiltration step enriches LMW peptides for

identification by LC-MS (Figure 1-6). The potential identified peptides can be assessed

using software programs that implement different approaches for characterizing and

sequencing, which then can be correlated with peptides that are found in

neurodegenerative disorders and peptides of special interests (i.e., potential

biomarkers)(106, 109-114).

Unfortunately, several potential biomarker candidates published in peptidomics

studies could not be validated further. Technological variances for biomarker discovery

approaches should be less than 10% to expect a decent probability of detection with

sets of clinical samples. However, the most crucial factor is not the detection but the

type, quality, and quantity of the clinical samples being researched as well as the study

design(110, 114-117).

37

Current Mass Spectrometric Peptidomic Technologies

Mass spectrometry technology has undertaken a tremendous development since

the invention of biopolymer ionization. All critical parameters of mass spectrometry have

been improved dramatically including ionization, quantification, resolution, time needed

for sample MS and identification methods, driven by innovation in analytical chemistry.

A considerable increase in the sensitivity of ESI-MS was achieved by the development

of micro and nano-electrospray, allowing the analysis of mass spectra from samples of

femto to a picomolar concentration of peptides. Nano-electrospray ionization has

become the standard platform in peptidomic. MALDI-MS studies also delivered similar

sensitivity with a very fast screening for hundreds of samples, allowing a new way of

assaying peptides(118).

The use of tandem MS (MS/MS) was another major pivotal breakthrough for the

identification of peptides and to emerge the ideas of peptidomics. Chemical

derivatization of peptides was overcome by using the mass spectrometric methodology

known as collision-induced dissociation (CID) for sequencing peptides. The combination

of nano-LC-ESI-MS/MS and CID delivered more robust data without the need of extra

chemical sample preparation and significantly improved throughput and speed of

peptide fragmentation and sequencing processes. N-terminal chemical sequencing still

complements these methods for proteolytic peptide or neuropeptide discovery(105).

Electron transfer dissociation (ETD) is another applied technique that transfers a

“soft” electron from singly charged anthracene anions to multiply protonated peptides to

trigger fragmentation, avoiding vibrational energy as in CID(119). Another technique is

the higher energy collisional dissociation (HCD), specific to the Orbitrap mass

spectrometer in which fragmentation occurs external to the trap. One advantage of HCD

38

is that it does not suffer from the low mass cutoff of resonant-excitation and therefore

used for isobaric tag-based quantification as reporter ions can be observed(120). The

combination of HCD and ETD were effectively used for peptidomics experiments. The

development of the orbitrap mass analyzer provided a further increase in the quality of

MS/MS data analysis by substantially increasing the resolution, mass accuracy and

speed for thousands of peptides identification(121).

The Scope of the Dissertation

The work presented in this dissertation is intended to highlight the use of tau

kinase inhibitors, post-translational modification and peptidomic approaches for studying

TBI and identifying novel biomarkers that can be used for possible diagnosis and

therapeutical (i.e., theranostic) strategies. This chapter introduced TBI, CTE, and tau

protein in normal and pathological functions. This chapter also gave a brief introduction

of the analytical strengths of using peptidomic platforms to provide useful data for

assessing disease state. The peptidomic approach presented will be used throughout

the remainder of this dissertation to assess tau peptidome from in vitro, mammalian

neuronal culture, animal TBI models, and human CSF-TBI samples. Chapter 2 will

focus on screening tau kinases as drug inhibitors in mouse neuroblastoma cells and

primary neuronal culture. Kinases that are relevant to tau proteolysis, phosphorylation,

and aggregation will be highlighted to be applied for future animal studies and

translational research approaches. Chapter 3 aims to characterize neurotoxic tau

peptide fragments produced in vitro from calpain-1 or under cell-based neurotoxic

challenges. We will perform in vitro calpain-1 digestion of purified human tau-441(non-

phosphorylated and phosphorylated) and naïve transgenic human tau mice cortices.

The samples will be subjected to ultrafiltration (10K MWCO) to study high molecular

39

weight (HMW, retentates) and low molecular weight (LMW, filtrates) tau products by

western blotting and nLC-ESI-MS/MS, respectively. In cell culture-based experiments,

okadaic acid (OA), a phosphatase 1A/2B inhibitor, will be used to induce tau

hyperphosphorylation and test susceptibility of tau to proteolysis when challenged by

the neurotoxic conditions. In chapter 4, we will employ a translational approach from our

findings (in vitro and cell-based studies) for analysis of TBI-induced animal models

samples. Chapter 5 will aim to characterize tau post-translational modification levels

and temporal profile in human cerebrospinal fluid from severe TBI and control subjects.

Those chapters will highlight potential tau biomarkers, cleavage sites,

phosphorylation sites, and fragments that can be used to differentiate TBI and normal

healthy populations. Finally, chapter 6 will provide a summary and conclusions of the

work presented in this dissertation with the future directions that will be conducted.

40

Figure 1-1. Human brain tau protein isoforms. Alternative splicing of exons 2, 3, or 10 produces a different combination of six tau isoforms. The N-terminal domain, N1 (green) and N2 (yellow), are generated from exon 2 and 3, respectively. Exon 10 encodes the second microtubule binding repeat domain, R2 (red). Tau proteins might be either 3R or 4R depending on the presence of R2.(122)

https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=2ahUKEwiV5bWV6d3bAhUE3FMKHf3SCn4QjRx6BAgBEAU&url=https://www.nature.com/articles/nrn.2015.1&psig=AOvVaw0syiTDMYzv3SYYGdpBk96h&ust=1529432110738050

41

Figure 1-2. Phosphorylation sites on tau protein and specific epitopes for tau antibodies. A). Orange color denotes to antibodies that recognized phosphorylated amino acid residues. Blue color donates to antibodies that recognize total tau. B) The accumulation of hyperphosphorylated tau in neurons (as neurofibrillary tangles (NFTs) and neurites) showing a different pattern in FD, AD and CTE.(123)

42

Figure 1-3. Tau phosphorylation events are sequential in the development of tau pathology. In the first stages, the first stages are those that decrease the affinity of tau protein to associate with the microtubule and increase of the ability of tau to self-associate. These events cause an imbalance between the activity of specific tau kinases and phosphatases. Phosphorylation of tau in specific pathological sites leads to the formation of pair helical filaments (PHF) and neurofibrillary tangles (NFT) causing neurodegeneration. Red block sign represents inhibition.

43

Figure 1-4. Schematic of tau proteolysis and its role in tauopathies. During tauopathy, tau is cleaved by several proteases into fragments that are neurotoxic and contribute to tau aggregation and NFT formation. Kinases phosphorylate tau proteolytic fragments at pathological sites, which increase their tendency to be hyperphosphorylated and aggregated. Tau can also be proteolytically cleaved when it is attached to the microtubule leading to its loss of function. Tau hyperphosphorylation, tau oligomers and tau proteolytic fragments all contribute to neurodegeneration.(79)

44

Figure 1-5. TBI-relevant pathways associated with neuronal death. Extracellular TBI

damage induces increased intraneuronal calcium influx through several different mechanisms, including channel opening and receptor activation. Calpain and caspase activation leads to tau proteolysis including both N- and C-terminal tau fragments and the activation of tau kinases. These fragments are vulnerable to aggregation and hyperphosphorylation resulting activation of neuronal death pathways.

45

Figure 1-6. Workflow for peptidomics platforms, which are divided into five main steps compromising different types of methods.

46

CHAPTER 2 SCREENING OF TAU PROTEIN KINASE INHIBITORS IN A TAUOPATHY-RELEVANT

CELL-BASED MODEL OF TAU HYPERPHOSPHORYLATION AND OLIGOMERIZATION

Introduction

Tauopathy is a multifaceted disorder associated with several neurodegenerative

diseases including traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE),

Alzheimer’s disease (AD). Significant tauopathy hallmarks are the presence of

hyperphosphorylated and aggregated forms of tau(16).

Tau is a structural protein that promotes microtubule stability and tubulin

assembly depending on its phosphorylation state(16, 17, 30). Various kinases and

phosphatases regulate tau phosphorylation state, and its ability to interact with other

microtubule proteins(37, 62). Imbalances in the activities of these kinases and

phosphatases can cause tau protein to be hyperphosphorylated at specific residues and

dissociated from microtubules. Dissociated tau from microtubules has increased

susceptibility to forming larger protein aggregates, filamentous assembly, and bundling

of pair helical filaments (PHF) into neurofibrillary tangles (NFT) causing cellular

neurotoxicity(16, 17, 30, 124, 125).

Pharmaceutical inhibition of tau kinases is one crucial strategy of Neurotherapy

(53). One hypothesis suggests that inhibition of tau kinases leads to decreased tau

hyperphosphorylation and thereby, less aggregated tau. A few protein kinase inhibitors

have been targeted clinically(4, 19, 55, 126-128). Examples of tau kinases being

targeted pharmaceutically are glycogen synthase kinase 3β (GSK-3β) and cyclin-

dependent kinase 5 (CDK5) tested in Alzheimer disease (AD) patients(56, 129).

47

Additionally, casein kinase I (CKI) is thought to play a role in hyperphosphorylation of

tau in AD(130, 131).

The role of CKI in neurodegenerative disease has been more extensively studied

than CKII in the recent years(130-132). Notably, a study reported that the hippocampus

and temporal cortex regions of the brain had increased levels of CKII in AD patients

when compared to controls(132). Furthermore, one kinase protein in the Src family

known as Fyn have been highly researched for its implications with tauopathy and

neurodegeneration in the post-synapse N-methyl-D-aspartate receptors (NMDAR)(54,

133-135).

Tau phosphorylation sites including: Ser202, Ser396/404, Thr108, Thr181, Thr205, and

Thr231 have been reported to affect tau association with microtubules(59, 60, 69, 130).

Analyzing these phosphorylation sites might help to show a pattern of relationships

between tau protein phosphorylation and pathology. Okadaic acid (OA) is a protein

phosphatase 1 and 2A (PP1/2A) inhibitor that can induce tau hyperphosphorylation at

pathological sites associated with diseases in both animal and cell-based models(40,

65, 66). In this study, we treated mouse neuroblastoma N2a cells and rat primary

cerebrocortical neuronal (CTX) culture with OA to induce tau hyperphosphorylation and

oligomerization, mimicking a tauopathy-relevant cell-based model. We used different

tau protein kinase inhibitors to test their efficacy in reducing tau hyperphosphorylation

and oligomerization.

We hypothesize that using OA-induced tau hyperphosphorylation and

oligomerization as a tauopathy-relevant cell-based model to screen for tau kinase

inhibitors might help to translate novel neurotherapeutic targets for clinical trials. Data

48

from our work have shown the various efficacies of the kinase inhibitors in inhibiting the

OA-induced tau hyperphosphorylation and oligomerization. These inhibitors need to be

assessed and validated further in different in vivo tauopathy-related models.

Materials and Methods

Inhibitors

Inhibitors used include: ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-

tetraacetic acid (EGTA) (Sigma-Aldrich, St-Louis, MO, USA), Dithiothreitol (DTT)

(Sigma-Aldrich), Lithium chloride (LiCl) (Sigma-Aldrich), N-(4-methoxybenzyl)-N'-(5-

nitro-1,3-thiazol-2-yl)urea (AR-A014418) (Sigma-Aldrich), (9S,10R,12R)-2,3,9,10,11,12-

Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-

kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (K252a) (Sigma-

Aldrich), (2R)-2-1-butanol (Roscovitine) (Sigma-Aldrich), 4,5,6,7-Tetrabromo-2-

azabenzimidazole (TBB) (Sigma-Aldrich), 1-(7-methoxyquinolin-4-yl)-3-(6-

(trifluoromethyl)pyridin-2-yl)urea (A-1070722) (Sigma-Aldrich), cyclosporine A (Sigma-

Aldrich), N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-

(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (Saracatinib) (Selleck Chemicals,

Houston TX), (5S,6R,7R,9R)-6-methoxy-5-methyl-7-(methylamino)-6,7,8,9,15,16-

hexahydro-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-

as-indacen-14(5h)-one (STS) (ab120056; Abcam, Cambridge, MA, USA), Z-Asp-2,6-

Dichlorobenzoyloxymethyl Ketone (Z-DCB) (Cayman Chemical, Ann Arbor Michigan)

and okadaic acid (Cell Signaling Technology, Danvers, MA). SNJ-1945 was a gift from

(Senju Pharmaceutical Co. Ltd., Kobe, Japan) (Table 2-2).

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Antibodies

The antibodies that were used in this study were: phospho-tau monoclonal

antibodies PHF-1 (pSer396/pSer404, 1/1000), CP13 (pSer202, 1/1000), RZ3 (pThr231,

1/1000), AT8 (pSer202/pThr205, 1/1000) , AT270 (pThr181, 1/1000) and total tau

monoclonal antibodies: DA9 (aa102-140, 1/1000), DA31 (aa150-190, 1/1000) (gifts from

Professor Peter Davies, Albert Einstein College of Medicine, Bronx, NY), polyclonal total

tau DAKO (aa243-441, 1/5000) (CiteAb, England). Mouse anti-αII-spectrin (ENZO Life

Sciences, Farmingdale, NY, USA, 1/5000). β-actin was used as protein loading control

(Abcam, Cambridge, MA, USA, 1/3000) (see Table 2-1).

Cell Line

Brain Mouse neuroblastoma N2a cells were purchased from American Type

Culture Collection (ATCC #CRL-2266, Manassas, VA, USA) and were grown as

recommended by the manufacturer. The cells were grown in 1:1 Dulbecco’s modified

Eagle medium: reduced serum Eagle’s minimum essential media (DMEM: Opti-MEM)

supplemented with 5% FBS (Thermo-Fisher), 100 units/mL penicillin and 0.1 mg/mL

streptomycin. Cells were incubated at 37°C in a humidified 5% CO2-containing

atmosphere.

Primary Cerebrocortical Neuronal Cultures

Cerebrocortical cells harvested from a homogenized pool of ten 1-d-old

Sprague–Dawley rat brains were plated on poly-L-lysine-coated (0.01% (w/v)) 12-well

culture plates (Erie Scientific, Portsmouth, NH, USA) similar to previously described

methods(71) at a density of 4.36 × 105 cells/ml. Cells were grown in Neurobasal® media

(Thermo Fisher), supplemented with 1% B-27 (Thermo Fisher), 1 mM Glutamine

50

(Thermo Fisher) and incubated at 37°C in a humidified 5% CO2-containing atmosphere.

The medium was replaced every three days.

Cell Treatments

For N2a cells treatments, complete media was replaced with serum-free DMEM

media. For primary cultures, all experiments were performed after ten days in culture,

and the media was replaced with Neurobasal® media supplemented with 0.5% B-27.

For both cell culture types, SNJ-1945 (100 µM) and Z-DCB (60 µM) were added to all

conditions before the treatment for 1 hour. This was followed by treatment with protein

kinase inhibitors K252a (10 µM), AR-A014418 (60 µM), A-1070722 (60 µM), Saracatinib

(100 µM), LiCl (5 mM) TBB (60 µM), EGTA (5 mM), Roscovitine (60 µM), STS (0.5 µM),

CsA (60 µM) (if added) (Table 2-2) for 1 hour followed by OA (100 nM) for 6 hours or 24

hours.

Cell Lysate Collection and Preparation

After the treatment, conditioned media were collected from each well, added into

separate tubes on ice, and centrifuged at 10,000 x g for 10 min at 4°C. Lysis buffer was

added to the attached cells on the 12-well plates (100 µl per well). The Triton-X lysis

buffer included 1mM DTT, 1% phosphatase inhibitors (Sigma), 1% Mini-Complete

protease inhibitor cocktail tablet (Roche Biochemicals) and 1% Triton X-100. The

attached cells were then scraped down into the lysis buffer and collected into separate

1.5 ml Eppendorf tubes. The insoluble pellets from the conditioned culture media were

combined with the lysed cells in the lysis buffer. The cell lysates were incubated for 90

minutes at 4°C and then centrifuged at 15,000 rpm for 15 minutes to remove cell debris.

51

SDS–PAGE and Western Blots

Protein concentrations of cell lysates were determined by bicinchoninic acid

microprotein assays (Pierce Inc., Rockford, IL, USA) against albumin standards. Equal

protein samples (20 μg) were prepared for SDS–PAGE in 8 x loading buffer containing

0.25 M Tris (pH 6.8), 2 mM DTT, 8% SDS, and 0.02% bromophenol blue. Each sample

was subjected to SDS–PAGE electrophoresis on 4-20% precast-gels (Bio-Rad), and

then transferred on to PVDF membranes.

The membranes were blocked in 5% milk for 1 hour and then incubated with

primary antibodies overnight. The secondary antibodies (Amersham Biosciences, UK,

1/10,000) anti-rabbit or anti-mouse IgG conjugated to alkaline phosphatase (Amersham,

Piscataway, NJ, USA), were then added for 1 hour at room temperature. The blots were

then washed with TBST, and immunoreactive bands were visualized by developing with

biotin, avidin-conjugated alkaline phosphatase, nitro blue tetrazolium, and 5-bromo-4-

chloro- 3-indolyl phosphate (BCIT/NBT) developer (KPL, Gaithersburg, MD, USA). A

250K to 14K-rainbow molecular weight marker (RPN800E, GE Healthcare, Biosciences,

Pittsburgh, PA, USA) was used to identify the protein. Quantitative evaluation of protein

levels was performed via computer-assisted densitometric scanning (NIH ImageJ,

version 1.6 software).

Statistical Analysis

Statistical analysis was performed with one-way ANOVA Tukey’s Test. For

multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was

performed. *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001, ns: non-significant. GraphPad

Prism 7.0 (GraphPad, La Jolla, CA).

52

Ethical Statement

All animal studies conformed to the guidelines outlined in the ‘Guide for the Care

and Use of Laboratory Animals’ from the National Institutes of Health and were

approved by the University of Florida.

Results

Okadaic Acid (OA) Induces Tau Hyperphosphorylation and Oligomerization at Different Time Points in Mouse Neuroblastoma N2a Cells.

Okadaic acid (OA), a protein phosphatase 1/2A inhibitor (PP1/2A), is reported to

induce tau hyperphosphorylation in mouse neuroblastoma cells (N2a)(136), human

neuroblastoma cells (SH-SY5Y)(137), and rat brain(65). Therefore, we treated N2a cells

with okadaic acid (100 nM) to induce tau hyperphosphorylation for six and 24 hours

(Figure 2-1a, b). Western blots were analyzed with total tau monoclonal antibody DA9

(aa102-140) and monoclonal phospho-tau antibodies including CP13 (pSer202) and

PHF-1 (pSer396/pSer404) (Table 2-1). β-actin was used as a loading control.

Untreated control showed that total tau antibody DA9 detected tau protein bands

at 46 kDa (non-phosphorylated tau), 48 kDa (phosphorylated tau) and high molecular

weight bands detected at 165 kDa and 170 kDa (red arrows in Figure 2-1a, b). The

band at 46 kDa (non-phosphorylated tau) was detected at higher densitometric levels

compared to the band at 48 kDa (phosphorylated tau) in control samples. Probing with

DA9 showed that treatment with OA (100 nM) for 6h and 24h dramatically decreased

levels of the 46 kDa (non-phosphorylated tau), increased the levels of 48 kDa

(phosphorylated tau) and increased levels of 170 kDa, presumably accounting for the

formation of tau oligomers (Figure 2-1a, b). This effect was seen to a lesser extent with

53

OA treatment for six hours, as tested by DA9 antibody compared to the 24 hours OA

treatment (Figure 2-1a, b).

Probing with CP13 (pSer202) antibody did not show any bands of tau protein in

control samples. However, with OA treatment, CP13 antibody detected a band formed

at 110 kDa (oligomeric tau form) observed at both 6h and 24h (Figure 2-1a, b). As for

probing with the PHF-1 antibody (pSer396/pSer404), control samples showed low levels of

240 kDa band and 260 kDa bands (PHF-1, indicated in red arrow) (Figure 2-1a, b).

Treatment with OA for 6h and 24h showed a major band detected at 220 kDa and a

dramatic increase of levels of 240 kDa and 260 kDa bands (Figure 2-1a, b).

Interestingly, the 260 kDa band (oligomeric tau; red arrow) (Figure 2-1a) was

detected with PHF-1 with OA treatment for 24 h only. β-actin protein as a loading control

showed no protein level changes across treatment. Taken together, these data suggest

that OA treatment induced tau hyperphosphorylation and oligomerization in N2a cells

and was detected by with different specific phospho-epitope tau antibodies including

pSer202 (CP13), pSer396/pSer404 (PHF-1), and total tau (DA9) antibody. For the following

experiments, OA treatment for 24 h was chosen since it achieved more robust tau

hyperphosphorylation and oligomerization as shown in the immunoblotting data (Figure

2-1a, b).

Testing Tau Kinase Inhibitors on OA-Induced Tau Hyperphosphorylation and Oligomerization in N2a Cells.

In order to test for potential tau protein kinase inhibitors as drug candidates,

mouse neuroblastoma N2a cells were pre-treated with or without various kinase

inhibitors including: LiCl (10 mM), AR-A014418 (AR) (60 µM), A-1070722 (A107)(60

µM), K252a (10 µM), STS (0.5 µM) 4,5,6,7-tetrabromobenzotriazole (TBB) (60 µM),

54

Roscovitine (60 µM), Saracatinib (100 µM), cyclosporine A (CsA) (60 µM) and EGTA (5

mM) for 1 hour followed by treatment with OA for 24 hours (Table 2-2; Figure 2-2a, b).

Cells were treated with or without SNJ-1945 (calpain inhibitor, abbreviated as S; 60 µM)

and Z-DCB (caspase, inhibitor abbreviated as Z; 100 µM) to avoid calpain and caspase-

mediated proteolysis(87, 138) (see table 2-2 for the kinase inhibitors effects against

their targets).

Casein kinase II (CKII) inhibitor: 4, 5, 6, 7-tetrabromobenzotriazole (TBB)

Probing with total tau DA9 showed that TBB treatment completely inhibited the

48 kDa band (phosphorylated tau), 170 kDa band (oligomeric tau) and increased levels

of the 46 kDa (non-phosphorylated tau) by 85% (Figure 2-2a, b and Table 2-3).

Furthermore, CP13 (pSer202) antibody showed that TBB caused complete

inhibition of the OA-induced 110 kDa band (oligomeric tau form). Additionally, probing

with PHF-1 antibody (pSer396/pSer404) showed that TBB restored the 220 kDa, 240 kDa,

and 260 kDa bands (tau oligomers) to control levels (Figure 2-2a, b). As a selective

casein kinase II (CKII) inhibitor, TBB showed robustness in inhibiting both OA-induced

tau hyperphosphorylation and oligomerization. Therefore, we aimed to test its dose-

response effect on OA-induced tau hyperphosphorylation and oligomerization in N2a

cells. To achieve this, cells were pretreated with various concentrations of TBB (10 nM,

30 nM, 100 nM, 300 nM, 1 µM, 3 µM, 10 µM, and 30 µM) for 1h followed by 24 h OA

treatment (Figure 2-3a, b).

Our result shows that treatment with ten micromolar of TBB resulted in a 50%

reduction of the 110 kDa (oligomeric tau form) probed by CP13. Moreover, total tau DA9

detected a 50% loss of 48 kDa (monomeric phospho-tau) and 170 kDa (oligomeric tau)

bands with ten micromolar of TBB treatment (Figure 2-3a, b). Increasing the

55

concentration of TBB up to 30 µM caused at least 90% reduction of 48 kDa (monomeric

phospho-tau, DA9), 170 kDa (oligomeric tau, DA9) and 110 kDa (oligomeric tau form,

CP13) (Figure 2-3a, b).

Calcineurin inhibitor: cyclosporine A (CsA)

Aside from its well-established inhibitory effect on calcineurin phosphatase

activity, CsA has been reported to inhibit Ca2+/calmodulin-dependent protein kinase II

(CaMKII) by blocking the Ca++ mitochondrial permeability(139). Therefore, CsA was

used in this study to test its effectiveness in the inhibition of Ca++-dependent kinases.

Interestingly, CsA fully restored the 110 kDa oligomeric phospho-tau band (CP13) and

240 kDa oligomeric tau form band (PHF-1) back to normal control levels and eliminated

the monomeric phospho-tau 48 kDa band (DA9) as well as oligomeric 170 kDa band

(DA9) (Figure 2-7, Table 2-3). Moreover, two additional antibodies were tested, RZ3

(pT231) and AT270 (pT181), to assess for different pathological phosphorylated tau

epitopes. Probing with RZ3 showed a significant band detected at 48 kDa with OA

treatment, which was completely inhibited when cells were treated with CsA (Figure 2-7,

Table 2-3). As for AT270 antibody, untreated samples showed a band detected at 48

kDa, presumably accounting for basal phosphorylated monomeric tau protein. AT270

antibody showed that CsA treatment completely inhibited the formation of OA-induced

170 kDa band (oligomeric tau form) (Figure 2-7, Table 2-3). β-actin protein levels

remained even in all experimental conditions.

Calcium chelator: EGTA

EGTA is a chelating agent that has a lower affinity for magnesium compared to

EDTA, making it more selective for calcium ions(140). Therefore, EGTA was used in

this study to block calcium-dependent kinases such as Ca2+/calmodulin-dependent

56

protein kinase II (CaMKII)(140). Total tau DA9 showed that EGTA treatment (with

calpain and caspase inhibitors added) caused: 11% reduction of the 48 kDa band

(phospho-tau), 85% increase of the 46 kDa band (non-phospho-tau) and 55% reduction

of the 170 kDa band (oligomeric tau form) (Figure 2-2a, b and Table 2-3). As for the

phospho-tau levels after OA treatment, EGTA caused 90% reduction of 110 kDa band

(oligomeric tau) detected at by CP13 (pSer202) and 85% reduction of 240 kDa

(oligomeric tau) detected by PHF-1 (pSer396/pSer404) (Figure 2-2a, b and Table 2-3).

Addition of caspase-3 and calpain inhibitors (Z DCB and SNJ-1945, respectively)

showed that part of the EGTA effect is due to apoptotic pathways as validated by the

αII-spectrin antibody (red arrows) (Figure 2-2a). We have reported in a previous study

that treatment with apoptotic-inducer EDTA cause caspase-mediated tau truncation(71).

Due to structural resemblance, EGTA might have a similar mechanism of inducing

neurotoxicity to the cultured cells.

GSK3 inhibitors: LiCl, A-1070722, and AR-1014418

To test the role of GSK3 in our tauopathy-relevant cell-model, LiCl, A-1070722

(A-107) and AR-1014418 (AR) were selected for the study. For phospho-tau levels after

OA treatment, LiCl showed an opposite effect by increasing levels of 110 kDa

oligomeric form (CP13; -14%) and levels of 240 kDa band (PHF-1; -9%) (Figure 2-2a, b

and Table 2-3). As for total tau DA9 antibody, LiCl showed an opposite effect of

increasing the 48/46 kDa (phospho/non-phospho-tau) band ratio by 20% and 170 kDa

oligomeric tau band by 12%. AR did not show a statistically significant effect on the

oligomeric form 240 kDa band (PHF-1) or 110 kDa oligomeric form band (CP13) (Figure

2-6, Table 2-3).

57

Moreover, probing with total tau DA9 showed that AR treatment caused an

opposite effect by increasing the 48 kDa/46 kDa ratio (monomeric tau form; 50%) and

reducing the 170 kDa oligomeric form band by 36% (Figure 2-6, Table 2-3). As for A-

107, phospho-tau levels after OA treatment showed 23% reduction of 110 kDa

oligomeric form band (CP13) and non-significant but partial effect on the 240 kDa

oligomeric form (PHF-1). Probing with total tau DA9 showed that A-107 caused 13%

reduction of 170 kDa band (DA9) and did not show a statistically significant effect on the

48 kDa/46 kDa ratio (Figure 2-6, Table 2-3).

Src/Fyn kinase inhibitor: Saracatinib

Saracatinib, a known potent Src/Fyn kinase inhibitor(54), was selected to

investigate the role of Fyn kinase on the tauopathy-relevant cell-based model. Probing

with total tau DA9 showed that Saracatinib did not affect the 48 kDa band

(phosphorylated tau) and 46 kDa (non-phosphorylated) monomeric tau band.

Saracatinib caused only a10 % reduction of the 170 kDa band (oligomeric tau form),

detected by DA9 (Figure 2-6, Table 2-3). As for the phospho-tau antibodies after OA

treatment, Saracatinib partially reduced the 110 kDa oligomeric form band (15%; CP13)

and did not affect the 240 kDa oligomeric form band detected by PHF-1 (Figure 2-6,

Table 2-3).

To investigate the synergistic effect of multiple protein kinase inhibitors on OA-

induced tau hyperphosphorylation and oligomerization, various protein kinase inhibitors

were combined following OA treatment for 24 h (Figure 2-6, Table 2-3).

Our results show that combining TBB and Saracatinib caused an additional 21%

reduction of the oligomeric form 170 kDa band (DA9) compared to TBB treatment alone.

Combination of Saracatinib and A107 showed 19% reduction of oligomeric form at 110

58

kDa band (CP13) and 71% reduction of oligomeric form 170 kDa band (DA9) (Figure 2-

6, Table 2-3). Surprisingly, combining AR with TBB caused opposite effect by reducing

the effectiveness of TBB inhibition at 110 kDa (CP13) by -20%, 240 kDa (PHF-1)

by`14%, and 170 kDa (DA9) by 20% compared to TBB treatment alone. AR and TBB

combination caused reduction of tau oligomeric form bands by 65% of 110 kDa (CP13),

86% of 240 kDa (PHF-1) and 80% of 170 kDa (DA9) (Figure 2-6, Table 2-3).

Combination of Saracatinib, A107, or AR, did not show a statistically significant effect on

the epitopes pSer202 (CP13) and pSer396/pSer404 (PHF-1) (Figure 2-6, Table 2-3).

CDK5 inhibitor: Roscovitine

Tau protein can be a hyperphosphorylated by cyclin-dependent kinase 5 (CDK5)

in pathological conditions(56, 60, 141). To investigate this, Roscovitine was selected in

this study to test its effect on OA-induced tau hyperphosphorylation and oligomerization.

Surprisingly, Roscovitine showed an opposite effect to what we expected by increasing

levels of oligomeric tau detected at 170 kDa (-51%; DA9), 110 kDa (-11%; CP13) and

240 kDa (-53%; PHF-1) at different epitopes tested (Figure 2-2a, b, Table 2-3). β-actin

protein levels remained even in all experimental conditions.

Baseline and OA-Induced Tau Hyperphosphorylation and Oligomerization: Effects of Various Kinase Inhibitors Treatment in Rat Primary Cerebrocortical Neuronal (CTX) Culture.

To expand our experimental paradigm in a cell-based model suitable for drug

candidate screening, we investigated the effectiveness of protein kinase inhibitors on rat

primary cerebrocortical neuronal (CTX) cultures. In contrast to N2a cells, we did not

observe oligomeric forms of tau protein when tested by total tau (DA31) and phospho-

tau antibodies in CTX cells. Notably, untreated control samples showed basal levels of

phosphorylated tau (67 kDa) detected by total and phospho-tau antibodies including:

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DA31 (aa150-190), CP13 (pSer202), RZ3 (pThr231), PHF-1 (pSer396/pSer404), AT8

(pSer202/pThr205), and AT270 (pThr205) (Figure 2-4, Table 2-1).

Therefore, CTX cells were pre-incubated with or without protein kinase inhibitors

for 1h (Table 2-2) followed by treatment with or without OA (100 nM) for 24h (OA-

induced hyperphosphorylation or baseline tau phosphorylation). SNJ-1945 and Z-DCB

were added to all experimental conditions to prevent calpain, caspase, or both

proteolytic action as a potential confound. Treatment with OA for 24 h caused a

dramatic increase of 67 kDa band (phospho-tau) at multiple phospho-tau epitopes

(CP13: 9x, RZ3: 9.8x, PHF-1: 13x, AT8: 3x, and AT270: 10x) (Figure 2-4).

Treating CTX cells with TBB reduced basal and OA-induced tau phosphorylation

(67 kDa) at CP13 (-OA: 91%, +OA: 98%), RZ3 (-OA: 100%, +OA: 100%), PHF-1 (-OA:

100%, +OA: 100%), AT8 (-OA: 91%, +OA: 100%), AT270 (-OA: 100%, +OA: 100%)

(Figure 2-4, Table 2-4). As for total tau DA31, TBB treatment caused reduction of the

phospho-tau band at 67 kDa (-OA: 41%, +OA: 91%) and an increase of non-phospho

tau band at 63 kDa (-OA: +53%, +OA: +81%) (Figure 2-4, Figure 2-5, and Table 2-4).

In contrast to N2a cell, LiCl caused considerable reduction of basal and OA-

induced tau phosphorylation (67 kDa) in CTX culture at CP13 (-OA: 94%, +OA: 50%),

RZ3 (-OA: 89%, +OA: 100%), PHF-1 (-OA: 98%, +OA: 100%), AT8 (-OA: 100%, +OA:

81%), AT270 (-OA: 100%, +OA: 100%) and total tau DA31 (-OA:93%, +OA:96%)

(Figure 2-4, Figure 2-5; Table 2-4). Similarly, AR showed a similar trend on basal and

OA-induced tau hyperphosphorylation (Figure 2-4, Figure 2-5; Table 2-4). Moreover,

LiCl and AR showed increased levels of the band 63 kDa (non-phosphorylated tau),

when probed by total tau DA31 (Figure 2-4, Figure 2-5; Table 2-4).

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Moreover, treating CTX cells with A107 showed substantial inhibition of basal tau

phosphorylation and OA-induced phosphorylation at CP13 (-OA: 92%, +OA: 36%), RZ3

(-OA: 79%, +OA: 70%), PHF-1 (-OA: 65%, +OA: 85%), AT8 (-OA: 82%, +OA: 21%),

AT270 (-OA: 100%, +OA: 100%) and total tau DA31 (-OA: 80%, +OA: 55%) (Figure 2-4,

Figure 2-5; Table 2-4). As for Roscovitine treatment, basal and OA-induced tau

hyperphosphorylation were reduced considerably at CP13 (-OA: 85%, +OA: 63%), RZ3

(-OA: 42%, +OA: 91%), PHF-1 (-OA: 63%, +OA: 81%) and total tau DA31 (30%).

However, Roscovitine had no effect on OA-induced tau phosphorylation at AT8 and

AT270 (Figure 2-4, Figure 2-5; Table 2-4).

On the other hand, CsA had dramatic inhibition on basal tau phosphorylation at

CP13 (90%), RZ3 (91%), PHF-1 (89%), AT8 (95%), AT270 (92%) and total tau DA31

(67 kDa, 95%) (Figure 2-6, Table 2-4). However, when OA added, CsA showed

considerable effect at the epitopes: CP13 (33%), AT8 (86%) and total tau DA31 (28%).

CsA had no effect on OA-induced tau hyperphosphorylation at PHF-1, AT270, and RZ3

(Figure 2-6, Table 2-4). Interestingly, Saracatinib caused reduction of basal and OA-

induced tau hyperphosphorylation at CP13 (-OA: 41%, +OA: 100%), RZ3 (-OA: 81%,

+OA: 100%), PHF-1 (-OA: 52%, +OA: 100%), AT270 (-OA: 0%, +OA: 84%) and total

tau DA31 (-OA: 5%, +OA: 20%). Saracatinib did not show any significant effect at AT8

phospho-tau epitope (Figure 2-4, Figure 2-5; Table 2-4).

Treatment with K252a caused substantial inhibition of basal and OA-induced tau

hyperphosphorylation at CP13 (-OA: 35%, +OA: 41%), RZ3 (-OA: 45%, +OA: 37%),

PHF-1 (-OA: 61%, +OA: 63%), and total tau DA31 (-OA: 41%, +OA: 45%) (Figure 2-4,

Figure 2-5; Table 2-4). K252a did not show any statistically significant inhibition at AT8

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and AT270 with both basal and OA-induced tau hyperphosphorylation (Figure 2-4,

Figure 2-5; Table 2-4).

Cells treated with STS showed dramatic loss of basal and OA-induced tau

phosphorylation at CP13 (-OA: 86%, +OA: 63%), RZ3 (-OA: 83%, +OA: 81%), PHF-1 (-

OA: 55%, +OA: 89%), AT8 (-OA: 88%, +OA: 12%), AT270 (-OA: 100%, +OA: 100%)

and total tau DA31 (-OA: 41%, +OA: 45%) (Figure 2-4, Figure 2-5; Table 2-4).

Unexpectedly, EGTA caused an opposite effect by further enhancing tau basal

and OA-induced tau hyperphosphorylation in CTX cells at CP13 (-OA: -51%,+OA:-

12%), RZ3 (-OA:-63%,+OA:-22%), PHF-1 (-OA:-22%,+OA:-13%), AT8 (-OA:-

64%,+OA:-5%), AT270 (-OA:-68%,+OA:-63%) and total tau DA31 (-OA:-73%,+OA:-

69%)( Figure 2-4, Figure 2-5;Table 2-4).

β-actin protein levels remained even in all experimental conditions. Taken all

together, treatments with CKII inhibitor TBB, GSK3 inhibitors LiCl and AR-1014418,

and Src/Fyn Kinase inhibitor Saracatinib showed the most robust effect of inhibition

leading to different reduced basal and OA-induced tau phosphorylation profiles

demonstrating the specificity of inhibitors tested in our cell-based models.

Pan kinase inhibitor: K252a and STS

K252a and staurosporine (STS) are very potent cell permeable pan-specific

protein kinase inhibitors that act through the prevention of ATP binding to the kinase

(Table 2-2). In our study, K252a and STS treatment showed 30% increase of 46 kDa

(monomeric non-phosphorylated tau) and 35% decrease of 48 kDa (monomeric

phosphorylated tau) when probed with total tau DA9 antibody (Figure 2-2a, b and Table

2-3). K252a treatment caused 40% reduction of 170 kDa (DA9; oligomeric tau)

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compared to OA treatment (average of n=3) (Figure 2-2a, b and Table 2-3). For

phospho-tau detection, probing with CP13 antibody showed 60% and 32% reduction in

110 kDa (oligomeric form) with K252a and STS treatment, respectively.

PHF-1 showed 70% and 80% reduction in levels of 240 kDa (oligomeric tau form)

with K252a and STS treatment respectively (Figure 2-2a, b, Table 2-3). Like EGTA, the

addition of STS without caspase and calpain inhibitors (Z-DCB and SNJ-1945,

respectively) caused loss of tau bands at the epitope tested (Figure 2-2a), suggesting a

role of the apoptotic enzyme in causing the proteolysis. The effect of the apoptotic

inducer STS is consistent with our finding that showed tau fragmentation and increased

αII-spectrin breakdown products (145K/120K), suggesting the activation of calpain and

caspase respectively(71).

Discussion

In this study, we used OA to induce tau hyperphosphorylation and

oligomerization in mouse neuroblastoma and rat primary cerebrocortical neuronal

cultures to screen for various tau kinase inhibitors as potential drug candidates. N2a cell

line has been widely used to study mechanisms of neurodegeneration because they are

homogenous culture system that is convenient to handle and can multiply quickly to

produce a significant amount of neuron precursor cells(136).

However, CTX primary cultures were used in this study as they represent a

healthier form of cortical cells as opposed to cell lines, which are cancerous, in a sense

that gene expression in primary cortical culture could represent and mimic the actual in

vivo expression. Additionally, primary culture has the advantage in representing the

complexity of the central nervous system by better translating into in vivo models used

for screening pharmaceutical drug candidate’s compounds(142). OA results in robust

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tau hyperphosphorylation at multiple pathological epitopes in animal and cell culture

studies(65, 66, 143-146).

To our knowledge, there are no studies that have used OA for studying tau

oligomerization process, in either cell culture or animal models. In this study, OA

treatment induced tau hyperphosphorylation and oligomerization at various phospho-tau

epitopes in N2a cells tested by various tau epitope antibodies. In contrast to N2a cells,

we have only observed the monomeric form of tau protein (ranging from 63 kDa – 67

kDa) in CTX culture. A plausible reason for such effect would be that our separation of

tau proteins by SDS-PAGE was carried out under reducing conditions (Dithiothreitol

(DTT) and -ME) that could have the ability to reduce tau oligomers to the monomeric

form in primary culture. Moreover, our serum-free combination of neurobasal media

contains vitamin E, glutathione, catalase, superoxide dismutase, and transferrin, which

increase neuronal resistance to biological oxidation, e.g., damages by free radical.

Increase in reactive oxygen species activates caspase and calpain which cleaves

cytoskeletal proteins (e.g., spectrin) and contribute to the formation of pathologic tau

(e.g., post-translational modification and aggregation)(27). Thus, the presence of these

antioxidants may have blocked these processes from occurring.

To test the feasibility of our approach we used pan kinase inhibitors (K252a and

STS), GSK3 inhibitors (LiCl, AR-A014418 and A1070722), calcineurin inhibitor

(cyclosporine A), CDK5 inhibitor (Roscovitine), and calcium chelator EGTA (Figure 1-3,

Table 2-1) and were able to detect specific profiles of tau hyperphosphorylation and

oligomerization inhibition indicative of kinase inhibitor specificity. We have also

extended this study to include two more novel concepts of Fyn kinase (tyrosine kinase,

64

indirectly involved) and CKII kinase inhibitors. To our knowledge, these two kinase

inhibitors (TBB and Saracatinib) have not been used previously in studying tau

hyperphosphorylation and oligomerization in cell culture in vitro models.

Moreover, among all the phospho-tau epitopes studied here, the Thr231 epitope is

believed to be associated in the initiation of tau hyperphosphorylation in tauopathies

while other epitopes such as Thr181, Ser202/Thr205, and Ser396/Ser404 are phosphorylated

far ahead during the tauopathy process and the progression of the disease(68). The

phosphorylation sites Thr231 and Thr181 have been proposed as biomarkers in AD

patients while Ser202/Thr205 are used to determine the stage of AD progression(70, 77,

78). Therefore, we selected these phosphorylation sites in our study, to associate the

effectiveness of protein kinase inhibitors with tauopathy-related phosphorylation sites.

PP2A is the primary enzyme responsible for dephosphorylation of tau protein in the

brain, controlling all tau phosphorylation sites. PP2A activity is decreased in AD and TBI

brains(62, 147). Thus, the OA-induced inhibition of PP2A is a highly relevant model to

test various tau protein kinase inhibitors as modulators of tau hyperphosphorylation and

oligomerization targeting tau pathology in CTE (Figure 1-3).

In the N2a and CTX cell culture, CKII inhibitor, TBB, surprisingly provided the

most profound reversal of tau phosphorylation and oligomerization at all the epitopes

analyzed. TBB is a selective, cell-permeable, reversible and ATP/GTP-competitive

inhibitor of casein kinase II (IC50=900 nM for rat liver CKII)(148). CKII function is

aberrant in AD, and its alteration precedes hyperphosphorylated tau accumulation in

NFT formation(132). Moreover, CKII can phosphorylate tau purified from human brain

and neuroblastoma cell line(33, 36, 132, 148).

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As for the GSK3 inhibitors, AR gave robust suppression of tau

hyperphosphorylation in CTX culture at all tau epitopes tested (Figure 2-4, Figure 2-5;

and Table 2-4) and was less effective in N2a cells (Figure 2-2a, b, and Table 2-3). We

also observed that the effect of AR is more prominent compared to another GSK3

inhibitor, A-107 in primary culture. This could be attributed, in part, to the high selectivity

and specificity of AR to GSK3β(149) compared to A-107 which display selectivity for

both GSK3α and GSK3β (Ki= 0.6 nM for both)(150) thereby diluting the effect of

inhibition of GSK3β, which is considered to be one of the central tau kinase inducing tau

hyperphosphorylation in AD(129).

LiCl is well-known to inhibit GSK3 and other kinases(146). Therefore, the

inhibition of basal and OA-induced tau hyperphosphorylation in the primary culture

could be attributed to the collective effect of LiCl on GSK-3β and other kinases.

However, LiCl showed opposite effect in the N2a cells by increasing OA-induced tau

hyperphosphorylation and oligomerization at multiple tested tau epitopes. A possible

explanation for such differences is the decreased activity of GSK3 at CP13, PHF-1, and

DA9 phosphorylation sites(146). Also, the primary cultures are differentiated cells that

represent the complex nature of the central nervous system (CNS) having potentially

more active GSK3β compared to the cancerous N2a cells.

The use of Roscovitine in CTX culture reduced substantially basal and OA-

induced tau hyperphosphorylation at CP13 (pSer202), RZ3 (pT231, PHF-

1(pSer396/pSer404) and AT270 (pThr181). Roscovitine reduced basal phosphorylation at

AT8 (pSer202/pThr205) but did not affect the OA-induced tau hyperphosphorylation,

reflecting its specificity and the selectivity to our cell models. Also, Roscovitine resulted

66

in opposite effects in the N2a cells by increasing phosphorylation at CP13 (pSer202) and

PHF-1 (pSer396/pSer404). These results could be attributed to the decreased activity of

CDK5 at CP13 and PHF-1 phosphorylation site in the neuroblastoma cells(56, 141, 151,

152).

EGTA treatment caused almost complete inhibition of OA-induced tau

hyperphosphorylation and oligomerization at 110 kDa of CP13 (pSer202) and partial

inhibition at PHF-1 (pSer396/pSer404) in N2a cells but showed opposite effect in primary

culture by increasing basal and OA-induced tau phosphorylation at all tested tau

epitopes. EGTA is well known to chelate intracellular Ca2+ that could potentially inhibit

calcium-dependent kinases such as CaMKII(139, 153). CaMKII plays a significant role

in tau phosphorylation in neuroblastoma cells(153). On the other hand, Saracatinib

reduced both basal and OA-induced tau hyperphosphorylation (67 kDa) in CTX primary

cultures at the epitopes: CP13 (pSer202), RZ3 (pThr231), PHF-1 (pSer396/pSer404) and

AT270 (pThr181).

Saracatinib is a small molecular inhibitor that has a high potency for Src and

Fyn(54, 133-135). Fyn kinase is involved in normal tau phosphorylation(54, 133-135).

Fyn can physically associate with tau and phosphorylate some residues by interacting

through its SH3 domain with SH3-binding domains on tau (PXXP motifs). Calcineurin

inhibitor, CsA caused complete inhibition of OA-induced tau hyperphosphorylation and

oligomerization in N2a cells but was less effective in doing so with CTX culture at the

analyzed epitopes. The differences in effect between N2a and CTX culture might be

due to the lower protein levels of p35, and CDK5 in primary neuronal cultures(154).

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Moreover, CsA can inhibit calcineurin, which interrupts its binding to calcium-dependent

calmodulin, required for CaMKII activation (Figure 1-3).

Limitations and Future Directions

OA is a potent PP2A/PP1 inhibitor and is known to induce hyperphosphorylation

of tau. However, the idea that OA-induced tau hyperphosphorylation in cells might not

be directly specific to TBI or CTE because the phosphorylation sites checked in the

study are general in tauopathies rather than TBI or CTE-specific.

Other biochemical assessments will be needed to validate our finding in OA-

treated N2a cells and CTX other than western blotting. Supporting data from other

immunologically based methods, such as ELISA and immunocytochemistry will be

added in the future. The different inhibition results between N2a and CTX cultures

suggest differences in the dose responsiveness, the physiological mechanisms, or both.

Going forward, we will need to test these drugs on cell lines that are more

representative of the human CNS to show a consistent trend. Data on dose-response

effects of all the protein kinase inhibitors using both the mouse and rat cultures need to

be included in future studies. Different cell types can have remarkably different dose

ranges and optimizations.

Conclusions

In summary, we identified two novel potential drug candidates (TBB and

Saracatinib) that warrant further test design, possibly involving animal model

tauopathies. Testing the response of TBB and Saracatinib in animal TBI models is

crucial as recent studies implicate pre-fibrillary hyperphosphorylated tau as the toxic

species in tauopathy-related disorders, and therefore, re-establish the interest in tau

68

kinase inhibitors development at putative neurotherapies, which could translate in

human clinical trials.

69

Table 2-1. Antibodies used in this study

*Epitope based on human Tau-441 sequence.

Clone name Epitope* Supplier (Catalog#) MAb/ PAb

AT8 pSer202/pThr205 Fisher-Thermo (MN1020) Mouse MAb

AT270 pThr181 Fisher-Thermo (MN1050) Mouse MAb

RZ3 pThr231 Peter Davies, Albert Einstein College of

Medicine, Bronx, NY Mouse Mab

CP13 pSer202 Peter Davies, Albert Einstein College of

Medicine, Bronx, NY Mouse MAb

PHF-1 pSer396/pSer404 Peter Davies, Albert Einstein College of


DA9 aa102-140 Peter Davies, Albert Einstein College of


DA31 aa150–190 Peter Davies, Albert Einstein College of


Total Tau aa243-441 Dako (A0024) Rabbit PAb

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Table 2-2. Phosphatases, kinase inhibitors, and pharmacological agents used in the study.

Agent Full Name / Function Target Affinity in vitro (Ki)

Cell–based

inhibition (IC50)

OA Okadaic Acid, phosphatase Inhibitor PP1, PP2A 32pM-150 nM(155)

0.1 µM(156)

CsA Cyclosporine A/calcium dependent

protein phosphatase-immunosuppressant PP3 7 nM(157)

1-10 µM(158)

AR-A014418

N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl) urea, Inhibits in an ATP

competitive manner GSK3β

38-104 nM(159)

1-10 µM(159)

A-1070722

1-(7-methoxyquinolin-4-yl)-3-(6-(trifluoromethyl) pyridin-2-yl) urea/ Brain penetrant. Inhibits in an ATP

competitive manner.

GSK-3 0.6

nM(160) 1-10

µM(160)

AZD0530 Saracatinib, anti-invasive and anti-tumor

activities

Src/Fyn Tyrosine Kinase

2.7 nM(161)

0.1-10 µM(162)

K252a Staurosporine analog, non-selective cell

permeable Protein Kinase Inhibitor

GSK3, Erk2, CDK1, PKA,

and PKC

10-30 nM(163)

10 µM(164)

LiCl Lithium Chloride–acts by competing for

magnesium. GSK3β

2 mM(165)

20 mM(165)

Roscovitine Seliciclib, competes for the ATP binding

sites, apoptotic and antineoplastic activity CDK5

0.7 µM(165)

20 µM166

STS Staurosporine, highly non-selective cell

permeable Protein kinase Inhibitors

Pan protein kinase,

PKC,cAMP-dependent

kinases

3-8 nM(166)

0.5 µM(164)

TBB

4, 5, 6, 7-tetrabromobenzotriazole, Acts in an ATP/GTP-competitive manner by

binding to the Val66 residue of casein kinase-2.

CKII 900

nM(167) 10 µM

EGTA Ethylene glycol-bis (β-aminoethyl ether)-N, N, N’, N'-tetraacetic acid, a chelator of

divalent cations.

Ca2+-dependent

kinases

2-5 mM(168)

32 mM(169)

SNJ-1945 Amphipathic ketoamide – neuroprotective

cell-permeable calpain inhibitor Calpain-1 and

2 100 nM

20-30 µM(170)

Z-DCB

Z-Asp-2,6-Dichlorobenzoyloxymethyl Ketone, Inactivates the interleukin-1β-

converting enzyme

Pan kinases and caspases

1-10 µM 4 µM(171)

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Table 2-3. Composite effects of tau kinase inhibitors on OA-induced tau hyperphosphorylation in N2a cells.

*Negative value shown in brackets stand for reverse effect by increasing tau hyperphosphorylation and oligomerization above the basal level induced by OA alone.

Inhibitor

Inhibition %

CP13 (110 kDa form)

PHF-1 (240 kDa form)

DA9 (170 kDa oligomer)

K252a 62 70 40

STS 32 82 -10

LiCl (-14) (-9) (-12)

EGTA 90 85 55

CsA 98 90 100

Roscovitine (-11) (-53) (-22)

TBB 100 100 100

Saracatinib 15 0 10

A107 23 0 13

AR-A014418 0 0 36

TBB + Saracatinib 100 100 100

TBB + A107 100 100 100

TBB + AR-A014418 65 86 100

Saracatinib + A107 19 0 71

Saracatinib + AR 6 0 14

A107 + AR-A014418 0 0 0

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Table 2-4. Composite effects of tau kinase inhibitors on basal and OA-induced tau hyperphosphorylation in rat primary cerebrocortical neuronal cells.

* Negative value shown represent reverse effect by increasing tau hyperphosphorylation above the basal

level induced by OA alone.

Inhibitor

Inhibition %

CP13 RZ3 PHF-1 AT8 AT270 DA31

(67 kDa)

K252a - OA 35 45 61 10 17 41

+ OA 41 37 63 0 0 45

STS - OA 86 83 55 88 100 79

+ OA 63 81 89 12 100 77

LiCl - OA 94 89 98 100 100 93

+ OA 50 100 100 81 100 96

EGTA - OA -51 -63 -22 -64 -68 -73

+ OA -12 -22 -13 -5 -63 -69

Roscovitine - OA 85 42 63 91 100 32

+ OA 63 91 81 0 18 29

TBB - OA 91 100 100 91 100 41

+ OA 98 100 100 100 100 91

Saracatinib - OA 41 81 52 0 0 5

+ OA 100 100 100 0 84 20

A-1070722 - OA 92 79 65 82 100 80

+ OA 36 70 85 21 100 55

AR-A-14418 - OA 100 89 100 100 100 100

+ OA 89 100 100 100 100 100

CsA - OA 90 91 89 95 92 95

+ OA 33 26 0 86 11 28

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Figure 2-1. OA induced tau hyperphosphorylation and oligomerization at different time points in mouse neuroblastoma N2a cells. A. Immunoblots of N2a cells extracted protein (20 µg) using total and phospho-tau antibodies: DA9, CP13, and PHF-1. Blue arrows show phosphorylated and oligomerized tau. Red arrows show minor bands of oligomerized tau. Black arrows show non-phosphorylated tau band. SNJ-1945 (calpain inhibitor) and Z-DCB (caspase inhibitor) were added for 1h before the treatment with OA for 24h. A reverse time course followed OA treatment, and all cells were collected at the same time and conditions. B. Immunoblots quantifications. All data are normalized to β-actin and are expressed as a percentage of control. Data are presented as ± SEM for n=3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant.

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Figure 2-2. Screening of protein kinase inhibitors on OA-induced tau hyperphosphorylation and oligomerization in N2a cells (with or without cell-death linked protease inhibitors (calpain/caspase inhibitors). a. Immunoblots of N2a cells extracted protein using phospho-tau antibodies (CP13, PHF-1), total tau (DA9) and αII-Spectrin. αII-Spectrin was probed to assess cell apoptosis monitored with spectrin breakdown products (SBDP- red arrow). Kinase inhibition of phosphorylation and oligomerization was monitored by evaluating the levels of p-tau antibodies and total tau (blue arrows) and non-phospho tau (black arrows). For all conditions, SNJ-1945 (abbreviated as S, calpain inhibitor) and Z-DCB (abbreviated as Z, caspase inhibitor) were added for 1-hour prior the treatment with OA. Actin was probed as a loading control. B. Immunoblot quantifications. Conditions that included (S+Z) were quantified only. All data are normalized to β-actin and are expressed as a percentage of control. Data are presented as ± SEM for n=3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post-hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant.

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Figure 2-2. Continued.

76

Figure 2-3. Dose-response of TBB on OA-induced tau hyperphosphorylation and oligomerization in N2a cells. N2a cells were pre-treated with 0, 1, 3, 100, 300 nM and 1, 3, 10, 30 µM of TBB for one hour and then treated with OA for 24 hours. A. Immunoblots of cell extracted proteins using phospho-tau antibodies including CP13 and total tau DA9. Blue arrows represent phosphorylated and oligomerized tau. β-actin antibody was used as a loading control. All conditions included SNJ-1945 (calpain inhibitor) and Z-DCB (caspase inhibitor). B. Dose-response treatment curve following TBB treatment. Statistical analysis was performed with one-way ANOVA followed by Bonferroni’s post-test. *p<0.05, **p<0.01, ***p<0.001. Data are presented as ± SEM for n=3.

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Figure 2-4. Screening of protein kinase inhibitors on basal tau phosphorylation in rat primary cortical neuronal culture (CTX). Rat primary neuronal cultures at 15 DIV were pre-treated with various kinases inhibitors including K252a (30 μM), STS (20 μM), LiCl (10 μM), EGTA, (5 mM), Roscovitine (60 μM), TBB, (30 μM), A-1070722, (20 μM), AR-A104418, (60 μM) for 24 h. A. Immunoblots of cell lysates analyzed for phosphorylated tau at the epitopes CP13, PHF-1, AT8, RZ3, AT270. Total tau was probed with DA31 antibody. Spectrin Break down products (SBDP) were analyzed with alpha-II-spectrin antibody. Dashed gray arrows represent the position of tau oligomers found in N2a, which is absent in CTX. B. Immunoblot quantification of basal tau phosphorylation. Ratios of phospho-epitope levels over β-actin ± SD are represented as a percentage. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. n=3 per condition.

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79

Figure 2-5. Screening of protein kinase inhibitors on OA-induced tau hyperphosphorylation in rat primary cortical neuronal culture. Rat primary neuronal cultures at 15 DIV were pre-treated with various kinases inhibitors including (K252a, 30 μM), (STS, 20 μM), LiCl (10 μM), (EGTA, 5 mM), Roscovitine (60 μM), TBB, (30 nM) and (A-1070722, 20 μM), (AR-A104418, 60 μM) followed by OA (100 nM) for 24 h. A. Immunoblots of cell lysates analyzed for phosphorylated tau at the epitopes CP13, PHF-1, AT8, RZ3, AT270. Total tau was probed with the DA31 antibody. Spectrin Break down products (SBDP) was analyzed with the alpha-II-spectrin antibody. Dashed gray arrows represent the position of tau oligomers found in N2a, which is absent in CTX. B. Immunoblot quantification of OA-induced tau phosphorylation. Ratios of phospho-epitope levels over β-actin ± SD are represented as a percentage. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. n=3 per condition.

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81

Figure 2-6. Effect of combining protein kinase inhibitors on OA-induced tau hyperphosphorylation and oligomerization in N2a cells. A. Immunoblots of N2a cells extracted protein using phospho-tau antibodies (CP13, PHF-1, and RZ3), total tau (DA9), and αII-Spectrin. αII-Spectrin was probed to assess cell apoptosis monitored with spectrin breakdown products. For all conditions, SNJ-1945 (calpain inhibitor) and Z-DCB (caspase inhibitor) were added for 1h before the treatment with OA (abbreviated as S+Z). Actin was probed as a loading control. B. Immunoblot quantifications. All data are normalized to β-actin and are expressed as a percentage of control. Data are presented as ± SEM for n=3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant.

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83

Figure 2-7. Different effects of cyclosporine A on basal and OA-induced tau hyperphosphorylation in mouse N2a vs. rat CTX culture. A. Immunoblots of N2a cells protein extracts and B. CTX cells using antibodies directed against major tau phosphorylation sites. Total tau levels were probed using DA9 in N2a cells and DA31 in CTX cells. C. Immunoblots quantification of N2a and D. CTX cells. The ratio of phosphorylation epitopes levels over β-actin levels ± SD are represented as a percentage of control. n=3 per condition. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: non-significant.

84

Figure 2-7: Continued.

85

CHAPTER 3 IDENTIFICATION OF LOW AND HIGH MOLECULAR WEIGHT TAU FRAGMENTS FOLLOWING IN VITRO CALPAIN DIGESTION AND CELL-BASED NEUROTOXIC

CHALLENGES BY BIOCHEMICAL AND PEPTIDOMIC APPROACHES

Introduction

Microtubule-associated protein tau (MAPT) is a soluble protein whose function is

to control the stability of the axonal microtubules(42). Tau is expressed in the central

nervous system (CNS) and mainly present in neurons and at lower levels in astrocytes

and oligodentrites(42). Tauopathies of the CNS including AD, TBI and Parkinson’s

diseases associate with defective tau protein that can no longer stabilize the

microtubule,(16, 18, 39). Processes occurring in tauopathies disrupt tau physiological

function of microtubule stabilization and formation. Examples of post-translational

modifications (PTMs) that occur in tauopathies include hyperphosphorylation,

acetylation, deamination, glycation, glycosylation, isomerization, methylation, nitration

sumoylation, ubiquitination, and proteolysis(79). Phosphorylation is one of the most

well-characterized and researched PTM of tau, due to its direct ability to disrupt binding

to microtubules and contribution to aggregation as well as neurofibrillary tangles (NFT)

formation(43).

Tau proteolysis has generated a considerable amount of research interest

because of its involvement with tauopathies, mainly through calpain activation(172).

Calpain family consist of 14 different-cysteine-dependent proteases that are regulated

by calcium binding to a specific conserved site in all members of the family(80). Tau

proteolysis by calpain-1 and 2 contribute to opposing functions of regulating synaptic

plasticity and neurodegeneration. Calcium-mediated proteolytic cleavage of tau by

calpain can lead to the production of truncated tau break down products (tau-BDP)

86

which are susceptible to hyperphosphorylation and aggregation processes. Moreover,

calpain can degrade a wide variety of cytoskeletal proteins such as microtubule-

associated protein 2 (MAP2) and αII-spectrin(80, 173, 174).

Calpain-mediated αII-spectrin cleavage products are used to detect post-TBI

axonal injury and neurodegeneration following TBI(84). Calpain degradation of tau

produces a 35K tau fragment and a 17K tau fragment that can contribute to neurotoxic

events within the brain cells.(175) In AD tauopathy, increased calpain activity and

decreased levels of endogenous calpain inhibitor, calpastatin, have been reported(176).

A study of 17K tau-BDP have shown decreased microtubule binding ability of tau and

increased susceptibility to accumulation in the perikarya(175). Another study showed

that amyloid-β (Aβ) could activate calpain in cultured hippocampal neurons, producing

the 17K tau-BDP and cause neurodegeneration properties, which are delayed by

inhibition of calpain activation.(177) Calpain is also an upstream activator of

extracellular-regulated kinase (ERK), which can phosphorylate tau protein(178).

Increase in ERK expression leads to early accumulation of tau in neurons and glia in

various tauopathies(179).

Most proteomics studies nowadays follow the bottom-up approach where protein

samples are digested with trypsin, followed by different fractionation techniques for

peptide identification. This approach results in the generation of tryptic peptides and

leads to fragmentation of native endogenous peptides present in the samples; thus,

making their identification a challenge. Although structural and functional studies of tau

protein have been researched extensively in the past, studies characterizing the

endogenous low molecular weight (LMW, ≤10K) tau peptides are of interest due to their

87

association with a disease state. These natural peptides could reflect several active

processes occurring in vivo in the human brain such as enzymatic/proteolytic activity,

exocytosis, and tau aggregation. The standard analytical proteomics platforms utilize

trypsin digestion and produce tryptic peptides that can be used for quantification using

mass spectrometric analysis. One of the major disadvantages of proteomic studies is

the loss of valuable natural peptide information present in the biological samples leading

to the generation of artificial tryptic peptides ending with Lysine and Arginine residues.

This might be useful for increasing the identification and proteome coverage given the

abundance of Lys and Arg residues in a particular protein. However, the natural

released peptides following traumatic brain injuries and other neurodegenerative

diseases provide valuable use for biomarker studies that can be applied in diagnosis

and therapy “theranostic”. This is defined as peptidomics and any peptidomic approach

involves studying the entire set of naturally occurring endogenous peptides in biological

samples. To our knowledge, there are no peptidomic studies performed systematically

to identify low molecular weight tau peptides as biomarker candidates for

neurodegenerative diseases.

Thus, this chapter aims to characterize tau peptides generated from calpain or

under different cell-based neurotoxic conditions. To achieve this goal, we performed an

in vitro calpain-1 digestion of purified human tau-441(non-phosphorylated and

phosphorylated) and naïve transgenic human tau (htau) mice to study intact or high

molecular weight (HMW) and LMW tau products by western blotting and nLC-ESI-

MS/MS, respectively. We have also used non-phosphorylated and phosphorylated tau

to survey for unique LMW peptidome generated by calpain-1. To take this a step further,

88

we have used rat primary cerebrocortical neuronal culture (CTX) to study HMW and

LMW tau fragments generated when challenged with neurotoxic conditions that trigger

necrosis (MTX), apoptosis (STS), calcium influx (A23187). Moreover, CTX culture was

treated with okadaic acid (OA) to induce tau hyperphosphorylation, test the differential

susceptibility of tau to proteolysis, and analyze the release of LMW proteolytic

fragments in conditioned cell media.

OA is well known to specifically inhibit protein phosphatases 1/2A, which results

in tau hyperphosphorylation within the afflicted cells(180). Therefore, OA was used to

serve as a tauopathy-relevant cell-based model. The processed biological samples

were subjected to ultrafiltration devices with molecular weight cut-off (MWCO) value of

10K. This method was used to enrich LMW fractions that were analyzed by nanoLC-

tandem mass spectrometry. We were able to detect novel proteolytic tau peptides that

might be linked to neurodegeneration, as they were exclusively present in treatments

but not in controls. We were also able to derive calpain-1 cleavage sites using our MS

data. Moreover, we identified phosphorylated sites that that could be critical to the

pattern of calpain-1 proteolysis of tau protein. Our findings might aid in developing

promising future neurotherapeutic strategies with an emphasis on the suppression of

tau proteolysis and biomarker candidates for neurodegenerative diseases.

Material and Methods

Ethics Statement




89

Mouse Brain Collection and Samples Preparation

In our study, all mice used aged three months. For naïve transgenic human tau

mouse brain samples, mice were anesthetized and killed by decapitation. Subsequently,

the cortex and hippocampus part of the brains were removed and flash-frozen in liquid

nitrogen. The brain samples were pulverized to a fine powder using a mortar and a

pestle set in dry ice and chilled with liquid nitrogen. The fine powder was then

transferred to microcentrifuge tubes (Eppendorf). The brain powder was lysed with 1%

Triton X-100 lysis buffer containing 20 Mm Tris HCl, pH 7.0, 5 mM EDTA, protease and

phosphatase inhibitor cocktail and 1 mM DTT in LC-MS grade water. The brain lysates

were incubated at 4°C for 120 minutes kept at a tube revolver (Thermo scientific) at low

speed. Following the incubation, the samples were centrifuged at 10,000 x g for 15

minutes at 4°C, and the supernatant was transferred to new tubes. The protein

concentration was determined by performing protein assay using bicinchoninic acid

microprotein assays (Pierce Inc., Rockford, IL, USA) against albumin standards. The

brain lysates were then stored at – 80°C until further use.

In Vitro Calpain-1 Digestion of Purified Tau and Naïve Mouse Brain Lysate

Purified recombinant human tau-441 protein (non-phosphorylated and

phosphorylated; five µg each; rPeptideTM) or mouse cortex naïve brain lysate (10 µg)

were used for the study. The reaction buffer contained 1 mM of DTT (Sigma), 1 mM of

CaCl2 and 100 mM Tris/HCl (pH 7.4). The samples were diluted with ultra-pure water

for a final volume of 100 µl per reaction. The samples were incubated with calpain-1

(Enzo Life Sciences; 1µg/µL) for 1 hour at 30oC followed by addition of 1µM of SNJ-

1945 to stop the reaction (calpain inhibitor; Senju Pharmaceutical Co. Ltd., Kobe,

Japan). Calpain-1 was titrated in the ratio of 1:10, 1:25, 1:50, 1:100, and 1:250 (enzyme

90

to protein ratio). The control contained only the purified protein or naïve mouse brain

sample without the addition of calpain enzyme.

Rat Primary Cerebrocortical Neuronal (CTX) Culture

Rat primary cerebrocortical neuronal culture (CTX) were harvested from a

homogenized pool of 1-day old Sprague–Dawley rat brains and were plated on poly-L-

lysine-coated 12-well culture plates (Erie Scientific, Portsmouth, NH, USA) at a density

of 4.36 × 105 cells/ml. Cultures were maintained in Dulbecco’s modified Eagle’s medium

(DMEM) with 10% fetal bovine serum in a humidified incubator in an atmosphere of

10% CO2 at 37°C. After three days, the DMEM solution was replaced with another

DMEM solution containing 1% cytosine arabinoside (ARC). Two days later, the solution

was replaced with DMEM. The cells were cultured for an additional ten days before use.

Subsequent media changes were done twice a week.

Neurotoxic Challenges

Complete media was replaced with Serum-free medium, Neurobasal-A (Gibco)

consisting of 1% (v/v) B-27 supplement (Gibco), 1 mM L-glutamine (Sigma) and 1%

(v/v) penicillin-streptomycin (Sigma) in a total volume of 500 µl per well in a twelve-well

plate. In addition to untreated controls, the following conditions were used: apoptotic

inducer STS (staurosporine; 0.5 µM; Sigma) that activate calpain and caspase-3,

calcium ionophore A23187 (calcimycin; 20 µM; Sigma, St. Louis, MO, U.S.A.), and

maitotoxin (MTX; 10 nM; Sigma) for 16 hours. A23187 and MTX both acts by activating

extracellular calcium channels leading to an increase of cytosolic Ca2+ ions, and

indirectly can activate calpain-1 and -2. The conditions mentioned above were

pretreated with or without okadaic acid (OA; 100 nM; Cell Signaling) for 6 hours prior

the additions of STS, A23187, and MTX challenges.

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Preparation and Collection of Cell Lysate and Conditioned Media

After the treatment, conditioned media were collected from each well, added into

separate tubes on ice, and centrifuged at 10,000 x g for 10 min at 4°C. The

supernatants were collected frozen at –80°C until further analysis. As for the attached

CTX cells, 100 µl per well of lysis buffer that included:1 mM DTT, 1x phosphatase

inhibitors (Sigma), 1% Mini-complete protease inhibitor cocktail tablet (Roche

Biochemicals) and 1% Triton X-100 (Sigma) was added. The attached cells were then

scraped down into the lysis buffer and collected into a separate 1.5 ml Eppendorf tubes.

The cell lysates were incubated for 90 minutes at 4°C and then centrifuged at 15,000

rpm for 15 minutes to remove cell debris. The supernatants were collected and frozen at

–80°C until analyzed by SDS-PAGE followed by western blotting.

Sample Preparation and Ultrafiltration

The calpain-1 digested purified tau protein, mouse brain lysate, and CTX

conditioned cell media were loaded into 10K molecular weight cut-off (MWCO)

membrane filter (Vivacon 500 HY, Sartorius Stedim Biotech, Goettingen, Germany) and

centrifuged at 3000g at 4°C for 60 min. Each condition was prepared in three biological

replicates (n=3). The filtrates were evaporated until dryness using speed vacuum

(Thermo Scientific) and reconstituted in 20 µL of LC-MS grade 0.1% formic acid in

water. The samples were stored at –80°C pending analysis. The retentate fractions

were saved for SDS-PAGE described in the following section. As for the cell conditioned

media from CTX culture, the same protocol mentioned above was followed except

subjecting the filtrates to ZipTip cleanup (Millipore Sigma) before nLC-ESI-MS/MS

analysis. The ZipTip C18 (0.6 μL bed volume; 5 μg) were conditioned with 3 x 10 μL of

100% ACN and equilibrated by 3 x 10 μL of 0.1% FA, after which the samples were

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aspirated ten times for maximum binding. The samples were then washed 1 x 10 μL

0.1% FA and subsequently eluted with ten microliters of 70% ACN, 0.1% FA. Finally,

the eluate solvents were evaporated in speedvac until dryness and stored at –80°C.

The samples were re-suspended in 10 μL 0.1% FA.

Gel Electrophoresis and Western Blotting

Equal amounts of purified protein (5 μg), CTX lysate and mouse brain lysate

proteins (20 μg) were prepared for SDS–PAGE in 8 x Laemli loading buffer containing

0.25 M Tris (pH 6.8), 5% beta-mercaptoethanol (BME), 8% SDS and 0.02%

bromophenol blue. Samples were subjected to SDS-PAGE on 4-20% precast-gels for

60 min at a constant voltage of 200V. After separation, the gels were transferred on to

PVDF membranes using iBlot transfer (Invitrogen, Life Technologies).

The membranes were blocked in 5% milk prepared in TBST for 1 hour. After

blocking. The membranes were incubated at 4°C overnight with continuous shaking with

primary antibodies. Primary antibodies used include PHF-1, CP-13, RZ3 (a gift from

Peter Davies, Albert Einstein College of Medicine, Bronx, NY) and total tau (DA9 and

DAKO). αII-spectrin antibody (BML-FG6090, ENZO Life Sciences, Farmingdale, NY,

USA) was used to assess cell injury.

β-actin and UCH-L1 antibody were used as loading control. After overnight

primary antibody incubation, membranes were washed three times with TBST and

probed using anti-rabbit or anti-mouse IgG conjugated to alkaline phosphatase

(Amersham, Piscataway, NJ, USA), for 1 hour at room temperature and

Immunoreactive bands were detected by developing with nitro blue tetrazolium, and 5-

bromo-4-chloro- 3-indolyl phosphate (BCIT/NBT) (KPL, Gaithersburg, MD, USA). A

250K to 14K-rainbow molecular weight marker (RPN800E, GE Healthcare, Biosciences,

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Pittsburgh, PA, USA) was used to estimate the molecular weight of the proteins.

Quantitative evaluation of protein levels was performed via computer-assisted

densitometric scanning (NIH ImageJ, version 1.6 software).

Nano-LC-ESI-MS/MS

The filtrate fractions were vortexed for 15 s and centrifuged at 15,000 rpm for 2

min. This step was repeated twice to make sure all the residues has been dissolved.

The samples were transferred into auto-sampler microvial tubes avoiding any air-

bubbles. Five microliters of each sample were injected into the nanoLC system (Waters

NanoAcquity UPLC system). The separation column used was C18, (130Å, 1.7 µm, 100

μm X 100 mm; Waters) fitted with a C18 trap column (5 µm Symmetry 180 µm x 20 mm)

coupled to linear ion trap mass spectrometry (LTQ-XL; Thermo Scientific). The flow rate

was set to 300 nL/min. Peptide separation was achieved using a 120 min linear

gradient, running from 1—50% of solvent B (0.1% formic acid in 100% acetonitrile).

Tandem mass spectrometry was performed on LTQ-XL (Thermo, San Jose, CA, USA).

The mass spectrometer was calibrated using the positive ion standard calibration

solution (Thermo) to maintain the accuracy of the instrument. The peptide samples

eluted from the reversed-phase column were introduced into the mass spectrometer

using a nano-spray ionization source (ADPC-IMC adaptor, New Objective). The mass

spectrometer was operated in a data-dependent acquisition mode with a spray voltage

of 2.1 kV, ion transfer tube voltage at 35 V, and an ion transfer tube temperature at

275oC. The fragmentation mode was set to collision-induced dissociation (CID) with

Helium gas as the collision gas.

The sheath and auxiliary gases were set to zero. The ion signal threshold was

set to 1000 for MS/MS. The normalized collision energy was set to 35%, activation of

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q=0.25, and activation time of 30 ms for MS/MS acquisitions. The instrument setting for

MS scan was: m/z 300-1800, max injection time 200 ms; AGC target 1e6. Data-

dependent acquisition mode with automatic switching between MS and MS/MS modes

was employed. CID MS/MS was set to collect the top ten most abundant ions for

fragmentation. The isolation width was set to m/z 1.5. The CID target value was set to

10,000 ions for fragmentation. The following dynamic exclusion setting was applied to

precursor ions chosen for MS/MS analysis: repeat count—1; repeat duration—30 s; and

exclusion duration—120 s. The neutral loss experiment where data-dependent settings

were chosen to trigger an MS3 scan when a neutral loss of 97.97, 48.99 or 32.66 m/z

units (corresponding to singly, doubly or triply charged phosphorylated daughter mass

ions, respectively) was detected among the most abundant ten product-ions to enhance

the fragmentation of phosphopeptides. Multistage activation feature was enabled on X

caliber 2.4.1 for neutral loss detection. Each analysis was repeated three times to

confirm the reliability of the mass spectrometric analysis.

Thermo Orbitrap Fusion Tribrid Mass Spectrometer Analysis

Filtrate peptides were desalted with C18-solid phase extraction according to

manufacturer protocol. For high-resolution analysis, we used a Thermo Orbitrap Fusion

tribrid mass spectrometer with high energy collisional dissociation (HCD) in each MS

and MS/MS cycle (Thermo Scientific, Bremen, Germany). The instrument was run in

data-dependent mode with a full MS (m/z 400–2000) resolution of 70 000 and ten

MS/MS experiments (HCD NCE = 28%, isolation width = 3 Th, first mass = 105 Th, 5%

underfill ratio, peptide match set to “preferred”, and an AGC target of 1e6). Dynamic

exclusion for 10 s was used to prevent repeated analysis of the same peptides, and a

lock mass of m/z 445.12003 (polysiloxane ion) was used for real-time internal

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calibration. The MS system was interfaced with an automated Easy-nLC 1000 system

(Thermo Fisher Scientific, Bremen, Germany). Each sample fraction was loaded onto

an Acclaim Pepmap 100 precolumn (20 mm × 75 μm; 3 μm-C18) and separated on an

Easy-Spray analytical column (500 mm × 75 μm; 2 μm-C18) at a flow rate at 300 nL/min

during a linear gradient from solvent A (0.1% formic acid (v/v)) to 25% solvent B (0.1%

formic acid (v/v), 99.9% acetonitrile (v/v)) for 280 min, followed by ramping up to 98%

solvent B for an additional 20 min. Peptides were sprayed into the orifice of the mass

spectrometer, which was operated in an information-dependent data acquisition mode.

Data Analysis

The MS/MS data analysis was done using Proteome Discoverer 2.2. (Thermo).

The MS and MS/MS spectra were searched against mouse tau (UniProt Identifier:

P10637), rat tau (Identifier: P19332) and human (Identifier: P10636) tau isoforms (all

accessed Sept 2017 from UniProtKB/Swiss-Prot) using their fasta file through Sequest

HT 2.5.0 search engine. Since we selected non-specific enzymatic digestion in Sequest

HT, it would take a longer time to analyze the whole rat, mouse, and human proteome

database. Therefore, we created a subset database that includes only tau isoforms from

the mentioned species above. To validate that the identified peptides are only present in

tau and not another protein, we searched the raw files using the protein BLAST

database. Moreover, we did compare one sample search against the whole human

proteome and tau-441 to make sure there are no discrepancies in the search algorithms

between and no differences in the detected peptides (Figure 3-18).

The minimum and the maximum peptide length were set to 6 and 30,

respectively. The search was performed with no enzyme specificity; oxidized

methionine; phosphorylated serine, threonine, and tyrosine (+79.9663 Da) as dynamic

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modifications. For the LTQ-XL instrument the precursor mass error tolerance was set to

1.5 Da, and product ion tolerance was set to 0.8 Da.

For the Orbitrap Fusion Tribrid instrument the precursor mass error tolerance

was set to 10 ppm, and daughter ion tolerance was set to 0.6 Da. The tandem mass

spectra and product-ions were inspected manually to make sure the quality of the CID

fragmentation and phosphorylation site identified. The following peptide scores selected

were: ΔCn >0.1, PSM>20 and XCorr>2. A target FDR value of 1% was set for decoy

searches. The phosphorylation sites probabilities were assigned using ptmRS node on

Proteome Discoverer 2.2, which localize phosphorylation sites based on the search

engine identification.

Statistical Analysis

Statistical analysis was performed GraphPad Prism 7.0 (GraphPad, La Jolla, CA)

with one-way ANOVA Tukey’s Test. For multiple comparisons, one-way ANOVA

followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01,

***p<0.001, **** p<0.0001, ns: non-significant. Peptides presented had at least an XCorr

value of 2, two PSM, and high confidence to be included for the analysis. Peptides with

p-values<0.05 were reported.

Results and Discussion

Tau proteolytic fragments produced by calpain and other proteases following TBI

can potentially play a role in hyperphosphorylation and aggregation (43, 71, 79, 181) In

this study, we designed a simple protocol to collect and enrich the endogenous peptides

from proteolytic cleavages induced by calpain-1 or cell-based neurotoxic activation

system to mimic the conditions of neurodegenerative diseases such as TBI. Figure 3-1

shows the overall workflow utilized in this chapter. In the initial step of this workflow, we

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added calpain-1 to purified tau-441 (non-phosphorylated and phosphorylated forms)

protein (Figure 3-1A) and transgenic mouse brain lysate (Figure 3-1B). We have

included the non-phosphorylated and phosphorylated tau-441 (tau and p-tau) protein in

our study to account for the differential proteolytic peptides generated by calpain

digestion to mimic pathological processes that occur in the neurodegenerative brain.

The ultrafiltration technique allows simultaneous monitoring of intact parent

protein, high molecular weight tau breakdown products (HMW tau-BDP; retentate

≥10K), and the low molecular weight tau products (LMW tau-BDP; filtrate ≤10K). We

implemented the use of 500 μL capacity ultrafiltration devices with a molecular weight

cut-off (MWCO) value of 10K membrane filter. After centrifugation at 3500 x g for 30

min, we subjected the ultrafiltrate fraction containing the enriched proteolytic peptide

pool from the samples to nano-LC-ESI-MS/MS analysis (Figure 3-1A, 1B, 1C). We

analyzed the retentate fraction (≥10K) that contained the enriched intact protein and

higher molecular weight breakdown products (HMW-BDP) of the bio-sample by SDS-

PAGE followed by western blotting analysis. The reason behind choosing the 10K

MWCO was to reduce the possibility of losing peptides that had a complex secondary

structure or tertiary structure and thus might not pass through lower MWCO due to the

smaller filter pore size (e.g., 3K MWCO). Moreover, the ultrafiltration techniques allow

isolation of high abundance large fragments, which make up the bulk of the protein

content and lower abundance proteolytic peptides; thus, enabling detection of lower

molecular weight peptide fragments (Figure 3-1A, 1B, 1C).

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Immunoblot Analysis

Recombinant purified human tau/p-tau protein

We subjected recombinant human tau and p-tau (441-residue; 4R isoform) to

calpain-1 in the ratios of 1:100, 1:50, 1:25 and 1:10 (Figure 3-2A and 3A). A few

proteolytic fragments of tau and p-tau were observed with Coomassie Brilliant Blue

staining (data not shown). For validation of the proteolysis, we ran the retentate

fractions on SDS-PAGE followed by Western blotting.

From our western blot analysis, we observed that the amount of intact non-

phospho-tau (intact 63K; Figure 3-2A) and phosphorylated tau-441 protein (intact 65K;

Figure 3-3A) diminished considerably with increasing concentration of calpain-1

enzyme. Moreover, tau-BDPs (40K, 38K, 24K, and 12K) elevated considerably with

increasing concentration of calpain-1 and were reduced considerably with 1:10 ratio,

with tau and p-tau (Figure 3-2A, Figure 3-3A). This observation was detected with both

total tau antibody (DA9: a.a:102-145) and p-tau antibody RZ3 (pThr231) (Figure 3-3A).

We were able to observe the tau-40K, tau-24K and tau-12K fragments of p-tau with 1:10

ratio of calpain/p-tau using DA9 and DAKO, suggesting that there is some resistant to

calpain-1 by the phosphorylated tau (Figure 3-3A). We have reported that p-tau-BDP-

24K corresponds to the exposed N-terminal NH2-PTREPKKVAVV that suggests a

cleavage site between Gly157/Ala158 of the full-length tau (71).

Transgenic mouse brain lysate

To validate the fidelity of tau proteolysis in a more complex biological system, we

performed calpain-1 digestion in mouse transgenic htau cortex brain lysate (5 μg) and

analyzed the samples by SDS-PAGE followed by Western blotting (Figure 3-4A). The

mouse model we used can express the six isoforms of human tau, including 3R and 4R,

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but cannot express mouse tau. Neither htau mice nor tau knockout mice have been

shown to develop tau pathology in the CNS (72, 73, 91, 151).

Coomassie stain of the gel showed several fragments generated from calpain

digestion, suggesting the vulnerability of multiple substrates in the mouse brain to

calpain (data not shown). Western blot analysis of the retentate fractions showed that

total intact tau (63K; DA9) was highly vulnerable to calpain-1 from brain lysate source

(Figure 3-4A). With calpain-1 treatment, we observed a cluster of immunoreactive

bands at tau-BDP-42K, 35K, and 12K (DAKO and DA9; Figure 3-4A). Moreover,

probing with RZ3 showed that naïve control mouse had a considerable amount of p-tau,

which was vulnerable to calpain, reducing the 35K band entirely with the 1:100 ratio

(Figure 3-4A). β-actin was used as loading control and showed equal amounts of

protein. With DAKO antibody, we were able to observe the 12K tau-BDP that diminished

gradually with increasing concentration of calpain in brain lysate samples, suggesting

the high vulnerability of these proteolytic fragments (Figure 3-4A). These tau-BDPs are

consistent with what has been reported in our earlier study (71). With this knowledge,

we confirmed that tau from various sources is cleaved and fragments of multiple HMW

sizes are generated. We also observe slight differences in the molecular weight

breakdown products detected using immunoblotting between tau, p-tau and brain

lysate, suggesting that there might be a differential fragmentation pattern by calpain-1.

Primary Rat Cerebrocortical Neuronal (CTX) culture Subjected to Neurotoxic and Neurodegenerative Conditions

To test tau vulnerability to calpain or caspase in a system that represents the

sophisticated structure of the CNS, we used neurotoxic agents on the differentiated rat

CTX culture (Figure 3-7A, 7B). We added pro-necrotic calcium ionophore A23187, pro-

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apoptotic staurosporine (STS), calcium ionophore maitotoxin (MTX) for 16h, with or

without okadaic acid (OA; 6h) in CTX culture to mimic intracellular fluid in the CNS after

neuronal injury (Figure 3-7A, 7B). These conditions were selected based on their ability

to induce necrosis/apoptosis that activates calpain and caspase to trigger events that

mimic neurodegenerative diseases(71). Okadaic acid (OA) is a potent inhibitor of

phosphatase 1/2A, resulting in hyperphosphorylation of specific proteins including tau

(65, 66, 136, 182, 183). OA was added to test cellular hyperphosphorylated and

oligomerized tau vulnerability for fragmentation by activated proteolytic enzymes.

Using the total tau DAKO antibody (a.a. 243-441), control lane showed a band

detected at 54K (non-phosphorylated tau) and minor band around 60K (p-tau). We did

not observe high molecular weight band with DAKO antibody (Figure 3-7A, 7B). As for

the RZ3 (pThr231) and PHF-1 (pSer396/pSer404) antibodies, control lane showed a band

at 54K (monomeric p-tau) and a minor band at 110K with PHF-1 (presumably oligomeric

p-tau; Figure 3-7A, 7B). Utilizing αII-spectrin antibody showed considerable levels of

intact 240K band and minor levels of spectrin breakdown products (SBDP-

150K/145K/120K), suggesting a healthy neuronal culture. Treatment with STS, A23187,

and MTX for 16h showed a dramatic reduction of intact tau-54K as detected by total tau

(DAKO, DA31, DA9), and p-tau antibodies including RZ3 and PHF-1 compared to

control (Figure 3-7A, 7B). Probing with αII-spectrin, we observed a considerable loss of

intact spectrin-240K, and an increase of calpain-mediated SBDP-150K/145K, with STS,

A23187, and MTX treatments.

OA treatment caused a considerable increase of tau-54K (DAKO, RZ3, and PHF-

1) monomeric p-tau band and formation of a 102K band (presumably corresponding to

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oligomeric p-tau). Interestingly, treatment with OA followed by STS protected the

complete loss of 54K (RZ3 and PHF-1), but not the 102K band (oligomeric p-tau; PHF-

1) suggesting that tau is less vulnerable to proteolysis with OA-induced

hyperphosphorylation (Figure 3-7A, 7B). When samples were probed with αII-spectrin,

we observed both, the SBDP150K/145K and SBDP-120K, suggesting calpain and

caspase activation. In a similar pattern observed to OASTS treatment, MTX and

A23187 treatment in the presence of OA, a cluster of low molecular weight fragments

(tau-BDP-24K) was observed with 16h treatment when probed with DAKO (Figure 3-7A,

7B). Thus, STS, A23187, and MTX treatment cause a neurodegenerative paradigm that

involves a dual contribution of calpain-1 and caspase-3 with the phosphorylated tau

being partially protected from proteolysis induced by the neurotoxic challenges.

nLC-ESI-MS/MS Analysis of Purified Tau and Mouse Brain Lysate

First, we analyzed the ultrafiltrate fractions of purified tau-441 protein (tau and p-

tau) by nanoLC-MS/MS analysis. We have included several dilutions of calpain-1

(1/250, 1/100, 1/50, 1/25, and 1/10 enzyme to protein ratios) to account for the different

LMW fragments that could potentially be generated under neurodegenerative

environments and be able to identify these fragments as potential biomarkers in bio-fluid

samples. It should be noted that identification of non-specific proteolytic peptides by

sequence database is more challenging than identification of peptides using a specific

protease (e.g., trypsin). These challenges are due to unpredictable enzyme cleavage

specificity, cleavage by more than one protease in biological environments, and

increased length of peptide sequences, thereby increasing the search algorithms

complexity by a factor of 100-1000 compared to tryptic peptides.

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To increase the confidence of our matches, we performed the database searches

using false discovery rate (FDR) of 1%, searched against a decoy database, used

minimally five peptide sequence matches (PSMs) based on MS/MS, and an XCorr value

of 2. We identified peptides relating to the protein turnover. All peptides, which are

found in control, were excluded from our bioinformatics analysis.

Since we are interested tau protein fragments in this study, our database search

included only the tau protein (based on UniProt, Human Proteome Org, and Human

Protein Atlas organ distribution datasets). To make sure there are no discrepancies

between the matches, we selected one representative sample of tau digest and ran a

global human proteome search as well as a targeted search against tau-441

(accession# P10636-8). We did not observe any difference in peptide identifications

between the global human proteome search and targeted tau search (Table 3-5; Figure

3-18).

We also were able to observe exclusive tau peptide fragments that were only

present in either the non-phosphorylated or the phosphorylated form of tau but not in

both fractions (Table 3-1 and 3-2). We also observed a variation in the length of the

core peptide identified, suggesting the ability of calpain to multi-process tau residues

further as it becomes more active (Figure 3-2C, 3-3C).

We have implemented the use of PSM counts (spectral count based on MS/MS)

to account for the abundance of each proteolytic peptide in the different samples. This

method can be applied with confidence since each peptide reported was identified at

least five times. Only peptides with high confidence were considered. Figure 3-2B, 3-3B,

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are plots of peptides with the highest PSM number versus m/z observed in the tau and

p-tau filtrate fractions.

The N-terminal core peptide AEPRQEFEVMEDHAGTYG (a.a.: 2-19) with m/z

689.625 was observed in tau and p-tau with varying lengths on the C-terminal,

extending 4-5 amino acid residues (Figure 3-2C; Figure 3-3C;). This N-terminal

fragment might be linked to Johnson et al. group reported fragmented species which

predominantly consisted of the N-terminus tau using epitope-specific antibodies in

human CSF samples (79). Figure 3-13 shows the product spectrum of

AEPRQEFEVMEDHAGTYG. Other fragments of particular interest are the C-terminal

peptides: SPRHLSNVSSTGSIDMVDSPQLATLADEVS (a.a 404-433) with m/z 1038.75

and TLADEVSASLAKQGL (a.a. 427-441) which are both observed with tau and p-tau

samples. We were able to identify these peptides in varying lengths from the N-terminal

and C-terminal domains (Figure 3-2C; Figure 3-3C). Figure 3-14 shows the product

spectrum of SPRHLSNVSSTGSIDMVDSPQLATLADEVS peptide released from

calpain-1 digestion.

As for brain Lysate filtrate fractions, we have identified two clusters of N-terminal,

the middle region, and C-terminal domain peptides with a fragmentation pattern that is

very closely similar to the recombinant purified tau-441 calpain-mediated proteolysis

pattern (Table 3-3; Figure 3-4B, 3-4C). We have noted that the core sequence region

a.a.:413-441 protein coverage of brain lysate considerably overlaps with the purified tau

protein coverage (Figure 3-2C, 3-3C, 3-4C), suggesting that calpain-1 digested the

cellular htau similar to purified htau protein. Around 110 peptides were exclusively found

from p-tau, 202 peptides from non-phosphorylated tau, 20 peptides from brain lysate,

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and 24 peptides were found in all samples (Figure 3-5B). Of particular interest as

biomarkers are the C-terminal domain peptides from brain lysate that overlap with the

purified tau fragments including SPRHLSNVSSTGSIDMVDSPQLA (a.a. 404-426),

ASLAKQGL STGSIDMVDSPQLA (aa 413-426), and TLADEVSASLAKQGL (aa 427-

441) (Figure 3-4C and 3-6C). Figure 3-16 shows the product spectrum of the

phosphorylated peptide STGSIDMVDSPQLA released from calpain-1 digestion. Figure

3-4B summarizes the top 10 highest PSM score of calpain-mediated tau peptides from

purified and brain lysate source. Collectively from our bioinformatic analysis, we

identified a total of 404 peptides from tau, 312 from p-tau and 45 peptides from brain

lysate with 24 there are in common between the three samples (n=3). Figure 3-5A

shows a composite summary of representative calpain-mediated proteolytic peptides

identified from tau, ptau, and mouse brain lysate. Figure 3-5B shows a Venn diagram

with the total number of peptides released in each of the biological replicates from the

different samples. The identified peptides are usually spanning either the N-terminal

domain, middle domains or C-terminal domain. Figure 3-5C shows a schematic of

human tau-441 and summarizes the predicted calpain-1 cleavage sites and

phosphorylation sites detected in our study.

Another well-known approach for monitoring peptide abundance for peptidomics

studies is implementing the use of peak intensity. Peak intensity involves the calculation

of the ion current of the peptide as it elutes from LC/MS usually integrating signal over

the entire elution period, measuring the peak value. Figure 3-2D, 3-3D, and 3-4D

demonstrate the peptidome profile of purified human tau, p-tau, and mouse brain lysate

digested by calpain-1 respectively based on their intensity. At each amino acid residue

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along the protein, the height of the green bars is proportional to the count of peptides

overlapping this position. The intensity of the color (green) is proportional to the sum of

the peptide intensities overlapping this position. The proteolytic peptide profile of tau

and p-tau shows a similar distribution of count and intensity. The brain lysate tau

peptides showed the highest intensity around the C-terminal region. Such differences

might be due to the sample heterogeneity and tau tertiary/quaternary structure

complexity for allowing different access for the enzyme.

Figure 3-6 shows a peptigram of longest tau isoform (P10636; a.a. 758) identified

peptides alignment map generated from ProViz web-based visualization tool (184). The

map alignment was created using a compiled excel file that contains the highest ranked

calpain-mediated peptides (total of 50 peptides) from tau, p-tau, and brain lysate to

determine the potential cleavage sites of calpain. A cluster of varying lengths of the

peptides AEPRQEFEVMEDHAGTYG (a.a. 2-19) and

LGDRKDQGGYTMHQDQEGDTDAGLK (a.a. 20-44) has been identified with high

intensity, suggesting a cleavage site between G19/L20, spanning the N-terminal region of

tau (Figure 3-6A). The tau fragments 26-44, 26-44, 1-44 and 45-441 have been

reported as calpain-1-mediated fragments causing NMDAR-mediated cell death in

cerebellar granule cells (79). Moreover, a cluster of varying lengths of the peptides

SETSDAKSTPTAEDVTAPLVDEGAPGKQA (a.a. 61-89) and

AAQPHTEIPEGTTAEEAGIGDTPSLE (a.a. 90-115) has been identified with high

intensity, suggesting a cleavage site between A89/A90 (Figure 3-6B). The peptides

spanning the C-terminal domain RENAKAKTDHGAEIVYKSPVVSGDT (a.a.696-720)

and SPRHLSNVSSTGSIDMVDSPQLATLADEVS (a.a. 721-750) suggest calpain

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cleavage site at Thr720/Ser721 (Figure 3-6C). To our knowledge, these calpain-1

cleavage sites have not been reported previously. Figure 3-11 shows sequence

coverage of human tau-441with phosphorylation sites identified using proteome

discoverer 2.2. The coverage of Tau-441 included the N-terminal, mid domain, and C-

terminal domains. Figure 3-12 shows sequence coverage of peptides identified from

human Tau-776 isoform. Unique peptides and phosphorylation sites were identified

from tau-776 isoform and not in tau-441 (Figure 3-12). Figure 3-17 shows a general

protein alignment from the human tau-441 isoform and human longest isoform tau-776,

highlighting the overlapping amino acids in red. The alignment was generated using

BLAST software from NCBI website. The tau-776 is subjected to genetic alternative

splicing leading to the production of tau-441.

Conditioned Media of CTX Culture Analysis by nLC-ESI-MS/MS

Cell conditioned media were a starting material for the identification of

neuropeptides, prohormones growth factors and cytokines (185). With technological

advances in mass spectrometry, conditioned media received attention as a source for

discovering novel primary cleavage sites for peptides processing, ectodomain, and

regulated and neurotoxic proteolytic peptides. Therefore, conditioned media from CTX

culture treated with necrotic and apoptotic agents were collected for analysis by nLC-

ESI-MS/MS to search for tau proteolytic fragments released in response to neurotoxic

challenges.

Cells within a biological system secrete tau as full-length or proteolytic fragments,

depending on cellular environments. Table 3-4 shows the list of peptides identified from

CTX culture conditioned media. We did not observe N-terminal tau peptides in our

analysis (Figure 3-8A). All peptide clusters were identified starting around a.a. 200-700

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range on the rat tau isoform. One of interest is the C-terminal peptide,

PRHLSNVSSTGSIDMVDSPQLA (a.a. 716-737; accession number P10637),

corresponding to the amino acid 405-426 on human tau-441 isoform, which we have

seen with our in vitro experiment with tau (Figure 3-6B, 3-6C). Figure 3-15 shows the

product spectrum of the peptide PRHLSNVSSTGSIDMVDSPQLA released from

conditioned cell media of primary cerebrocortical cultures. We also observed unique

peptides from the high molecular weight form of tau (rat Tau-752; accession number

P19332-1) (Figure 3-8C and 3-9A). A cluster of similar peptides was observed in htau

mouse brain digested by calpain-1. The presence of the LMW C-terminal tau fragments

agrees with the work showing that synaptosomes from AD brains had high levels of tau

fragments (20-22K), with the majority lacking the C-terminal domain (79). Our peak

intensity analysis overlaps with the PSM counts showing high peptide intensity around

a.a. residue 210-250, 400-450, and a.a. 700-737 of rat tau (Figure 3-8B). We did not

observe peptide released with high confidence from control or OA treatment for 16h.

One might expect such outcome as OA treatment causes hyperphosphorylation of tau

making it less vulnerable to proteolysis.

With STS treatment, we observed the highest number of hits of proteolytic tau

peptides when compared to other tested conditions. One peptide observed in our in

vitro digestion is STGSIDMVDSPQL (a.a.724-736), with a phosphorylation site

probability of 99% at Ser724 residue (corresponding to pSer413 on human tau441)

(Figure 3-10). The rat tau sequences including a.a. 114-201, and a.a. 241- 444 are

missing from the human ta-441 isoform (Figure 3-10A). Due to these variances in amino

108

acid sequences, one might expect different cleavage by proteolytic enzymes. Figure 3-

10B shows rat tau-752 and tau-441 equivalent phosphorylation sites.

STS is a pan-kinase inhibitor that is known to induce apoptosis by activating

caspase-3(164). Others and we have identified a.a. 413-414 as caspase-3 cleavage site

on the human tau-441, suggesting the involvement of caspase-3 in the generation of

this fragment (71, 79). When cells were pretreated with OA followed by STS, we

observed unique fragments not found with STS treatment alone (Figure 3-10A). As for

A23187, no high confidence fragments were detected with and without OA pretreatment

(n=3). An explanation might be that A23187 did not produce LMW (<5K) fragments

released into the conditioned media during the 16h treatment; therefore, a longer time-

course treatment time is needed to confirm this observation. Lastly, MTX (with and

without OA) treatment resulted in peptides of varying length ranging from a.a. 377 to

441 on the rat tau-752 (Figure 3-8B and Figure 3-9A).

Phosphorylation Sites Analysis

As for the phosphorylation sites, we were able to identify novel sites on the p-tau

that are not found in the non-phosphorylated tau-441, particularly close to the C-

terminal domain including Thr63, Thr101, Ser400, Thr403, Ser412, and Tyr394 (Figure 3-7C).

Phosphorylation sites such as Thr69, Ser199, Ser202, Ser396, and Ser404 are associated

with AD brain (59, 60, 68). These phosphorylation sites might cause a differential

pattern of calpain recognition of tau sequence, producing unique peptides fragments.

As for CTX conditioned media, phosphorylated sites that are detected with and

without OA pretreatment are shown in Figure 3-9B. Phosphorylation at pSer724 on

STGSIDMVDSPQL (a.a.724-736) is of interest. It might be possible that

phosphorylation on pSer724 is required for the process of cleaving tau at a.a.

109

Ser723/Ser724 site, as it has been suggested that tau has a dynamic secondary structure

that demands a stereospecific angle, affected by the phosphorylation state to allow the

proteolytic enzyme to access the cleavage site. Alternatively, phosphorylation of

pSer724 might have resulted after tau cleavage as a requirement for the enzyme to

access this site.

Conclusions

In this study, we used in vitro analysis of calpain-mediated tau fragments as a

template to mimic the proteolysis of tau protein post-TBI mimicking other

neurodegenerative disorders including the CTE and FTD. Based on the data from our

immunological and peptidomic study that calpain-1 produced distinct clusters of N-

terminal (e.g., 2-19) and C-terminal peptides (e.g., 413-441) with varying lengths from

purified tau, mouse brain lysate, and cell-based neurotoxic challenges. To our

knowledge, this is the first systematic approach to characterize calpain-mediated

proteolytic fragments of tau protein for a unique peptidome biomarker library using

ultrafiltration-based nanoLC-MS/MS platform. Peptidomic data derived from this work

can serve as a novel approach in clinical diagnosis and therapy approach defined as

“theranostic”.

Our overall data in this study shows that tau is not only degraded into sizeable

molecular weight fragments by calpain but also into small fragments which are low in

molecular weight peptides that could simultaneously play a pathological role in the

neurodegenerative diseases such as Alzheimer’s disease, schizophrenia, CTE, and

TBI. Our results also were consistent with studies that showed the high vulnerability of

tau to calpain producing BDPs of varying length depending on the stage of the injury.

Thus, these results may be used in parallel in clinical studies to monitor the activity of

110

calpain and to investigate if similar patterns of tau peptides are generated in a disease

state, or to monitor the effect of pharmaceutical drugs. A future direction of our work is

to perform functional immunoassays to assess the potential role of these endogenous

peptides in tauopathies disorders, which may include aggregation and

hyperphosphorylation processes.

111

Table 3-1. Selected human tau-441 calpain-mediated proteolytic peptides from purified protein digestion identified by nLC-MS/MS.

Annotated Sequence #

PSMs Positions

Theo.MH+

[Da]

Sequence

Length Charge m/z [Da] XCorr

AEPRQEFEVMEDHAGTYG 65 2-19 2065.89 18 3 689.62 4.71

LSKVTSKCGSLG 46 315-326 1179.64 12 1 1179.61 3.73

SPRHLSNVSSTGSIDMVDSPQLA 81 404-426 2398.17 23 2 1199.91 5.69

TLADEVSASLAKQGL 37 427-441 1502.81 15 3 502.35 4.47

SASLAKQGL 32 434-441 787.467 8 2 394.37 2.51

SPRHLSNVSSTGSIDMVDSPQLATLADEVS 29 404-433 3113.51 30 3 1038.75 5.35

KAKTDHGAEIVYKSPVVSGDT 28 383-403 2202.14 21 3 735.03 4.05

RENAKAKTDHGAEIVYKSPVVSGDT 26 379-403 2672.36 25 3 891.93 5.3

AEPRQEFEVMEDHAGTYGLG 24 2-21 2236.31 20 2 1118.87 4.05

These measurements were obtained using LTQ-XL mass spectrometer.

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Table 3-2. Selected human calpain-mediated proteolytic peptides from purified p-tau protein digestion identified by nLC-MS/MS.


PSMs

Positions in

Proteins

Theo.

MH+ [Da]

Sequence

Length m/z [Da] XCorr

STGSIDMVDSPQLA 77 413-426 1500.63 14 751.098 4.18

KSPVVSGDTSPRHLSNVS 17 395-412 2106.87 18 1054.21 5.4

STGSIDMVDSPQL 16 413-425 1349.63 13 675.64 3.98

KPVDLSKVTSKCGSLG 7 311-326 1618.88 16 810.24 3.69

AAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVT 4 89-123 3472.56 35 1158.62 6.22

KPVDLSKVTSKCG 2 311-323 1361.75 13 681.41 3.44

KSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLA 2 395-426 3508.51 32 1170.50 5.01


113

Table 3-3. Selected tau-441 calpain-mediated proteolytic peptides from mouse htau transgenic brain lysate digestion identified by nLC-MS/MS.



PSMs

Positions in

Proteins

Theo. MH+

[Da]

Sequence

Length Charge

m/z

[Da] XCorr

TLADEVSASLAKQGL 9 427-441 1502.80 15 2 752.17 3.5

STGSIDMVDSPQLA 6 413-426 1420.66 14 1 1420.76 2.83

LSKVTSKCGSLG 4 315-326 1179.64 12 2 590.70 2.89

TDHGAEIVYKSPVVSGDT 4 386-403 1874.91 18 2 938.46 2.9

SPRHLSNVSSTGSIDMVDSPQLATLADEVS 3 404-433 3113.50 30 3 1039.01 4.38

ESPLQTPTEDGSEEPGSETSDAK 3 45-67 2391.03 23 2 1196.55 3.55

SPRHLSNVSSTGSIDMVDSPQLA 3 404-426 2398.16 23 2 1199.91 4.53

ASLAKQGL 2 434-441 787.46 8 2 394.57 2.15

TPPKSPSSAKSRLQT 2 231-24 1584.87 15 2 793.316 2.53

NIHHKPGGGQVEVKSEKLDFKDRVQ 2 327-351 2845.50 25 3 949.78 3.86

114

Table 3-4. Selected rat Tau-752 peptides identified from conditioned primary CTX culture media by nLC-MS/MS.


Sequence # PSMs Positions Sequence Length Charge m/z [Da] XCorr

PRHLSNVSSTGSIDMVDSPQLA 55 716-737 22 3 849.14 3.5

RSVSASGVSGETTSI 39 238-252 15 1 1677.78 2.07

VSASGVSGETTSIPGF 25 240-255 16 1 1736.90 2.09

FSKVSAETQASPPEGPG 16 268-284 17 1 1767.22 2.07

HLSNVSSTGSIDMV 15 718-731 14 1 1685.57 2.26

DLSNVQSKCGSKDN 12 594-607 14 2 868.42 2.77

AKTTPSPKTPPGSGEPPKSGERSGYSSPGSPGTPGSRSRTPSLPT 8 484-528 45 3 1552.74 4.33

SSQESPPSQASLAPGTATPQARSVSASGVSGETT 7 217-250 34 2 1734.38 2.53

HKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNV 6 685-722 38 3 1476.76 3.85

TRIPAKTTPSPKTPPGSGEPPKSGERSGYSSPGSPGTP 2 480-517 38 2 1995.92 2.7

115

Table 3-5. Global Homo sapiens database search using in vitro calpain-mediated tau-441 digested sample.

These measurements were obtained using Orbitrap fusion Tribrid mass spectrometer.

Accession Description Coverage

[%] #

Peptides #

AAs MW [kDa]

calc. pI

Score Sequest HT:

P10636-8 Isoform Tau-F of Microtubule-associated protein tau 90 499 441 45.8 8.25 3375.23

P04632 Calpain small subunit 1 47 25 268 28.3 5.2 134.57

P07384 Calpain-1 catalytic subunit 10 5 714 81.8 5.67 26.83

Q9UNZ2 NSFL1 cofactor p47 9 2 370 40.5 5.1 20.17

Q9NP31-2 Isoform 1 of SH2 domain-containing protein 2A 9 1 399 44.1 7.9 4.4

Q9NYQ8 Protocadherin Fat 2 1 1 4349 479 5.16 4.2

O15067 Phosphoribosylformylglycinamidine synthase 1 1 1338 144.6 5.76 3.95

Q9Y4P8-1 WD repeat domain phphoinitide-interacting protein 2 3 1 454 49.4 6.46 3.63

Q99650-1 Onctatin-M-specific receptor subunit beta 1 1 979 110.4 5.82 3.55

Q92552-2 Isoform 2 of 28S ribomal protein S27, mitochondrial 4 1 428 49.1 6.33 3.44

Q8N1W1-6

Isoform 6 of Rho guanine nucleotide exchange factor 28

1 1 1731 194.4 6.09 3.4

P50990 T-complex protein 1 subunit theta 2 1 548 59.6 5.6 3.37

Q8IVI9-4 Isoform 4 of Ntrin 4 1 563 64.3 9 3.36

P0DOX4 Immunoglobulin epsilon heavy chain 6 1 547 60.3 7.99 3.35

Q6L8Q7-1 2',5'-phphodiesterase 12 2 1 609 67.3 6.57 3.33

Q7Z7G8-1 vacuolar protein sorting-associated protein 13b 0 1 4022 448.4 6.46 3.29

Q92530 proteasome inhibitor PI31 subunit 5 1 271 29.8 5.74 3.29

Q6ZS46 Putative uncharacterized protein FLJ45840 11 1 218 22.1 10.55 3.26

116

Figure 3-1. Peptidomic sample preparation workflow for the nLC-ESI-MS/MS for the analysis of purified Tau protein,

conditioned cell media, and mouse brain lysate. This workflow can be applied to the preparation of several other types of biological fluid and samples. Subjecting the proteins to in vitro calpain-1 digestion results in both HMW protein fragments and LMW proteolytic peptides production. The LMW fragments were cleaned up and analyzed using nano-LC-ESI-MS/MS with the data-dependent acquisition on a Linear Ion Trap. In biological environments, these LMW peptides can be released into biofluid and can be used as a biomarker for neurotoxicity. SDS-PAGE and western blotting was used to analyze the HMW fragments.

117

Figure 3-2. Identification of calpain-mediated tau peptides by immunoblotting and mass spectrometry. (A) Western blot is displaying Tau-BDP (HMW) with different calpain-1 concentrations. The right panels show the quantification of the intact tau (63K) and tau-BDP (40K/38K) respectively from the immunoblots. Densitometric quantification of the intact and BDP of tau was performed using image-J. Data are presented as ± SEM for n=3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant. (B) Shows the selected peptides that had the highest PSMs plotted on the y-axis and m/z on the x-axis for non-phosphorylated-tau-441 calpain treatment. For the criteria of each peptide selection, each had at least 20 PSM, XCorr value from Sequest HT at least 3, and showed high confidence. (C) Schematic representation for top selected PSM tau-441 peptides generated from calpain-1 digestion. The core peptides are shown in blue, while the variability of the different lengths of peptides is represented as error bars. These error bars were calculated from all the peptides that are high in confidence, an XCorr value of 3, and at least had PSM value of 20 from Sequest HT search engine. Peptides found in control are not shown. The panel shows tau (P10636-8) sequence. Residues in blue represent the peptides detected. The underlined residues represent the overlapping peptide detected (D) Peptide profile of purified human tau-441 (P10636-8) digested by calpain-1. At each amino acid residue along the protein, the height of the green bars is proportional to the count of peptides overlapping this position. The intensity of the color (green) is proportional to the sum of the peptide intensities overlapping this position.

118


119

Figure 3-3. Identification of calpain-mediated phosphorylated tau peptides by immunoblotting and mass spectrometry. (A) Western blot demonstrating phosphorylated Tau-BDP (HMW) with different calpain-1 concentrations. The right panels show the quantification of the intact tau (63K) and tau-BDP (40K/38K) respectively from the immunoblots. Data are presented as ± SEM for n=3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant. (B) Shows the selected phosphorylated tau peptides that had the highest PSMs plotted on the y-axis and m/z on the x-axis for phosphorylated-tau-441 calpain treatment. For the criteria of each peptide selection, each had at least 20 PSM, XCorr value from Sequest HT at least 3, and showed high confidence. (C) A schematic representation of top-selected PSM phospho-tau-441 peptides generated from calpain-1 digestion. The core peptides are shown in red, while the variability of the different lengths of peptides is represented as error bars. The panel shows tau (P10636-8) sequence. Residues in red represent the peptides detected. The underlined residues represent the overlapping peptide detected. The yellow color represents phosphorylated peptides (D) Peptide profile of purified human phospho-tau-441 (P10636-8) digested by calpain-1. At each amino acid residue along the protein, the height of the green bars is proportional to the count of peptides overlapping this position. The intensity of the color (green) is proportional to the sum of the peptide intensities overlapping this position.

120


121

Figure 3-4. Identification of calpain-mediated tau peptides from transgenic htau mouse brain lysate by immunoblotting and mass spectrometry. (A) Western blot is displaying total Tau-BDP (DA9) and phosphorylated tau (RZ3; pThr231) with different calpain-1 concentrations. The right panels show the quantification of the intact tau (63K) and tau-BDP (35K) respectively from the immunoblots. Data are presented as ± SEM for n=3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant. (B) Shows the selected tau peptides from brain lysate that had the highest PSMs plotted on the y-axis and m/z on the x-axis for phosphorylated-tau-441 calpain treatment. For the criteria of each peptide selection, each had at least 5 PSM, an XCorr value from Sequest HT of 3, and showed high confidence. (C) A schematic representation for selected PSM tau peptides generated from calpain-1 digestion. The core peptides are shown in orange, while the variability of the different lengths of peptides is represented as error bars. The panel shows tau (P10636-8) sequence. Residues in orange represent the peptides detected. The underlined residues represent the overlapping peptide detected. (D) Peptide profile of mouse brain tau-441 (P10636-8) digested by calpain-1. At each amino acid residue along the protein, the height of the green bars is proportional to the count of peptides overlapping this position. The intensity of the color (green) is proportional to the sum of the peptide intensities overlapping this position.

122


123

Figure 3-5. Composite summary of calpain-mediated tau proteolytic peptides and phosphorylation sites. (A) Spreadsheet showing representative calpain-mediated proteolytic peptides identified from tau, ptau, and mouse brain lysate samples. This table listing shows the confidence of each peptide in each fraction. Not found peptides are represented as white. Green represents high confidence peptides. Yellow represents medium confidence peptides. Red represents low confidence peptides. (B) Venn diagram created using the proteome discoverer 2.2 software, showing the total number of peptides released in each of the biological replicates from the different samples. The common peptides (24) identified from the three replicates will be used for further analysis. (C) A schematic representation showing the domains of tau protein and location of phosphorylation identified by nano-LC/MS/MS and predicted calpain cleavage sites derived from in this study.

124


125

Figure 3-6. View of a customized Peptigram peptide alignment map compiled from tau, p-tau, and mouse brain lysate. The peptides were aligned to fit the longest human tau precursor protein (P10636; 758 a.a.). The representative peptides aligned were obtained by filtering and compiling peptides identified from tau, p-tau, and brain lysate digested by calpain-1 from 1; 100, 1:50, 1:25, and 1:10 dilutions. The dashed lines and the table on the right show our predicted calpain cleavage sites. The alignments represented are retrieved by ProViz(184) and were further modified. Peptides from the input data are displayed in green boxes, where the color intensity of the peptide box is proportional to the peptide intensity. (A) This alignment is generated from tau-758 starting from the N-terminal domain: a.a. 1-57 (B) covers amino acid 60-128 and (C) cover amino acid 698-744.

Residue number

Compiled identified peptides from tau, p-tau and brain Lysate

Tau SequenceResidue number

Tau Sequence

Tau SequenceResidue number

Compiled identified peptides

from tfau, p-Tau and brain Lysate

Compiled identified peptides from Tau, p-Tau and brain Lysate

TAU_HUMAN_758 – P10636

A

B

C

Cleavage sites

G19↓L20

Q49↓T50

A89↓A90

Q124↓E125

T720↓S721

126

Figure 3-7. Basal and OA-induced tau proteolysis following neurotoxin challenges in rat cerebrocortical neurons. (A) Rat cerebrocortical cultures (CTX) were either untreated (control) or pre-treated with phosphatase inhibitor (OA, 100 nM) for six hours. For tau proteolysis analysis, neurotoxin challenges included: STS (0.5μ M), A23187 (20 μM) and MTX (10 nM) or 16 h. After the treatment, cell lysates were harvested for protein and immunoblotting analysis with total tau monoclonal antibody (DAKO; top panel), phospho-tau antibodies (RZ3 and PHF-1), and αII-spectrin monoclonal antibody. The UCH-L1 antibody was used as loading control. Results are representative of three independent experiments. (B) Show densitometric quantifications from the immunoblots. Data are presented as ± SEM for n=3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant.

127


Co

ntr

ol

ST

S

A23

18

7

MT

X

OA

OA→

ST

S

OA→

A23

18

7

OA→

MT

X 0.0

0.5

1.0

1.5

To

tal-

Tau

/UC

H-L

1 levels DAKO (tau-BDP-50K)

***

***

****

******* *

Co

ntr

ol

ST

S

A2

31

87

MT

X

OA

OA→

ST

S

OA→

A2

31

87

OA→

MT

X 0.0

0.5

1.0

1.5

pT

hr2

31/U

CH

-L1 levels

RZ3 (Intact p-tau 54K)

***

********

******

******

*****

*

Co

ntr

ol

ST

S

A2

31

87

MT

X

OA

OA→

ST

S

OA→

A2

31

87

OA→

MT

X 0.0

0.5

1.0

1.5

To

tal-

Tau

/UC

H-L

1 levels

DAKO (Intact tau-54K)

**** ***

******** **

***

Co

ntr

ol

ST

S

A23

18

7

MT

X

OA

OA→

ST

S

OA→

A2

31

87

OA→

MT

X 0.0

0.5

1.0

1.5p

Ser3

96/S

er4

04/U

CH

-L1 levels

PHF-1 (Intact-p-tau-54K)

********

****

*

*****

**

****

****

****

Co

ntr

ol

ST

S

A23

18

7

MT

X

OA

OA→

ST

S

OA→

A2

31

87

OA→

MT

X 0.0

0.5

1.0

1.5

Sp

ectr

in-2

80K

/UC

H-L

1 levels

Intact a-II-Spectrin (280K)

********

***

************

****

B

128

Figure 3-8. Identification of tau neuropeptides from conditioned media following neurotoxin challenges in rat cerebrocortical neurons. (A) Schematic representation of tau peptides generated and released into conditioned media. Duplicate peptides found are not shown. None of the peptides shown were found in non-treated control samples. Green box represents the identified peptide. Panel (B) shows the selected tau peptides released from CTX conditioned media. Peptides that had the highest PSMs were plotted on the y-axis, and m/z was plotted on the x-axis. The criteria of peptide selections were: minimum value of 3 PSMs, an XCorr value of 3, and high confidence only. (C) Rat tau accession # P19332 amino acid sequence. Residues in green show coverage of tau peptides released into the conditioned media. Underlined residues shown represent two overlapping peptides. Residues in red show the phosphorylation (D) Peptide profile of -rat Tau-441 (P19332) digested by calpain-1. At each amino acid residue along the protein, the height of the green bars is proportional to the count of peptides overlapping this position. The intensity of the color (green) is proportional to the sum of the peptide intensities overlapping this position.

129


130

Figure 3-9. Composite summary of tau proteolytic peptides and phosphorylation sites from CTX conditioned cell media. (A) Spreadsheet showing representative proteolytic peptides identified rat CTX conditioned media. This table listing shows the confidence of each peptide in each fraction. Peptides not found are represented as white color. Green color represents high confidence. Yellow color represents medium confidence. Red represents low confidence. (B) Schematic representation showing the domains of rat tau protein (Accession# P19332) and location of phosphorylation sites identified and predicted calpain cleavage sites derived from in this study as detected by nano-LC/MS/MS.

131

Figure 3-10. Tau protein sequence alignments of rat and human. (A) Human tau is the placed on the query lane while rat tau is on the subject lane. The amino acid sequences 114 to 201 and 241 to 444 of rat tau are missing on the human ta-441 isoform. Panel (B) is a table showing rat tau-752 phosphorylation sites and their location on tau-441.

132

Figure 3-11. Representative tau-441 sequences coverage of the human protein (P10636-8) detected. The figure was extracted from proteome discoverer 2.2 software.

133

Figure 3-12. Representative tau-776 sequences coverage of the human protein (P10636-9) detected. The figure was extracted from proteome discoverer 2.2 software

134

Figure 3-13. Product ion spectrum of the tau peptide AEPRQEFEVMEDHAGTYG released from calpain-1 digestion. (A) Product ion spectrum for the tau-441 peptide AEPRQEFEVMEDHAGTYG (amino acid residues 2-19), charge, +3, monoisotopic m/z 689.62 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions for the tau peptide shown in red and blue identified from the database search results. The figure shows b and y ions table with the theoretical fragment ions monoisotopic masses. The y ions are the series that extend from the N-terminus, or the front of the peptide. The b ions are series of amino acids that extend from the C-terminus, or the back of the peptide. The residue number are indicated in #1 column and #2 column. The masses presented in the table are theoretical calculated monoisotopic masses of each fragment or amino acid expected from the identified peptide. The blue and red highlighted colors numbers are b ions that are identified from the peptide and fall within the specified value of the allowed mass threshold to be matched.

135


136

Figure 3-14 Product ion spectrum of the tau peptide SPRHLSNVSSTGSIDMVDSPQLA released from calpain-1 digestion. (A) Product ion spectrum for the tau-441 peptide SPRHLSNVSSTGSIDMVDSPQLA (amino acid residues 404-426), charge, +2, monoisotopic m/z 1199.91 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions for the tau peptide shown in red and blue identified from the database search results.

137


138

Figure 3-15. Product ion spectrum of the rat tau peptide PRHLSNVSSTGSIDMVDSPQLA released from conditioned cell media of primary cerebrocortical cultures. (A) Product ion spectrum for the phosphorylated tau-752 peptide PRHLSNVSSTGSIDMVDSPQLA (amino acid residues 716-737), charge +3, monoisotopic m/z 849.14 Da, displaying the fragment ions for this peptide. The phosphorylation losses are shown in yellow boxes of the b and y ions (B) Identified b+ and y+ type ions for the tau peptide shown in red and blue identified from the database search results.

139


140

Figure 3-16. Product ion spectrum of the phosphorylated tau peptide STGSIDMVDSPQLA released from calpain-1

digestion. (A) Product ion spectrum for the phosphorylated tau-441 peptide STGSIDMVDSPQLA (amino acid residues 413-426), charge +3, monoisotopic m/z 751.09 Da, displaying the fragment ions for this peptide. The phosphorylation losses are shown in yellow boxes of the b+ and y+ ions (B) Identified b+ and y+ type ions for the tau peptide shown in red and blue identified from the database search results

141


142

Figure 3-17. Human tau-441 and tau-776 isoforms amino acid sequence alignments. Human tau-441 is in the top lane while tau-776 is on the bottom lane. The red color amino acid sequences represent amino acid sequences are found in both isoforms. The dashed lines mean the absence of amino acids.

143

Figure 3-18. Comparison between targeted database search against Human tau-441 versus Homo sapiens proteome. Panel (A) shows a list of peptides starting from N-terminal domain to amino acid 64 on the tau protein. Panel (B) cover peptides starting from a.a. 50 to124. Panel (C) cover peptides from amino acid 96 to 345. Panel (D) cover tau peptides starting from amino acid 341 to 426. Panel (E) shows a list of peptides covering tau from 404 to 441. These peptides were identified using Orbitrap Fusion Tribrid Mass spectrometer.

144


145


146


147


148

CHAPTER 4 ANALYSIS OF TEMPORAL PROFILE OF TBI-INDUCED TAU

HYPERPHOSPHORYLATION AND PROTEOLYSIS IN MOUSE BRAIN BY IMMUNOBLOTTING AND MASS SPECTROMETRY

Introduction

Traumatic brain injury (TBI) is caused by damage from external force resulting

from fast acceleration or deceleration, penetration by a projectile, crush, impact,

potentially leading to short-term or permanent deficiencies of cognitive, physical, and

psychosocial functions(123). TBI is a complicated pathophysiological process that leads

to structural and functional damages resulting from primary and secondary injury

events(186).

The primary injury occurs after the instant physical disturbance of brain tissue

that happens at the moment of exposure to the mechanical force such as contusion,

damages to blood vessels (hemorrhage), and shearing of axons, in which neuronal

axons are torn and stretched. The secondary injury develops over minutes to months

after exposure to the primary injury, in which a cascade of metabolic reactions, cellular

and molecular processes that finally cause apoptosis, tissue injury and

neurodegeneration.(25)

Several biochemical reactions are responsible for the secondary injury, such as

glutamate excitotoxicity, disturbance of cellular calcium levels, elevated free radical

production, lipid peroxidation, mitochondrial damages, cell death, and diffuse axonal

injury. The reaction events of secondary injury cause neuronal, endothelial, glial

apoptosis and white matter disintegration. Apoptosis happens within a couple of

minutes after the injury and could lengthen over an interval of days to months.(187)

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The medical heterogeneity seen in TBI patients depend on the site and degree of

the initial injury(188). Moreover, preexistent factors and patient condition may also

subsidize the pathophysiological heterogeneity of TBI, for example, age, health, gender,

alcohol, drug consumption and genetic factors(189). Animal studies of TBI are

structured to generate a reasonably homogenous mode of injury considering age,

gender, genetics and the injury parameters(190).

Therefore, it is challenging to fully reproduce all characteristics of secondary

injury seen in human TBI in any one animal model, which potentially could explain why

a lot of pharmaceutical drugs that showed promise in animal studies failed in clinical

studies. Animal models of TBI are undoubtedly critical for understanding the

biochemical and molecular functions of human TBI that clinical studies cannot test.

Moreover, animal studies are vital to develop and characterize novel diagnostic and

therapeutic strategies to translate into preclinical and clinical studies (190).

Several animal models of TBI injuries have been developed in the view of the

heterogeneous nature of the clinical environments. Rodents are used most of the time

instead of larger animals in TBI research because of their cost, small size, and

consistent result measurements(190).

Most of the recent TBI studies are focused on addressing the complex molecular

processes that occur after the head injury(188). The four animal TBI models that are

used extensively are fluid percussion injury (FPI), cortical impact injury (CCI), weight

drop–impact acceleration injury, and blast injury(191). We will cover in this chapter the

CCI animal model of TBI and will not discuss other models since CCI only was selected

for the study.

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Cortical Impact Injury (CCI)

In order to mimic the cortical tissue loss, subdural hematoma, neuronal axonal

damage, mild TBI, blood-brain barrier (BBB) dysfunction, and even coma(192), the CCI

model was built to use electromagnetic impact or pneumatic devices to drive the

impactor onto the exposed intact dura(191). The physical impact delivered to the

exposed dura through a unilateral craniotomy, most often distort the underlying cortex.

Studies have shown that a wide-ranging neuropathological characterization of the CCI

animal model can include acute cortical, hippocampal, and thalamic

neurodegeneration(193).

One of the significant advantages of this type of injury over the other TBI models

is the convenience, at which the mechanical factors can be controlled, such as time,

velocity, depth of the impact. Therefore, it could be argued that CCI is more beneficial

than FPI model for studies that involve biomechanical factors. Another feature of the

CCI model is the absence of risk of rebound injury when compared with TBI models that

involve gravity-driven devices. The histopathological damages of CCI increase with

increasing the cortical disruption and the velocity of the impact allowing the control of

TBI severity, required for the experimental condition.(194)

The cognitive dysfunction of the animal model is highly related to the depth of the

cortical deformation and the velocity of the impact as measured in the Morris water

maze test in mice and rats. The cognitive impairment might persist up to one year after

the impact and could be associated with brain atrophy. The CCI animal model produces

injury that is consistent with the pathological characteristics of human TBI. Using large

animal TBI models might provide the chance to collect physiological information after

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the injury in an environment that resembles the intensive care unit and thus could help

in translational studies of animal models into clinical practice.(74, 190)

Most of the animal TBI experiments have been performed on a short-term basis,

in the range of hours to days and seldom beyond one month after the injury(190). The

short-term studies have provided information on the pathophysiology and molecular

function outcomes during the acute stage after the TBI. However, the short-term TBI

studies might not generate a fair assessment of long-term TBI and cannot be

implemented alone in assessing strategies for clinical therapy for long-term therapy.

Studies that involve long-term injury response are critical for evaluating the outcomes of

TBI (three months and more after TBI). The use of different types of injury and longer-

term evaluation after TBI is essential for future preclinical studies.(190)

Analysis of tau phosphorylation levels after TBI models is essential since tau

levels profoundly influence the pathophysiology of TBI, both in short-term and long-term

experiments. Tau can provide highly informative biomarkers for post-TBI. In this

chapter, we implement the use of immunological methods including phospho-specific

tau antibodies to assess the temporal profile of tau phosphorylation and proteolysis

(including LMW and HMW) following TBI. We processed the samples using the

peptidomic approach described in chapter 3 for nano-UPLC-MS/MS. We have identified

novel peptides fragments of tau with different levels of phosphorylation resulted from

TBI mouse injury, which might provide a prognostic biomarker potential for patient

recovery in a tauopathy-relevant disease.

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Ethical Statement




Mouse Brain Samples Preparation

TBI in human tau transgenic mice (3-4-month-old; Charles River Laboratories;

USA) was induced using controlled cortical impact (CCI) device. The mice were

anesthetized using 4% isoflurane in oxygen as a carrier gas for four minutes and

maintained under anesthesia with 2-3% isoflurane in oxygen. Surgery was performed

on mice by mounting in a stereotactic frame in prone position and secured by ear and

incisor bars. A 3 mm unilateral (ipsilateral) craniotomy was performed midway between

bregma and lambda with the dura mater remaining intact over the cortex. A brain

trauma was induced by using a PSI TBI-0310 Impactor (Precision Systems and

instrumentation, USA) by impacting the right cortex (ipsilateral cortex) with 2 mm

diameter impactor tip at velocity of 3.5 m/second, 1.5 mm compression depth, and a

200 ms dwell time (compression duration).

After one, three, and seven days post-CCI, the animals were euthanized with a

lethal dose of pentobarbital; whole brains were removed, and snap frozen using liquid

nitrogen. For naïve brain samples, mice were euthanized, and subsequently, brains

were removed and flash-frozen in liquid nitrogen and did not receive an impact injury.

The brains were stored at – 80°C until further use. The brain samples were pulverized

to a fine powder using a mortar and a pestle set in dry ice using liquid nitrogen. The fine

powder was transferred to microcentrifuge tubes (Eppendorf). Powdered brain samples

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were stored at – 80°C until further use. The brain powder was then lysed with 1% Triton

X-100 lysis buffer containing 20 mM Tris HCl, pH 7.0, 5 mM EDTA, and 1 mM DTT in

double distilled water. The brain samples were incubated at 4°C 90-120 minutes kept at

a tube revolver (Thermo scientific) at low speed (10,000 x g). Following the incubation,

the samples were centrifuged at 14,000 rpm for 15 minutes at 4°C, and the supernatant

was transferred to new tubes. The protein concentration was determined by performing

protein assay using bicinchoninic acid microprotein assays (Pierce Inc., Rockford, IL,

USA) against albumin standards.

Ultrafiltration Method and Ultrafiltrate Processing for Mass Spectrometry

Ultrafiltration step is introduced to partition small peptides from higher molecular

weight protein or protein fragments using an ultrafiltration unit with a molecular weight

cut-off (MWCO) value of 10 kDa (Sartorius Stedim Biotech, VS0102). A sample of 250

µL of brain tissue lysate was loaded into the ultrafiltration device and centrifuged at 4°C

for 20 min, 5000 x g [Eppendorf 5415R]. Twenty micrograms of the retentate were used

for SDS-PAGE and western blot analysis (described in the following section below). The

200 μL of filtrates were dried using speed vacuum (Thermo Scientific). The ultrafiltrate

samples (containing peptides) were reconstituted with 15-µL LC-MS grade water with

0.1% formic acid, and five µL was used for each injection into nanoLC-MS/MS.

Immunoblot Analysis

The retentate fractions of (ipsilateral and contralateral cortex brain lysate) were

analyzed using SDS-PAGE followed by western blotting. The samples (20 µg of protein

for tissue lysate) were mixed with SDS-containing sample buffer along with rainbow

molecular weight marker full-range rainbow (RPN800E, GE Healthcare, USA). Tris-

glycine gels (4-20%, 1 mm X 18 well) were run on 200 V, for about one hour.

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For immunoblotting, the gel was placed in deionized water for 5 minutes and then

in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 5 minutes before

electrotransfer to PVDF membrane on the iBlot Dry Blotting system (Carlsbad, CA,

USA) at 20 V for 13 min. The PVDF was blocked with non-fat milk (5%) in Tris-buffered

Saline Tween (TBST) [20 mM, Tris pH 7.5, 15 mM NaCl, and 0.02 % Tween 20] and

probed with specific primary antibodies prepared in 5% skim milk in TBST overnight at 4

°C, followed by secondary goat anti-Rabbit IgG alkaline phosphatase conjugate (Fc

specific) or goat anti-mouse IgG alkaline phosphatase conjugate (69266, EMD Millipore,

USA] for two hours.

Immunoreactive bands were then observed by adding 5-bromo-4-chloro-3-

indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) [Product Code: 50-81-10].

Dried membrane was scanned with Expression 8836XL (Epson) and the UN-SCAN-IT

software (version 6.1, Silk Scientific Corporation). Quantitative evaluation of the protein

levels was performed with the computer-based densitometric NIH Image J (version 1.6)

software.

Reversed-Phase Nano-UPLC-MS/MS

Separation of peptides from tissue lysate samples was performed on

NanoAcquity UPLC (Waters, Milford, MA) by reversed phase chromatography. Five

microliters of each sample were loaded onto a NanoACQUITY UPLC symmetry C18 trap

column, 100 Å, 5 µm, 180 µm x 20 mm followed by separation on an ACQUITY UPLC

BEH (C18) column, 130 Å, 1.7 µm, 100 μm X 100 mm. The mobile phase consisted of

solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic

acid) (Honeywell, Muskegon, MI). Separation was achieved within a run time of 135 min

at a flow rate of 300 nL/min. The first linear gradient was run from 1% to 50% B over 90

155

min; the second linear gradient was from 50% to 85% B over 5 min and held for 5 min

before returning to initial mobile-phase composition (1% B). Tandem mass spectra were

collected on LTQ-XL (Thermo, San Jose, CA, USA) using a data-dependent acquisition

method in Xcalibur 4.0 (Thermo).

The analysis was set up for a full scan recorded between m/z 300–1800, and an

MS/MS scan to generate product-ion spectra to determine the amino acid sequence in

consecutive instrument scans of the ten most abundant peaks in the spectrum MS

(scan event 1) with dynamic exclusion enabled. The following dynamic exclusion

settings were applied to precursor ions chosen for product spectra analysis: repeat

count—1; repeat duration—30 s; and exclusion duration—30 s. All MS/MS spectra were

analyzed using Proteome Discoverer 2.2 (Thermo), SEQUEST HT (version: 2.1.1.21).

Database search engines were set up to search with no enzyme UniProt-Tau-441

(P10636-8), FASTA file version 2018_07. The search was achieved using the average

mass for matching the precursor with a fragment ion mass tolerance of 0.8 Da and a

parent ion tolerance of 1.5 Da. Phosphorylation of serine, threonine, and tyrosine was

selected as a dynamic modification, while the oxidation of methionine and cysteine were

selected as a static modification, using the output from SEQUEST HT.


Controlled cortical impact (CCI) is a model that implements a mechanical force to

mimic traumatic brain injury (TBI)(191). CCI was invented 30 years ago with the goal of

devising a mechanical platform to study brains subjected to direct physical deformation.

The CCI model quickly altered into a standardized method to analyze different TBI

models and evaluate different strategies for therapy. In this study, we have used the

CCI model to produce mouse TBI at the proper time-points (one, three, and seven

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days). The five different brain parts in the right and left hemisphere (cerebrocortex,

subcortical white matter, hippocampus, and corpus callosum) were at once dissected

after decapitation.

The cerebrocortex was further dissected into the ipsilateral and contralateral

cortex to study the different effect of the injury on these two regions. After collecting the

brain parts, all samples were snap-frozen with liquid nitrogen at once to minimize

unwanted protein degradation that is not due to CCI. Moreover, phosphatase and

protease inhibitors were added to the lysis of buffer. Since the controlled cortical impact

device targets directly the cortex, there was a focal injury in the ipsilateral (IC) and

contralateral cortex (CC) tissue than in the hippocampal tissue, which was impacted

indirectly by contusive force.

Our mouse models were designed to express only human tau isoforms(195). It

was made by breeding two existing line of tau mice and tau knockout mice(196, 197).

The tau mice express a tau transgene through P1-derived artificial chromosome (PAC)

cloning vector controlled by the tau promoter and generate all human tau isoforms. A

stretch of complementary DNA (cDNA) expressing the enhanced green fluorescent

protein (EGFP) was introduced into exon 1 of the MAPT gene.

As for the neuropathology, htau mice develop age-related tau pathology that

includes rearrangement of tau to cell bodies and dendrites, hyperphosphorylated tau,

aggregation of pair helical filaments (PHF), and detection of neurofibrillary tangles using

thioflavin S at nine to fifteen months. Tau pathology is maximally severe in the cortex

and hippocampal region of the brain and least in the brain stem and spinal cord(195,

198). In our study, all mice used for CCI aged from three to six months. After nine

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months minimally, the aggregated tau and PHF can be seen by immunoelectron

microscopy. However, insoluble tau aggregates can be separated from the brain tissue

as early as two months. Hyperphosphorylation of tau starts to elevate by six months and

continue to increase by thirteen and fifteen months.(199) Figure 4-1 shows our

optimized peptidomic sample workflow for the nano-LC-ESI-MS/MS after CCI treatment

to htau mouse brain. Subjecting the mouse brain proteins to ultrafiltration results in the

separation of HMW protein fragments from LMW proteolytic peptides production.

Immunoblot Analysis of Tau in Transgenic Mouse Brain after Experimental CCI

To determine the phosphorylation, proteolysis, and oligomerization state of tau

following CCI, we analyzed our samples by western blotting using the tau phospho-

antibodies CP13 (pSer202), PHF-1(pSer396/pSer404), RZ3 (pThr231), AT270 (pThr181) AT8

(pSer202/pThr205), and total tau DAKO (Figure 4-2, 4-3 and Table 2-1).

In the naïve cortex, tau protein was detected as a doublets band around 52K—

54K, when samples were probed with DAKO, AT270, AT8, and PHF-1 (Figure 4-2A and

Figure 4-3A). These bands of tau observed could be attributed to the multiple isoforms

of tau protein, phosphorylation levels, or both. Additionally, the basal levels of

phosphorylation in naïve cortex observed with PHF-1, AT8, and AT270 (Figure 4-2A

and Figure 4-3A), could potentially be an effect of age-related phosphorylation resulting

from the transgenic htau expressed. As described above, tau hyperphosphorylation

starts to elevate by six months and continue to increase by thirteen to fifteen months.

Mouse naïve cortex (24h after craniotomy) did not produce any tau-BDPs (Figure 4-2

and Figure 4-3).

Our total tau antibody (DAKO) detected a considerable increase for tau-55K at

D3 and D7 with contralateral cortex (CC) and ipsilateral cortex (IC) of TBI samples

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(Figure 4-3). This observation is consistent with Rubenstein et al. group study where

they observed increased levels of total tau and p-tau after TBI in the brain and blood

that were associated with PrPc expression levels as measured immunologically(200).

We also observed with total tau DAKO antibody increased levels of tau-BDP-26K,

starting on day 1 and dramatically peaking at day 7 in both IC and CC samples (Figure

4-3A).

As for AT270 (pThr181) and AT8 (pSer202/pThr205), we observed an increase of

tau-56K considerably after seven days of TBI in the IC and CC (Figure 4-2A, B).

Interestingly, with AT270 and AT8, we were also able to detect a dramatic increase of

high molecular weight bands of tau-225K (accounting for tau oligomers) compared to

naïve cortex after one, three, and seven days of CCI in both cortex brain regions BDPs

(Figure 4-2A, B). Moreover, we observed that as 225K band increase in band intensity,

there was a simultaneous decrease of the tau-56K band as well (Figure 4-2A). One

might expect that p-tau polymerize into oligomers phase, thereby, reducing the total

levels of monomeric p-tau. Phosphorylation at Thr181 and Thr205 has been considered

major toxic sites and are associated with abnormal accumulation of p-tau in postmortem

human brain found after years of sustained repetitive mTBI.

Moreover, a study showed that oligomeric and monomeric p-tau were detected

after four hours, one day, two weeks post-TBI in a non-transgenic rodent model of

parasagittal FPI model(201).

Probing samples with PHF-1 (pSer396/pSer404) showed a dramatic increase for

the tau-56K band after D3 and D7 of TBI in both IC and CC samples (Figure 4-3). A

molecular weight shift was observed for p-tau-56K to p-tau-58K at D7 in both CC and

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IC. However, we did not observe any Tau-BDP or oligomeric-tau with PHF-1 antibody at

the different time point tested post-TBI (Figure 4-3A). Phosphorylation sites at

pSer396/pSer404 are known to associate with the pathological features of tauopathies in

AD(202) and TBI(200). Therefore, our observation suggests that modifications at the

PHF-1 site can coincide with other pathological sites in post-TBI.

In the mouse IC of the TBI group, probing with RZ3 (pThr231) showed a

considerable increase of p-tau-56K at D1, D3 of IC and D1, D3, and D7 of CC following

CCI (Figure 4-3). RZ3 showed the highest increase in p-tau-56K levels at D1 of IC and

D7 of CC. Interestingly, RZ3 showed a dramatic loss of ptau-56K at D7 in IC samples

and a significant increase in p-tau-BDP-24K (p-value<0.05) at D1 and D7 compared to

control in IC region (Figure 4-3). Like PHF-1, we did not observe any high molecular

weight tau (225K) post-CCI with the RZ3 antibody. We observed tau-BDP 24K only in

IC and not in CC samples. Phosphorylation at Thr231 is known to facilitate tau

hyperphosphorylation, aggregation, and initiate neurodegenerative mechanisms within

the cells. A recent study reported increased GSK3β levels, pThr175, pThr231, and

oligomerized tau in hippocampal neurons of moderate TBI rats(203). Moreover, the

pThr231-Pro232 motif can be either in the cis or trans configuration, but studies indicated

that cis configuration is the pathological form for AD(204).

As for CP13 antibody, tau-56K increased dramatically with D1, D3 D7 in IC and

D7 in CC and to a lesser extent in D1 and D3 of IC (Figure 4-3). Multiple clusters of tau-

BDP were observed as with CP13, concentrating mainly at 24K and 15K at D7 of IC and

CC. Less tau-BDP-24K was observed when samples were probed with CP13 after D1

and D3 of IC and CC post-CCI (Figure 4-3). Again, we did not observe any oligomeric p-

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tau form with the CP13 antibody. Phosphorylation site on Ser202 is one of the well-

known epitopes linked with tauopathies-relevant diseases and was found to be

hyperphosphorylated in a rat model of TBI (201).

To assess the cell integrity and calpain-mediated SBDP150K/SBDP145K and

caspase-3-mediated SBDP120, we have also included the αII-spectrin antibody (Figure

4-4). We observed with αII-spectrin antibody a major calpain-mediated

SBDP150K/SBDP145K bands, with a minor band of caspase-3 mediated SBDP120K

with all samples that were treated with CCI (Figure 4-4). The naïve mice did not show

any spectrin breakdown products. The role of calpain in TBI pathology is well-

known(205). Calpain activation occurs within hours following the contusive or diffuse

brain trauma in the animal. Ubiquitin carboxy-terminal hydrolase isozyme L1 (UCHL-1)

antibody was probed as a loading control. All blots showed an equal amount of UCH-L1

protein (Figure 4-4).

Identification of Tau Proteolytic Fragments from Mouse Brain after CCI using nLC-nESI-MS/MS

Tau fragments have sparked much research interest due to their ability to play a

role in tau aggregation. Post-translational modifications (PTMs) of tau can disrupt the

primary structure of tau and hinder its ability to fold correctly into the paper-clip like

tertiary structure, leading to the accumulation of an aggregated form of tau species

because of the disoriented quaternary structure(42).

Tau protein proteolysis is well established to produce large breakdown products

(45K, 35K, and 17K) relative to the size of the intact protein(79). Low molecular weight

(LMW) tau peptides (≤5K) released from tau proteolysis needs further characterization

for biomarker utilization and neurotherapeutic purposes. Peptidomics is the study of

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LMW peptide fractions from proteins including active biological neuropeptides, protein

fragments produced from proteolytic enzymes, or other small proteins such as cytokines

and signaling peptides. Technological advances in mass spectrometry have made MS-

based peptidomics a method of choice for characterization and quantification of novel

biomarkers. Some pathological tau peptides have been reported previously for

neurodegenerative diseases, such as Alzheimer’s and other pathologies(206, 207).

Others and we have studied calpain-mediated tau-BDPs using western blotting

analysis. However, to the best of our knowledge, there are no systematic studies of the

tau peptidome following TBI. The significant difference between proteomics and

peptidomics studies is the absence of the trypsin digestion step in vitro that produce the

artificial tryptic peptides that facilitate the identification process. In our study, because

we are dealing with natural low molecular weight peptides (≤5K), biological samples can

be directly subjected to LC-MS/MS analysis after ultrafiltration step without the

additional step of tryptic digestion.

Our focus in this part of the study is to examine the concept that TBI in mouse

activates multiple intracellular and extracellular proteases such as calpains, caspases,

cathepsins, and other tau proteases resulting universal brain tau breakdown and

release of neurotoxic peptides. We have used mouse brain lysate samples from two

different regions (IC and CC) after CCI. We have processed our samples by

ultrafiltration devices with a MWCO value of 10K to enrich LMW tau peptides and isolate

any HMW contaminants that could interfere with our LC-MS/MS analysis.

Figure 4-1 shows the optimized peptidomic sample preparation workflow for the

nano-LC-ESI-MS/MS after CCI treatment to transgenic htau mouse brain. Figure 4-5

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and Figure 4-6 display the total ion chromatograms for nLC-ESI-MS/MS for peptides

isolated from ipsilateral (IC) and contralateral (CC) mouse brain cortex lysate using

peptidomic workflow. We have observed a better separation of peptides using a 160

min gradient of 1-40%B; therefore, we ran the rest of the samples using this LC method.

One interesting observation shown in Figure 4-5 and Figure 4-6 chromatograms is the

presence of peaks that are separated in specific patterns which might accounts for

polymeric contaminations such as PEG or keratin as they might be included in our

sample preparation steps. Therefore, to avoid such contaminants, one might subject the

samples to ZipTip clean up, solid phase extraction, strong cation exchange

chromatography to further remove detergents effect in our chromatographic

separations.

We analyzed the samples by MS/MS performed in the order of the top ten most

abundant ions from MS spectra. The resulting raw data was searched using Proteome

Discoverer 2.2. Sequest HT database search engine. We started first by analyzing the

filtrate fractions of naïve mouse brain cortex followed by the injured (CCI) ipsilateral and

contralateral cortex (n=4 for technical and biological replicates) (Figure 4-1). Table 4-1

shows some identified CCI-induced proteolytic peptides from mouse IC and CC

samples.

We were able to identify multiple tau peptides fragments that are present in the

naïve cortex samples only, which might be resulting from protein turnover and samples

processing (Figure 4-17). Example of a peptide that has been detected in the naïve

mouse cortex fraction only is ATRIPAKTPPAPKTP (a.a.168-182).

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An interesting observation from our peptidome database search for the injured

cortex fraction is that we consistently found several tau proteolytic peptides derived from

the C-terminal domain (around amino acid 407-441) (Figure 4-10). There was a cluster

of different C-terminal domain peptides when samples were searched against the

human tau-441. These variants of the C-terminal tau peptides could be derived from

single or multiple protease actions.

One example of the top hits for peptides sequence matches (PSM) that is found

in CCI fraction and not found in the naïve fraction is

NVSSTGSIDMVDSPQLATLADEVSASLAKQGL (a.a.410-441), consisting of 26 PSM

and an average intensity of 3.778 X 104 (Figure 4-10 and Figure 4-13). Interestingly, we

have identified by nano-LC-MS/MS in our previous experiments with in vitro purified tau

and brain lysate calpain-1 digestion, a variety of different lengths of this peptide,

suggesting that calpain-1 might be playing a role in the generation of this fragment.

However, since there is a considerable difference in the length of some of these

peptides (~15 a.a. from the peptide N-terminal domain; Figure 4-10), one should not

exclude the possibility of other proteases such as caspase-3 that is well-known to

cleave tau in vitro and in vivo(208). Also, a recent study has shown that tau can be

cleaved by calpain-1 at the amino acid 243 producing a 24K C-terminal fragment(209).

It has been found in a tauopathy mouse model that C-terminal tau fragments were

present in brain samples, were truncated, and induced damage to other cells

expressing tau effectively. Our identified tau LMW peptide

NVSSTGSIDMVDSPQLATLADEVSASLAKQGL (a.a. 410-441) could be a further

processed form of the longer C-terminal peptide 24K identified by our immunoblotting

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and other studies(209, 210). Figure 4-15 shows a product ion spectrum of the tau

peptide NVSSTGSIDMVDSPQLATLADEVSASLAKQGL released from CCI-induced

mouse brain cortex.

Another released truncated form of tau C-terminal peptide is

MVDSPQLATLADEVSASLAKQGL (a.a. 419-441), which is very close to the caspase

cleavage site Asp421 reported(211). As mentioned above, tau is well known to be a

substrate of the caspase enzymes, which are recognized for their role in apoptosis.

Asp421 is the most well-characterized caspase cleavage site on tau, which produces a

tau fragment of 50K. Our immunoblots showed a band at tau-BDP-50K detected with

the CP13 antibody, which suggests a proteolyzed tau at Asp421. Caspase tau fragments

can be found in several tauopathies and are inversely correlated with formation of

Asp421 truncated tau which occurs early in the disease process(172, 179). It is

postulated that the accumulation of Asp421 fragments is the result of disruption of the

autophagy process, which is known to occur in tauopathies and AD(212). Moreover,

Asp421 fragments can induce tau hyperphosphorylation causing conformational

changes, thereby enhancing the assembly of PHF and aggregation processes(213-

215).

We were also able to identify the C-terminal peptides:

STGSIDMVDSPQLATLADEVSASLAKQGL (a.a. 413-441) and

HLSNVSSTGSIDMVDSPQLATLADEVSASLAKQ (a.a. 407-441), implying that tau is

processed by proteolytic enzymes mainly on the C-terminal end following TBI (Table 4-

1; Figure 4-10). Likewise, these peptides have been detected in our previous chapter

study using calpain-1 mediated tau proteolysis. We have concluded that a.a.

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Ser412/Ser413 is one of the top hits for possible calpain cleavage sites on tau, further

supporting the role of calpain-1 in the proteolysis process. However, we also reported in

a previously published study that caspase-3 cleave tau at Ser412/Ser413. It might be

possible that both enzymes can cleave the same tau site, taking into consideration the

different phosphorylation state of the amino acid residues, which might change the

secondary/tertiary structure leading to the dual vulnerability of tau by the enzymes.

Figure 4-14 shows another C-terminal variant of tau peptide

SPQLATLADEVSASLAKQGL that released from CCI-induced mouse brain.

On the other hand, we have identified a tau peptide in the acidic region (N2) that

was also detected from previous in vitro tau calpain digestion which is

EIPEGTTAEEAGIGDTPSLEDEAAGHVTQA (a.a. 96-125) (Table 4-1 and Figure 4-9, 4-

10- 4-11). Figure 4-12 shows a product ion spectrum of

EIPEGTTAEEAGIGDTPSLEDEAAGHVTQA peptide released from CCI injured mouse

brain cortex. This peptide was found only in the IC fractions. Calpain-2 produce tau

fragments of a.a. 3-124(216). Our identified LMW tau peptide (a.a. 96-125) might be a

truncated form of the HMW tau fragment with one amino acid difference from the C-

terminus. Another tau proteolytic fragment found in IC-D3 post-CCI sample is

EAGIGDTPSLEDEAAGHVTQA (a.a.105-125 which is a truncated form of the peptide

EIPEGTTAEEAGIGDTPSLEDEAAGHVTQA found from our in vitro tau digestion. A

Study suggested that cleavage of tau at Q124/A125 by calpain-2 and thrombin and not

calpain-1 suggested that these fragments might not be able to trigger microtubule

polymerization and aggregation into pair helical filaments (PHFs) (89, 216).

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Since our transgenic mice model can express the six isoforms of human tau,

including 3R and 4R, we included in our bioinformatics database search the longest tau

isoform tau-758 (canonical sequence, accession number P-10636-1) (Figure 4-7 and

Figure 4-8). Tau-441 differs from tau-758 by missing the amino acids 125-375 and 395-

460 due to alternative splicing mechanism. We were able to identify novel two peptides

from the canonical sequence tau-758 including TRQPSGTGPEDTEGGRHAP (a.a. 167-

185) and KYVSSVTSRTGSSGAKEMK (a.a. 440-458) that are only found in ipsilateral

cortex CCI fractions with 21 and 20 PSMs respectively (Figure 4-8). Furthermore, a

Venn diagram was constructed to show the average hits for the control samples versus

IC and CC-TBI fractions at different time points (Figure 4-16A, B). The CC fractions

showed five unique tau peptides, and IC fraction showed six unique peptides from TBI

with high confidence.

To identify phosphorylated tau sites on peptides identified using mass

spectrometry, we enabled the multistage activation mode on our linear ion trap mass

spectrometer. Multistage activation generates more structurally informative ions by

eliminating the ion isolation step between MS/MS and MS/MS/MS. The process starts

with a survey scan, and the trap is re-filled for MS/MS scan of a parent ion. The

resulting MS/MS spectrum is used to identify the neutral loss fragment that corresponds

to either singly, doubly or triply charged molecule of phosphate. The neutral loss ion is

collisionally-activated while the fragments from the precursor ion are still present in the

trap. The result is a composite spectrum that includes daughter ions from both the

precursor and the neutral loss product.

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Table 4-2 shows identified TBI-induced phosphorylated peptides from mouse

CCI cortex sample ultrafiltrate by nLC-MS/MS. Figure 4-16C, D, compares the number

of phosphorylated and non-phosphorylated peptides from IC and CC samples. We have

identified ten high confidence phosphorylated peptides from IC and twelve peptides

from CC post-TBI. High probability phosphorylation sites identified post-TBI includes

Thr101, Thr102, Thr111, Ser113, Ser210, Thr217, and Ser409. We have obtained high

confidence peptide coverage of these residues as detected by our MS protocol and the

probability of phosphorylation sites detected on these tau peptides ranged from 70% to

99%. Phosphorylation of tau at Thr101 and Thr102 has been reported to be associated

with by casein kinase 1δ (CK1δ) (217).

Interestingly, it has been revealed that phosphorylation of tau by CK1 at Ser210 or

Thr212 was detected only when phosphorylation at Ser214 or Thr217 was also

present(217). Our results agree with the hypothesis of tau phosphorylation in a specific

residue leads to subsequent phosphorylation at neighboring amino acid in the brain.

Monitoring phosphorylation sites are vital for understanding the tau mechanism in

initiating the aggregation process. Another interesting observation is the phosphorylated

site at Ser409, in which a study pointed that neurofibrillary tangle-associated kinase

phosphorylate Thr361 and Ser412 only after pre-phosphorylation of tau by PKA at the

neighboring amino acid residues Ser356 and Ser409 (218). However, to the best of our

knowledge, we are the first to report these phosphorylation sites in TBI animal models.

Conclusions

In this chapter, we have demonstrated that possible tau TBI biomarkers could be

identified from the CCI animal models. Others and we have studied proteolysis in rodent

TBI extensively and showed that TBI leads to a wide range of tau phosphorylation,

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oligomerization, and protein fragmentation from the N-terminal and mostly in the C-

terminal domain. Earlier studies have mostly focused on HMW break down products

with a molecular weight similar to the intact tau protein. Furthermore, the peptidome in a

normal healthy brain represents an array of bioactive neuropeptides and peptides

derived from a non-specific truncation at the N-terminal, the C-terminus, or both, of the

intact protein. Our results showed that TBI not only generated truncated tau fragments

but also produced LMW proteolytic peptides from either the intact protein or the

fragments. Local activation of proteolytic enzymes attacks unique non-specific sites on

the intact tau protein because of TBI. This results in the formation of HMW breakdown

products and LMW proteolytic peptides that could be released into the biofluids.

We implemented in this study a transgenic mouse model expressing human tau

isoforms. We collected the IC and CC on day one, three and seven from mouse TBI

models that represent severe focal injury. The novelty of the study resides in excluding

trypsin enzyme for our peptidome analysis and directly subjecting our samples to nLC-

MS/MS and bioinformatics searches to identify new tau neuro-toxic peptides. TBI

pathology produced all of the reported peptides herein. We have observed from our

data that caspase family, calpain-1, and 2, more closely relate the pattern of tau

fragmentation to those that are generated in our study (C-terminal domain fragments).

This truncated core peptide is also subjected to phosphorylation (based on our

immunoblotting and MS data) which might play a role in aggregation and activation of

neurodegenerative mechanisms.

In our in vitro studies, we have identified tau fragment from calpain-1 cleavage

that overlaps with our TBI animal experiments. From our peptidomics data in this

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chapter, we have identified multiple tau cleavage points from proteolytic enzymes

following TBI with varying lengths. Such differences could have remarkably different

biological mechanisms in enhancing the process of tau aggregation and neurotoxicity.

Possible protease candidates are calpain, caspase, cathepsin, thrombin, disintegrin,

metalloprotease 10, puromycin-sensitive aminopeptidase, and asparagine

endopeptidase. Our data also suggest that CK1 could have a role in the pathogenesis

of tau in TBI.

One direction for this study is the development of an optimized quantification

method of tau brain proteolytic peptides by isotopic labeling. We can use multiple

reaction monitoring (MRM) method to target the identified peptides and using a

synthetic targeted peptide as an internal standard for the quantification of natively

generated peptides. However, it is imperative to consider that the exact cleavage

specificity with the natively generated tau peptides is highly heterogeneous in mouse

and human TBI. Thus, it might be challenging to perform MRM-based quantification.

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Table 4-1. Identification of TBI-induced proteolytic peptides from mouse CCI cortex sample ultrafiltrate by nLC-MS/MS.

These measurements were obtained using LTQ-XL Mass spectrometer.

Annotated Sequence # PSMs Positions in Protein Sequence Length Charge m/z [Da] XCorr

NVSSTGSIDMVDSPQLATLADEVSASLAKQGL 26 410-441 32 3 1069.16 5.86

AQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARM 5 91-127 37 3 1326.31 4.58

HLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL 9 407-441 35 3 1234.37 4.48

SPQLATLADEVSASLAKQGL 28 422-441 20 2 1000.56 4.46

MVDSPQLATLADEVSASLAKQGL 20 419-441 23 2 1173.63 4.42

MVDSPQLATLADEVSASLAKQGL 5 419-441 23 2 1181.73 4.36

STGSIDMVDSPQLATLADEVSASLAKQGL 12 413-441 29 2 1453.84 4.16

EIPEGTTAEEAGIGDTPSLEDEAAGHVTQA 3 96-125 30 3 1052.82 4.1

VSSTGSIDMVDSPQLATLADEVSASLAKQGL 3 411-441 31 3 1031.69 4

GSIDMVDSPQLATLADEVSASLAKQGL 4 415-441 27 2 1359.18 3.94

SIDMVDSPQLATLADEVSASLAKQGL 4 416-441 26 2 1331.17 3.92

PQLATLADEVSASLAKQGL 5 423-441 19 2 956.20 3.68

EAGIGDTPSLEDEAAGHVTQA 1 105-125 21 2 1035.04 3.35

QLATLADEVSASLAKQGL 13 424-441 18 2 908.94 3.03

ADEVSASLAKQGL 18 429-441 13 2 645.21 2.96

MVDSPQLATLADEVSASLAKQ 2 419-439 21 2 1088.51 2.94

YVSSVTSRTGSSGAKEMKL 53 441-459 19 2 1074.50 2.93

GIGDTPSLEDEAAGHVTQA 4 107-125 19 2 934.93 2.92

LADEVSASLAKQGL 8 428-441 14 2 701.85 2.91

TRQPSGTGPEDTEGGRHAP 21 167-185 19 2 1096.46 2.84

GSPGTPGSRSRTPSLPTPPTR 23 201-221 21 2 1172.37 2.6

ATRIPAKTPPAPKTP 13 168-182 15 1 1705.71 2.43

ATLADEVSASLAK 33 426-438 13 1 1434.68 1.81

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Table 4-2. Identification of TBI-induced phosphorylated peptides from mouse CCI cortex sample ultrafiltrate by nLC-MS/MS. Adjacent amino acid are shown in brackets

These measurements were obtained using LTQ-XL Mass spectrometer.

Position Target Modification Classification Highest PTM

Score Highest Peptide Confidence

101 T Phospho Post-translational 100 High

102 T Phospho Post-translational 100 High

111 T Phospho Post-translational 77.7 High

210 S Phospho Post-translational 88.5 High

217 T Phospho Post-translational 84.4 High

409 S Phospho Post-translational 94.2 High

419 M Oxidation Artefact - High

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Figure 4-1. Peptidomic sample preparation workflow for the nano-LC-ESI-MS/MS for the analysis after CCI treatment to htau mouse brain. Subjecting the mouse brain proteins ultrafiltration results in both HMW protein fragments and LMW proteolytic peptides production. The LMW fragments were cleaned up and analyzed using nano-LC-ESI-MS/MS with the data-dependent acquisition and targeted peptides from in vitro tau digestion. The HMW was analyzed by SDS—PAGE followed by Western blotting.

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Figure 4-2. Characterization of tau phosphorylation, oligomerization, and fragments formation in ipsilateral and contralateral mouse cortical mouse lysate. The mice brains were collected at different time points (D1, D3, and D7) after TBI. (A) Western blots displaying intact, oligomeric tau and tau-BDP (fragment) using Total tau (DAKO) antibody and phospho-tau AT270, and AT8 antibodies. Intact tau appears as 54-55K bands, while major Tau-BDPs appear as 44K and 26K band. (DAKO panel). Oligomeric ptau appear as 225K bands (AT8 and AT270) (B) Densitometric quantification of the intact tau and BDPs for DAKO (C) for AT8 and AT270 phospho tau antibodies. Error bars represent the standard error of the mean (N=3). Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

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B

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Figure 4-3. Additional characterization of tau phosphorylation, oligomerization, and

fragments formation in ipsilateral and contralateral mouse cortical mouse lysate after TBI. The mice brains were collected at different time points (D1, D3, and D7) after TBI. (A) Western blots displaying intact, and tau-BDP (fragment) using PHF-1, RZ3, and CP13 antibodies. Intact tau appears as 54-56K bands (all panels), while major Tau-BDPs appear as 24K (RZ3 and CP13) and 15K band (CP13 panel). (B) Densitometric quantification of the intact tau and BDPs for PHF-1, RZ3, and CP13 phospho tau antibodies. Error bars represent the standard error of the mean (N=3). Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

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Figure 4-4. Assessment of cell injury using α-spectrin antibody in ipsilateral and contralateral mouse cortical mouse lysate

after TBI. (A) Western blots displaying intact-spectrin (240K), and spectrin break down products (SBDP-150K/145K and 120K). (B) Densitometric quantification of the intact 150K/145K SBDPs. Error bars represent the standard error of the mean (N=3). Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

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Figure 4-5. Representative TIC-LC chromatogram for peptides isolated from ipsilateral (IC) and contralateral (CC) mouse

brain cortex lysate using peptidomic workflow. The samples were run in 4 biological replicates and numbers correspond to the different ipsilateral cortex replicates. Replicate 1 and 2 are acquired from IC samples while 2 and 3 are acquired from CC. LC gradient was run in 130 min. These measurements were obtained using LTQ-XL Mass spectrometer.

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Figure 4-6. Representative TIC-LC chromatogram for peptides isolated from ipsilateral (IC) mouse brain cortex lysate

using peptidomic workflow. The samples were run in 4 biological replicates. D1, D3, and D7 numbers correspond to the different time point of mouse brain collection after TBI. LC gradient was run in 180 min. These measurements were obtained using LTQ-XL Mass spectrometer.

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Figure 4-7. Tau-441 sequences coverage (P10636-8) highlighting the peptides and phosphorylation sites detected. The green color highlighted represents the identified sequence. The peptides highlighted are compiled from all TBI samples excluding the control. Phosphorylation sites are labeled above the amino acids as “P”. The figure was extracted from proteome discoverer 2.2 software.

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Figure 4-8. Tau-758 sequences coverage (P10636-1) highlighting the peptides and phosphorylation sites detected. The green color highlighted is the identified sequence. The peptides highlighted are compiled from all TBI samples excluding the control. Phosphorylation sites are labeled above the amino acids as “P”. The figure was extracted from proteome discoverer 2.2 software.

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Figure 4-9. Top PSM TBI-induced tau peptides from mouse brain lysate. Peptides that had the top PSMs value plotted on the y-axis and their corresponding m/z on the x-axis. XCorr value is represented in color with the bar on the right panel as a reference. The brackets at the end of each peptide show adjacent amino acid residue.

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Figure 4-10. Schematic representation for the TBI-generated tau peptides recovered from ultrafiltrate fractions. Duplicate

peptides found are not shown. None of the peptides shown was found in non-injured control naive samples. Residue # shown on the X-axis. Peptides are ordered from N-terminal to C-terminal.

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Figure 4-11. Selected TBI-induced tau peptides from mouse brain lysate based on Intensity. Peptides Intensity value

obtained from raw data plotted on the y-axis and their corresponding peptides on the x-axis. Small letter amino acid residues correspond to modification. Error bars represent the standard error of the mean.

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Figure 4-12. Product ion spectrum of the tau peptide EIPEGTTAEEAGIGDTPSLEDEAAGHVTQA released from TBI-induced mouse brain. (A) Product ion spectrum for the tau-441 peptide EIPEGTTAEEAGIGDTPSLEDEAAGHVTQA (a.a. 96-125), charge +3, monoisotopic m/z 1052.82 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions for the tau peptide shown in red and blue.

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Figure 4-13. Product ion spectrum of the tau peptide NVSSTGSIDMVDSPQLATLADEVSASLAKQGL released from TBI-

induced mouse brain. (A) Product ion spectrum for the tau-441 peptide NVSSTGSIDMVDSPQLATLADEVSASLAKQGL (a.a: 410-441), charge +3, monoisotopic m/z 1069.16 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions for the tau peptide shown in red and blue.

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Figure 4-14 Product ion spectrum of the tau peptide SPQLATLADEVSASLAKQGL released from TBI-induced mouse brain. (A) Product ion spectrum for the tau-441 peptide SPQLATLADEVSASLAKQGL (a.a. 422-441), charge +2, monoisotopic m/z 1000.56 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions for the tau peptide shown in red and blue.

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Figure 4-15. Product ion spectrum of the tau peptide NVSSTGSIDMVDSPQLATLADEVSASLAKQGL released from TBI-induced mouse brain. (A) Product ion spectrum for the tau-441 peptide SPQLATLADEVSASLAKQGL (a.a. 410-441), charge +3, monoisotopic m/z 1069.16 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions for the tau peptide shown in red and blue.

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Figure 4-16. Venn diagrams comparing TBI-induced tau peptides with naive and phosphorylated versus non-phosphorylated peptides. Panel (A) diagram compares peptides that are detected in IC and Naïve. Panel (B) compare peptides detected between CC and control. Panel (C) compares the number of phosphorylated and non-phosphorylated peptides from IC. Panel (D) compares the number of phosphorylated and non-phosphorylated peptides from CC

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Figure 4-17. Composite summary of tau proteolytic peptides from TBI mouse brain samples. The spreadsheet is showing representative proteolytic peptides identified from ultrafiltrate samples. This table listing shows the confidence of each peptide in each fraction. Not found peptide is represented as white. Green color represents high confidence. Yellow color represents medium confidence. Red represents low confidence. The letters shown in brackets represent adjacent amino acid to the identified peptide. Each column represents a biological replicate of the labeled sample on the top of the panel

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Figure 4-18. View of a customized peptigram peptide alignment map from tau, p-tau, mouse brain lysate, and CCI mouse peptides. Panel (A) shows the peptides aligned to fit human tau protein (P10636-1; 758 a.a.). The top inset show peptides identified from CCI mouse. The bottom inset show peptides aligned by compiling peptides identified from tau, p-tau, and brain lysate digested from calpain-1 from 1:100, 1:50, 1:25, and 1:10 dilutions. The dashed lines show exact peptide found in CCI mouse model. Peptides from the input data are displayed in green boxes, where the color intensity of the peptide box is proportional to the peptide intensity. The alignments represented are retrieved by ProViz(184). Panel (B) shows a Venn diagram comparing in vivo TBI-induced tau peptides with naïve and in vitro tau-calpain peptides (phosphorylated and non-phosphorylated peptides).

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CHAPTER 5 CHARACTERIZATION OF TAU POST-TRANSLATIONAL MODIFICATION LEVELS AND TEMPORAL PROFILE IN HUMAN CEREBROSPINAL FLUID FROM SEVERE

TBI AND CONTROL SUBJECTS.

Introduction

Research evidence has shown that the pathological processes involved in TBI

are associated with a temporal profile that is linked with dynamic pathobiology during

the severe and sub-acute post-injury phase. Therefore, it is critical to address the

temporal distinctions for finding, classifying, or both, the chronic effects of TBI as an

independent disease.(188)

Bio-fluid biomarkers play a pivotal role in identifying and characterizing both TBI

severity and temporal profile. For most neurodegenerative and psychiatric diseases

including TBI and CTE, there are few or no biomarkers that can aid in the early

diagnosis or monitoring of disease progression.(4, 219)

The biomarkers in the blood that are currently used reflect the levels of neuronal

and glial damage and injury(220). These biomarkers are beneficial in characterizing the

severity of TBI, particularly in the acute post-trauma stage, for reflecting the severity of

the damage and predicting clinical consequences. However, several blood biomarkers

do not reflect pathological conditions, such as metabolic, vascular, and inflammatory

responses that are responsible for restoring and maintaining neuronal and glial damage

following TBI(220). Given the unsuccessful performance of blood biomarker in the

diagnosis of acute mild TBI (mTBI) as well as the lack of CNS specificity, researchers

began to look for alternative bio-fluid that is easily accessed, like the blood that would

be ideal for clinicians and researchers(221).

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Cerebrospinal fluid (CSF) is a clear body fluid produced by the specialized

ependymal cells in the choroid plexuses of the ventricles in the brain and absorbed in

the arachnoid granulation(222). About five hundred milliliters of CSF is produced every

day which acts as protective fluid or buffer for the brain, from physical damage,

immunological protection, or both, to the brain inside the skull(223). Due to the direct

contact with the extracellular space of the brain, CSF is a better reflection of the

biochemical and pathological changes that occur inside of the brain(224). The usage of

CSF for pre-clinical and clinical diagnosis of AD is well documented(78, 224-226). In

AD, there are three well-known cerebrospinal fluid biomarkers, which are used to aid

diagnosis of disease: amyloid-β (Aβ) 1—42, total tau protein, and phosphorylated

tau(226, 227). Moreover, CSF biomarkers have been studied taking into accounts the

temporal changes of mTBI and the chronic post-TBI state.

Several proteomics studies have been done in CSF using proteolytic digestion

followed by isobaric labeling for biomarker identification in AD, Parkinson, and multiple

sclerosis(228-230). Little attention has been paid for identifying natural peptidome in the

CSF following TBI. Low molecular weight peptides (LMW) in CSF represent a multitude

of the pathological process in the brain such as aggregation, oxidative stress, apoptosis

secretion, and inflammation. Natural tau peptidome in the CSF has rarely been studied

for potential biomarker, although tau is subjected to hyperphosphorylation and

proteolysis following TBI.

Studying more than one biomarker feature (e.g., tau hyperphosphorylation,

proteolysis, aggregation) is an essential strategy for detection and monitoring disease

development and progression(79). Therefore, in this study, we propose that monitoring

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LMW and HMW tau, as well as tau hyperphosphorylation/aggregation, could provide

insights into various potential biomarkers for TBI with an emphasis on CSF biomarker

for TBI diagnosis. These tau biomarkers are very promising for the early diagnosis of

TBI.


Human TBI and Control Cerebrospinal Fluid Procurement

The healthy control samples CSF (n=15) were purchased (Bioreclamation Co.,

USA). Archived de-identified CSF samples (n=12) from a severe TBI study were

collected from consenting adult subjects presenting to the emergency department of the

Ben Taub General Hospital, Baylor College of Medicine, (Houston, TX, USA)(231). The

study protocol was approved by the Baylor College of Medicine IRB, for subjects

sustaining blunt trauma to the head with a Glasgow coma scale of 12 or less at 12 to 48

hours. CSF samples were collected from the buretrol for up to 10 days or until an

intraventriculostomy was no longer clinically indicated. Timed CSF samples (10 ml) with

a total collection time not exceeding 1 hour were diverted to 15 mL conical

polypropylene centrifuge tubes (BD Falcon, USA) by a qualified and trained hospital

employee according to the hospital’s standard procedures. These CSF samples (5 to 10

ml) were then centrifuged at 4,000 x g with a tabletop centrifuge at room temperature for

5 to 7 minutes to remove loose cells and debris. A volume of 1 ml aliquots of cleared

CSF (supernatant) was pipetted into a 2 ml cryogenic tube, snap-frozen and stored at -

80˚C until further use. For this study, timed CSF samples collected within 48 hours of

injury, were used for the analysis. This procedure has been approved by the Ethics

committee at the University of Florida.

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Total-Tau-ELISA Kit

CSF total tau (T-tau) concentration was measured using a sandwich enzyme-

linked immunosorbent assay (ELISA) (Innotest hTAU-Ag, Innogenetics, Gent Belgium)

specifically constructed to measure all tau isoforms independently of phosphorylation

status.

SDS-PAGE and Immunoblotting

Samples (unfiltered) were subjected to SDS-PAGE. Ten microliters of the CSF

sample were mixed with μL SDS-containing sample buffer (10% β-ME). Tris-glycine pre-

cast gel (4-20%, 1 mm X 18 well; BioRad) were run on 200V, for about one hour. For

immunoblotting, the protocol was followed as described in chapter 4. The primary

antibodies used include total tau polyclonal (DAKO), phospho-tau primary antibodies

CP13, and PHF-1

Isolation of CSF LMW peptides

Five hundred microliters of CSF were thawed at room temperature, vortexed

gently, and transferred to 1.5 ml Lobind Eppendorf tubes. The rest of the protocol was

followed as described in chapter 4.

nLC-ESI-MS/MS

The high resolution mass spectrometric analysis was followed as described in

chapter 3.

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A total of 12 patients with TBI and 15 controls were used for the analyses. All

samples were collected from a severe TBI cohort and age-matched normal control. The

concentration of tau in CSF after TBI and for controls are shown in Figure 5-1. Total tau

concentration (ng/ml) in TBI patients were significantly higher than controls (p < 0.0249).

CSF total tau increased concentration did not correlate with the various time to sample

withdrawal points studied (6 hours to 48 hours). The increase in total tau concentration

after severe TBI in ventricular CSF is reported in other studies(224, 226). Tau protein

levels are physiologically higher in CSF withdrawn from ventricles than in samples

obtained by lumbar puncture(232).

To further characterize the temporal profile and post-translational modification of

tau, we analyzed the CSF samples using immunoblotting with antibodies that detect

total tau (a.a. 243-441; DAKO), and phosphorylated tau (pSer202, pSer396, and pSer404).

Human CSF samples were subjected to ultrafiltration (10K MWCO). The retentate

fractions were subjected to SDS-PAGE followed by western blotting. Our

immunoblotting data show that median levels of total tau (DAKO) are significantly

elevated (p < 0.0143) in TBI CSF patients (n=12) compared healthy controls within 48

hours (Figure 5-2A, B). Moreover, with DAKO antibody, we were able to observe two

distinct bands, one residing at 55K (intact tau) and the other at 225K (presumably

accounting for oligomeric tau), respectively. Both forms (55K and 225K) of tau were

significantly elevated in TBI subjects (p < 0.0143 and p < 0.0001, respectively) (Figure

5-2B).

Probing CSF samples with CP13 showed considerable increase in intact p-tau

55K and high molecular weight p-tau at 102K (n=12; p < 0.0009) (Figure 5-2A, B).

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Likewise, PHF-1 antibody detected significant increase in p-tau levels with TBI patients

compared to controls at 102K (n=10; p <0.0019) (Figure 5-2A, B). These results are

consistent with other studies showing elevation in total tau and p-tau in TBI

patients(219, 232).

To look for LMW proteolytic peptides, we identified from animal TBI, and in vitro

tau calpain digestion, we analyzed CSF samples collected withdrawn within 48 hours

from TBI patients. The ultrafiltrate fractions were analyzed by ultra-high-resolution mass

spectrometry (Orbitrap fusionTM tribrid). This instrument combines quadrupole, ion trap

and Orbitrap mass analysis with CID and HCD fragmentation. A set of ten CSF control

samples and ten TBI CSF samples were used for the analysis. Table 5-1 shows a list of

proteins that are found exclusively from TBI ultrafiltrate fractions. Proteins that are found

only in control CSF or in both control and TBI-CSF were filtered out from our analysis.

Proteins which were associated with TBI from our and other studies include

Amyloid-like protein 1, isoform 2 of the Major prion protein, serum amyloid A-1 protein,

serum amyloid A-2 protein, myelin basic protein, isoform 2 of neurofilament light and

medium polypeptide, and neurogranin. To the best of our knowledge, novel proteins that

are reported for the first time to be associated with TBI include Adenylate cyclase type

9, Desmoglein-1, Desmoplakin, glyceraldehyde-2-phosphate dehydrogenase, isoform 2

and 7 of Ras-specific guanine-nucleotide releasing factor (RalGPS1), and isoform beta

and gamma of poliovirus receptor (Table 5-1).

To further gain insight into the production and patterns of tau proteolysis, we

focused our analysis on tau-441 with dominant peptides listed in the peptidome

datasets. By employing the strategy from our in vitro and animal TBI study from chapter

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3 and 4, we were able to profile tau proteolytic peptides released from TBI CSF

exclusively (Table 5-2). A cluster of peptides starting from C-terminal domain 419 to 439

were consistently identified with high confidence. Figure 5-3 shows a schematic

representation of TBI-generated tau proteolytic peptides recovered from CSF ultrafiltrate

fractions. Figure 5-4 tau-441 sequences coverage (P10636-8) highlighting the peptides

and phosphorylation sites detected from TBI-CSF patients. Of interest, the N-terminal

domain tau peptide AEPRQEFEVMEDHAGTYGLGDRKDQGGYT (A.A. 2-30; Figure 5-

5) which was detected from our in vitro calpain digestion in a truncated form (i.e., 2-19).

Another N-terminal domain peptide is AGTYGLGDRKDQGGYTMHQD (A.A. 15-34)

released in TBI CSF samples (Table 5-2; Figure 5-6). Tyrosine 18, 29, and threonine 30

were found to be phosphorylated exclusively in TBI CSF. Interestingly, abnormal Src

family non-receptor tyrosine kinases Fyn and Src have been reported to phosphorylate

tau on Tyrosine 18 and 29 in AD brain(233).

Another mid-domain high confidence peptide is TREPKKVAVVRT (A.A. 220-231)

which was found to be phosphorylated on Threonine 220 and 231 (Figure 5-7). To the

best of our knowledge, phosphorylation sites at Threonine 220 are reported in this study

for the first time and association TBI. Two representative peptides are MVDSPQLATLA

(A.A. 419-429) and SPQLATLADEVSASLAK (A.A. 422-438) (Table 5-2, Figure 5-9;

Figure 5-10).

Taken together, it appears that there are multiple truncations on the tau protein

from the N-terminal, mid-region, and C-terminal domains leaving a molecular weight of

50K as validated from our immunoblots data.

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Conclusion

It is observed from the scatter plot that there is increased phosphorylation of both

intact tau (55K) and oligomeric tau (102K) from the CSF TBI cases compared to normal

control samples. Thus, tau and its LMW-BDP might be a promising candidate

biomarker. Tau is extremely sensitive to TBI-induced phosphorylation, oligomerization,

and proteolysis, and could become a new TBI disease tracker diagnostic marker. Also,

the use of TBI proteolytic biomarkers could be used as drug development tools, i.e., TBI

therapeutics to protect the brain should in principle attenuate the levels of such TBI

induced proteolytic biomarker levels. Thus, tau proteolytic biomarkers could be

considered “theranostic tools” with the utilities in augmenting the clinical trials for new

TBI drug development.

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Table 5-1. Brain proteins identified exclusively from human TBI-CSF ultrafiltrate samples by high-resolution nLC-MS/MS.

Accession Description Coverage

[%] #

PSMs #

AAs MW [kDa]

O60503 Adenylate cyclase type 9 1 1 1353 150.6

P51693-1 Amyloid-like protein 1 2 11 650 72.1

Q02413 Desmoglein-1 1 5 1049 113.7

P15924-1 Desmoplakin 0 6 2871 331.6

P04406-1 glyceraldehyde-3-phosphate dehydrogenase 3 4 335 36

P51693-2 Isoform 2 of Amyloid-like protein 1 2 11 651 72.2

P04406-2 Isoform 2 of Glyceraldehyde-3-phosphate

dehydrogenase 4 4 293 31.5

P04156-2 Isoform 2 of Major prion protein 6 13 246 26.9

P07197-2 Isoform 2 of Neurofilament medium polypeptide 2 3 540 59.4

Q5JS13-2 Isoform 2 of Ras-specific guanine nucleotide-

releasing factor RalGPS1 2 1 529 58.9

P0DJI9-2 Isoform 2 of Serum amyloid A-2 protein 12 12 83 9.2

P02686-3 Isoform 3 of Myelin basic protein 5 4 197 21.5




Q5JS13-7 Isoform 7 of Ras-specific guanine nucleotide-

releasing factor RalGPS1 2 1 537 59.8

P15151-2 Isoform Beta of Poliovirus receptor 5 2 372 40.1

P15924-2 Isoform DPII of Desmoplakin 1 6 2272 260

P15924-3 Isoform DSPIa of Desmoplakin 1 6 2428 278.7

P15151-3 Isoform Gamma of Poliovirus receptor 5 2 364 39.3

P04156-1 Major prion protein 6 13 253 27.6

P02686-1 myelin basic protein 3 4 304 33.1

P07196 Neurofilament light polypeptide 3 4 543 61.5

P07197 Neurofilament medium polypeptide 1 3 916 102.4

Q92686 Neurogranin 22 3 78 7.6

P15151-1 Poliovirus receptor 4 2 417 45.3

Q5JS13-1 Ras-specific guanine nucleotide-releasing

factor RalGPS1 2 1 557 62.1

P0DJI8 Serum amyloid A-1 protein 8 12 122 13.5

P0DJI9 Serum amyloid A-2 protein 8 12 122 13.5

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Table 5-2. Tau-441 proteolytic peptides identified from human TBI-CSF patients ultrafiltrate fractions by nLC-MS/MS.

Sequence Positions

in Proteins

Theo. MH+ [Da]

Sequence Length

Charge m/z [Da] ΔM

[ppm] XCorr

AEPRQEFEVMEDHAGTYGLGDRKDQGGYT 2-30 3512.354 28 2 1756.0238 -0.3 2.19

AGTYGLGDRKDQGGYTMHQD 15-34 2425.85571 20 4 607.2193 -0.14 2.52

TREPKKVAVVRT 220-231 1543.77572 12 2 772.40906 22.75 2.38

KNVKSKIGSTENL 254-266 1417.80092 13 2 709.37604 -39.5 2.77

KHVPGGGSVQIVYKPVDLS 298-316 2219.99028 19 3 740.68109 17.32 3.31

MVDSPQLATLA 419-429 1145.58709 11 2 573.30548 14.49 2

SPQLATLADEVSASLAK 422-438 1700.90651 17 2 850.95734 0.52 2.55

The data displayed in table are obtained from Thermo Orbitrap fusion Tribrid

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Figure 5-1. The plot for the concentration of CSF total tau in control patients (n=10) and

the clinically diagnosed patients with TBI (n=12). The solid lines represent the median with 95% confidence interval value ± standard errors (SE) of the concentrations of each group. The concentration of total tau in the TBI group was significantly higher than that in the control subjects (p=0.0249). Statistical analysis was performed with the Mann-Whitney U test. *p<0.05.

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Figure 5-2. Human CSF profile of tau phosphorylation (intact and oligomeric) for TBI and control patients. The CSF samples were collected between 6 and 48 hours after TBI. (A) Western blots displaying intact tau (50K-55K), and oligomeric tau (102K and 225K) using anti-total tau DAKO, phospho-tau CP13, and PHF-1. Loading β-actin controls were not applied as brain proteins are expected to be unevenly released into biofluid (i.e., more in TBI and less in normal healthy controls). Equal CSF volumes were loaded to mimic the ELISA-based diagnostic test (Figure 5-1), where tau concentration is reported as ng per mL (B) Densitometric quantification of the intact and oligomeric tau shown as scatter plots with solid line representing median and error bars representing the standard error of the mean (n=12). Statistical analysis was performed with two-tailed unpaired T-test. Statistical significance are represented as * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.

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Figure 5-3. Schematic representation for TBI-generated tau proteolytic peptides recovered from CSF ultrafiltrate fractions.

Duplicate peptides found are not shown. Peptide amino acid letters are shown on the X-axis. Sequence numbers are shown on the y-axis and are based on human tau-441. None of the peptides shown was found in control CSF samples. Peptides are ordered from N-terminal to C-terminal.

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Figure 5-4. Tau-441 sequences coverage (P10636-8) highlighting the peptides and phosphorylation sites detected from TBI-CSF patients. The green color highlighted is the identified sequence. The peptides highlighted are compiled from all TBI samples excluding the control. Phosphorylation sites are labeled above the amino acids as “P”. The figure was extracted from proteome discoverer 2.2 software.

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Figure 5-5. Product ion spectrum of the tau peptide AEPRQEFEVMEDHAGTYGLGDRKDQGGYT released in CSF samples in TBI subjects. (A) Product ion spectrum for the tau-441 peptide AEPRQEFEVMEDHAGTYGLGDRKDQGGYT (amino acid residues 2-30), charge +2, monoisotopic m/z 1756.02380 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions in red and blue, respectively. Tyrosine 18, 29, and threonine 30 were found to be phosphorylated.

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Figure 5-6. Product ion spectrum of the tau peptide AGTYGLGDRKDQGGYTMHQD released in CSF samples in TBI subjects. (A) Product ion spectrum for the tau-441 peptide AGTYGLGDRKDQGGYTMHQD (amino acid residues 15-34), charge +4, monoisotopic m/z 607.21930 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions in red and blue, respectively. Threonine 16, 29, and tyrosines 17 were found to be phosphorylated.

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Figure 5-7. Product ion spectrum of the tau peptide TREPKKVAVVRT released in CSF samples in TBI subjects. (A) Product ion spectrum for the tau-441 peptide TREPKKVAVVRT (amino acid residues 220-231), charge +2, monoisotopic m/z 772.40906 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions in red and blue, respectively. Threonine 220 and 231 were found to be phosphorylated.

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Figure 5-8. Product ion spectrum of the tau peptide KNVKSKIGSTENL released in CSF samples in TBI subjects. (A) Product ion spectrum for the tau-441 peptide KNVKSKIGSTENL (amino acid residues 254-266), charge +2, monoisotopic m/z 709.37604 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions in red and blue, respectively.

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Figure 5-9. Product ion spectrum of the tau peptide MVDSPQLATLA released from TBI-induced mouse brain. (A) Product ion spectrum for the tau-441 peptide MVDSPQLATLA (amino acid residues 419-429), charge +2, monoisotopic m/z 573.30548 Da, displaying the fragment ions for this peptide. (B) Identified b+ and y+ type ions in red and blue, respectively.

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CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS

Traumatic brain injury leads to secondary metabolic alterations that play a critical

role in subsequent tissue damage and neuronal cell death. Neurotherapeutic

approaches that interrupt the secondary metabolic pathways, recover behavioral

outcomes, or both, have been well documented on multiple animal models of TBI.

Neuroprotective approaches in human TBI have been unsuccessful, with the failure of

at least thirty clinical trials. The failure in finding successful neuroprotective therapy is

due to the heterogeneity in conceptual issues and methodological approaches between

preclinical and clinical injury. Peptidomic approaches have the potential to provide a

promising neurotherapeutic target and novel biomarkers that characterize the disease

states and might be incorporated into high-throughput clinical assays. Moreover, the

field of peptidomics can aid in the development of new tools to assess pharmacological

interventions for TBI and other neurodegenerative diseases.

Abnormal phosphorylation, aggregation, or both, of the tau protein, play a pivotal

role in the pathogenesis of TBI and other tauopathies such as AD.

Hyperphosphorylation of tau alone is sufficient to induce loss of function as

dephosphorylation of pathological tau by phosphatases restores microtubule stability.

Okadaic acid (OA), a protein phosphatase 1/2A inhibitor, was used to induce tau-

hyperphosphorylation and oligomerization aimed as a screening method for tau protein

kinase inhibitors in an in vitro model of neuroblastoma and rat primary neuronal cortical

cell cultures. CTE-relevant tau phospho-epitopes assessed included pSer202/pThr205

(AT8), pThr181 (AT270), pSer202 (CP13), pSer396/pSer404 (PHF-1) and pThr231 (RZ3).

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We showed that casein kinase II inhibitor, 4, 5, 6, 7-Tetrabromo-2-

azabenzimidazole (TBB) caused a robust inhibition of OA-induced tau

hyperphosphorylation and oligomerization in mouse and rat neuronal cultured cells.

Moreover, treatment with the calcineurin inhibitor (cyclosporine A), glycogen synthase

kinase inhibitors (LiCl, AR-A014418, and A-1070722, cyclin-dependent kinase 5

(Roscovitine), Fyn/Src kinase inhibitor (Saracatinib) and calcium chelator EGTA showed

robust inhibition of basal and OA-induced tau hyperphosphorylation in our in vitro cell-

based models.

In our work, we found two novel potential drug candidates (TBB and Saracatinib)

that showed efficacy against tau hyperphosphorylation and oligomerization processes.

These promising data warrant further experimentation, possibly including animal models

of tauopathies, which may provide a putative neurotherapy for CTE and other forms of

TBI-induced neurodegenerative diseases.

A possible way of taking these finding further would be pre-injecting TBB

followed by OA injection unilaterally into the amygdala of wild-type mice, and

pathological tau phosphorylation sites can be measured using phospho-tau specific

antibodies. Another future direction for chapter 2 is to use high-content siRNA strategy

for targeting the identified kinases in TBI-induced animal models. One can implement

the usage of immunofluorescence assay for the rapid detection and quantitation of total

tau and pathological tau phosphorylation sites.

Others and we have shown that tau is proteolyzed in vitro, in animal models and

human TBI extensively. The studies described tau protein as high molecular weight

break down products (HMW-BDPs) with a size that is close to the parent protein. To our

224

knowledge, this study is the first systematic approach to characterize TBI-based tau low

molecular weight break down products (LMW-BDPs), we termed this as tau proteolytic

peptidome. Overall, the results show that tau was highly vulnerable to proteolytic

enzymes (e.g., calpain) generating LMW and HMW-BDP following TBI, which can

equally contribute to pathogenesis. These highly active proteolytic enzymes can attack

target proteins and lead to the release of pathological tau peptides, which might in turn

leak, into the biofluid.

We have subjected purified tau protein (phosphorylated and non-phosphorylated)

and brain lysate from transgenic mouse overexpressing human tau (htau) to calpain-1

digestion to characterize the HMW and LMW tau peptide products. We have also

challenged rat primary cerebrocortical neuronal cultures (CTX) with neurotoxic

conditions including A23187, STS, and maitotoxin with or without protein phosphatase

inhibitor okadaic acid (OA) pretreatment, to mimic neurodegenerative conditions. The

samples were subjected to ultrafiltration (10K) and fractionated into HMW (retentate)

and LMW (filtrate) products. The HMW were analyzed by western blotting using total

and phospho-specific tau antibodies, while the filtrate fractions were analyzed using

nanoLC-ESI-MS/MS. We were able to detect novel proteolytic tau peptides that might

be linked to neurodegeneration, as they were exclusively present in treatments but not

in controls. We were also able to derive calpain-1 cleavage sites using our MS data.

Moreover, we identified phosphorylated sites that that could be critical to the pattern of

calpain-1 proteolysis of tau protein.

Our findings might aid in developing promising future neurotherapeutic strategies

emphasizing the suppression of calpain-mediated tau proteolysis and serve as

225

biomarkers for neurodegenerative diseases. In rat tau studies, we have previously

identified tau cleavage sites as Ser130Lys131, Gly157Ala158, and Arg380Glu381. Here we

found additional novel tau-calpain cleavage sites (the longest human tau isoform: Tau-

758) including G19L20, Q49T50, A89A90, Q124E125, and T720S721. These cleavage

points can lead to short peptide formation as evidenced by peptides found

(AEPRQEFEVMEDHAGTYG, SPRHLSNVSSTGSIDMVDSPQLATLADEVS,

TLADEVSASLAKQGL, SPRHLSNVSSTGSIDMVDSPQLA, ASLAKQGL,

STGSIDMVDSPQLA, and TLADEVSASLAKQGL). These peptides can be used for

“theranostic” translational studies to further validate their feasibility for clinical trials.

Such short peptides could have remarkably different kinetics when compared to

their larger tau protein-BDP counterpart we previously reported. One possible future

direction for this chapter would be to include high-resolution mass spectrometric data in

order to confirm and validate our findings. Another possible future direction is to

implement the of human SH-SY5Y neuronal culture in order to facilitate direct tau

peptidome comparison of the fragments as some amino acid sequences in the rat tau

protein is missing from the human tau. If our resources allow, one might also compare

the LMW-BDPs of tau generated using other critical TBI-related enzymes such as

cathepsins, Human high-temperature requirement serine A1 (HtrA1), Puromycin-

sensitive aminopeptidase (PSA), thrombin, caspase-3, disintegrin and metalloprotease

10 (ADAM10), Asparagine endopeptidase (AEP) using the same peptidomic platform. It

was also observed that there are several peptide variants of fragments. For example,

minor differences such as the deletion of one or two C- or N-terminal amino acids (or

both) of tau sequence.

226

Moreover, minor differences such as the addition of one or two C- or N-terminal

amino acids (or both) of sequences are also observed. These differences might be

attributed to the different tau isoforms or sometimes protein turnover. Therefore, going

forward, animal TBI models and human TBI clinical samples are best for pinpointing the

fragments, which are pathologically relevant.

In our experimental design, we approached these studies using a translational

strategy. Ipsilateral and contralateral cortices of transgenic htau mice brain lysates were

collected at different time points (day 1 to day 7) from a mouse model that represents

severe focal injury (CCI) with TBI and control subjects were used. Peptidomic approach

optimized from chapter 3 was used to identify the tau peptidome using nLC-MS/MS and

bioinformatic searches. We have identified natively released tau peptides using our high

throughput ultrafiltration method. From our injured mouse brain TBI ultrafiltrate samples,

based on the origin of proteolytic peptides release, we identified several proteolytic

peptides residing specifically in the C-terminal region and the mid-domain of tau protein.

To further gain insights into the generation and patterns of tau proteolytic

peptides, we focused our bioinformatic searches against a subset that include all tau six

isoforms. It was observed that there are several truncations on the C-terminal domain of

tau protein with varying lengths as detected by nLC-MS/MS in both mouse brain lysate

filtrate fractions. From the immunoblots analysis, we observed with total tau, a band

residing at a calculated molecular weight of 44K and intact tau band at 55K, thus

making it in the same size as the C-terminal core peptide of tau observed by MS/MS.

Another observation is related to the pattern of the C-terminal cleavage of tau, which

was more consistent with those that were generated in vitro by calpain-1. Therefore, it

227

can be concluded that these peptides might have pathological functions in mouse TBI

samples causing the neurotoxicity and cell death. This is consistent with proteomics

studies using mass spectrometry where they reported a distinct lack of C-terminal tau

peptide indicative of tau fragmentation.(234)

Taken together, we have used a combined immunoblot and peptidomic

approach, respectively to study tau-BDP and released peptides following in vitro protein

digestion, cell culture treatment, in vivo mouse and human TBI assisted with

ultrafiltration method (10K MWCO) to separate the larger tau fragments from, the

smaller released peptides. Phosphorylation of tau following TBI was elevated at many

reported pathological sites in mouse samples including pSer202, pThr205, pThr231,

pSer396, and pSer404 (when compared to controls). Another future direction is to develop

an optimized quantification method of these tau brain protein proteolytic peptides. For

classic tryptic peptide, one can use multiple reaction monitoring (MRM) method in

MS/MS to target the specific peptides using synthetic tryptic peptides as internal

standards for the quantification of natively generated peptides.

However, as our data in this study have shown multiple variants of the same core

peptide, thus showing more active proteolytic peptides in mouse and human TBI, and

leading to heterogeneity, which makes it challenging to perform MRM-based

quantification. Ultimately using antibody-based competitive enzyme-linked

immunosorbent assay (ELISA) will be the method of choice for the quantification tau

proteolytic fragments. We plan to build an antibody against the major core tau peptides

and implement the use of competitive ELISA for high precision quantification studies.

228

Another future direction for this study is to use the TBI sample with the more extended

time point of injury (6-9 month) as well as including mild repetitive TBI injury (rCHI).

Finally, the results in this dissertation present novel phosphorylation sites,

potential tau kinases, kinase inhibitor, tau cleavage sites, LMW and HMW proteolytic

peptides that can be used for pharmacological intervention and potential “theranostic”

tools for new TBI drug development.

Overall, phosphorylation sites associated with traumatic brain injuries identified

(pSer202/pThr205 (AT8), pThr181 (AT270), pSer202 (CP13), pSer396/pSer404 (PHF-1) and

pThr231 (RZ3)), calpain cleavage sites identified in this study (G19L20, Q49T50, A89A90,

Q124E125, and T720S721, and the tau proteolytic fragments

(AEPRQEFEVMEDHAGTYG, SPRHLSNVSSTGSIDMVDSPQLATLADEVS,

TLADEVSASLAKQGL, SPRHLSNVSSTGSIDMVDSPQLA, ASLAKQGL,

STGSIDMVDSPQLA, and TLADEVSASLAKQGL) can be used for “theranostic”

translational studies and warrants further investigation before proceeding to clinical

trials.

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BIOGRAPHICAL SKETCH

Hamad Yadikar was born in Kuwait in 1986 as the second oldest child to parents

Ahmad Yadikar and Fadila Behbehani. Hamad discovered his interest in science during

his undergraduate education when he attended his lab in biochemistry at Kuwait

University, under the supervision of Professor Jasim Al-Hasan. He worked on a

research project later with Dr. Amal Al-Saffar in studying antioxidant effects of camel

milk on streptozoan-induced diabetic rats using immunohistochemical approaches.

Hamad graduated from Kuwait University College of Science, Department of Biological

Sciences in 2009 with a Bachelor of Science in biochemistry.

Hamad’s later went on to pursue his master’s degree in the medical-biochemistry

field at the faculty of Medicine studying antioxidant effect of manganese

metalloporphyrin coupled with natural reducing agents on prokaryotic, eukaryotic and

mammalian systems. His mentor, Professor Ludmil Benov, was responsible for

supervising his research project. He obtained his master’s degree of Medical Science

from the college of Medicine majoring in biochemistry in 2013. After graduation, he

worked for one year at the forensic biochemistry lab in Kuwait where he was first

introduced to mass spectrometry and to the field of proteomics.

Hamad joined the Yost Research group and Dr. Kevin Wang team in McKnight

Brain Institute at UF in fall of 2015 and received his Ph.D. in chemistry in December

2018. During that time, Hamad mentored undergraduate students through the UF

summer program and four Wang group undergraduates in neuropeptidomics.

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