Selective Molecular Alterations in the Autophagy Pathway in Patients with Lewy Body Disease and in...

Selective Molecular Alterations in the AutophagyPathway in Patients with Lewy Body Disease and inModels of a-SynucleinopathyLeslie Crews1, Brian Spencer2, Paula Desplats2, Christina Patrick2, Amy Paulino2, Edward Rockenstein2,

Lawrence Hansen1,2, Anthony Adame2, Douglas Galasko2, Eliezer Masliah1,2*

1 Department of Pathology, University of California San Diego, La Jolla, California, United States of America, 2 Department of Neurosciences, University of California San

Diego, La Jolla, California, United States of America

Abstract

Background: Lewy body disease is a heterogeneous group of neurodegenerative disorders characterized by a-synucleinaccumulation that includes dementia with Lewy bodies (DLB) and Parkinson’s Disease (PD). Recent evidence suggests thatimpairment of lysosomal pathways (i.e. autophagy) involved in a-synuclein clearance might play an important role. For thisreason, we sought to examine the expression levels of members of the autophagy pathway in brains of patients with DLBand Alzheimer’s Disease (AD) and in a-synuclein transgenic mice.

Methodology/Principal Findings: By immunoblot analysis, compared to controls and AD, in DLB cases levels of mTor wereelevated and Atg7 were reduced. Levels of other components of the autophagy pathway such as Atg5, Atg10, Atg12 andBeclin-1 were not different in DLB compared to controls. In DLB brains, mTor was more abundant in neurons displaying a-synuclein accumulation. These neurons also showed abnormal expression of lysosomal markers such as LC3, andultrastructural analysis revealed the presence of abundant and abnormal autophagosomes. Similar alterations wereobserved in the brains of a-synuclein transgenic mice. Intra-cerebral infusion of rapamycin, an inhibitor of mTor, or injectionof a lentiviral vector expressing Atg7 resulted in reduced accumulation of a-synuclein in transgenic mice and ameliorationof associated neurodegenerative alterations.

Conclusions/Significance: This study supports the notion that defects in the autophagy pathway and more specifically inmTor and Atg7 are associated with neurodegeneration in DLB cases and a-synuclein transgenic models and supports thepossibility that modulators of the autophagy pathway might have potential therapeutic effects.

Citation: Crews L, Spencer B, Desplats P, Patrick C, Paulino A, et al. (2010) Selective Molecular Alterations in the Autophagy Pathway in Patients with Lewy BodyDisease and in Models of a-Synucleinopathy. PLoS ONE 5(2): e9313. doi:10.1371/journal.pone.0009313

Editor: Tsuneya Ikezu, University of Nebraska Medical Center, United States of America

Received December 22, 2009; Accepted January 28, 2010; Published February 19, 2010

Copyright: � 2010 Crews et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by NIH grants AG18440 and AG5131. The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the

most common causes of dementia and movement disorders in the

elderly [1,2]. While progressive accumulation of Ab dimers and

oligomers has been identified as one of the central toxic events in

AD leading to synaptic dysfunction [3,4], accumulation of a-

synuclein (a-syn) resulting in the formation of oligomers has been

linked to the pathogenesis of PD [5,6,7,8,9].

The pathology of AD and PD overlap in a heterogeneous group

of conditions denominated jointly Lewy body disease (LBD)

[10,11,12,13,14]. While in patients with dementia with Lewy

bodies (DLB) the clinical presentation is of dementia followed by

parkinsonism, in patients with PD dementia (PDD) the initial signs

are of parkinsonism followed by dementia [15,16,17,18]. In DLB,

Ab promotes a-syn aggregation and toxicity in vivo [19], and Aband a-syn might directly interact [20] to form hybrid channel like

structures [21].

Alterations in the rate of synthesis, aggregation and clearance of

these proteins might be responsible for the formation of toxic Aband a-syn oligomers in DLB [22]. Impaired clearance of the a-syn

aggregates might play an important role in the pathogenesis of PD

and DLB [23,24]. Among the lysosomal pathways involved, the

autophagy signaling cascade has emerged as a key mechanism for

the removal of a-syn aggregates. Autophagy is the major pathway

involved in the degradation of long-lived proteins and organelles,

cellular remodeling, and survival during nutrient starvation

[25,26]. There are three distinct autophagic pathways [27,28]: i)

macroautophagy, ii) microautophagy and iii) chaperone-mediated

autophagy (CMA). Autophagy has been linked to neuronal cell

death [29,30] and is abnormally activated in mouse models of

neurodegeneration and in neurodegenerative disorders such as

AD, PD and Huntington’s disease (HD) [31,32]. Macroautophagy

is constitutively active and highly efficient in healthy neurons and

recent studies indicate that the autophagic pathology observed in

AD most likely arises from impaired clearance of autophagic

PLoS ONE | www.plosone.org 1 February 2010 | Volume 5 | Issue 2 | e9313

vacuoles (AVs) rather than strong autophagy induction alone [33]

suggesting selective alterations in molecular components of the

autophagy pathway. For example, in the brains of patients with

AD, levels of the AV protein Beclin-1 are severely down

modulated [34].

In PD recent studies have suggested that a-syn aggregates might

interfere with the autophagy mechanisms and lead to neurode-

generation [23,35,36,37,38,39]. Mutant forms of a-syn found in

familial PD patients [23] as well as oxidized forms of a-syn [40]

found in sporadic PD and DLB have been shown to block

autophagy, and a-syn contains a consensus sequence for CMA

targeting. In neuronal cell cultures [41] and in transgenic (tg) mice,

a-syn overexpression is associated with impaired autophagy and

neurodegeneration that is reversed by Beclin-1 [42]. Further

supporting a role for lysosomal dysfunction in LBD, previous

studies have shown that in lysosomal storage disorders such as

Gaucher disease [43,44] and Niemann-Pick disease [45], there is

increased susceptibility to develop parkinsonism and a-syn

accumulation.

Taken together, these lines of evidence suggest that in DLB and

PD, specific molecular defects in the autophagy pathway might

play a role in the pathogenesis of these disorders. In this context,

the main objective of the present study was to investigate

alterations in components of the autophagy pathway in DLB

and in a-syn tg models. We found that levels of mTor were

increased and Atg7 levels were reduced in the brains of patients

with DLB and a-syn tg mice. Moreover, activating autophagy by

rapamycin treatment or viral-mediated delivery of Atg7 amelio-

rated a-syn accumulation and the related neuropathology. This

supports the notion that alterations in the autophagy pathway play

a role in DLB/PD and supports the possibility that modulators of

the autophagy pathway might have potential therapeutic effects.

Materials and Methods

Ethics StatementThis study was conducted according to the principles expressed

in the Declaration of Helsinki. For studies utilizing human tissues,

all tissues were obtained from the University of California, San

Diego Alzheimer’s Disease Research Center (ADRC). Written

informed consent for neurobehavioral evaluation, autopsy, and for

the collection of samples and subsequent analysis was obtained

from the patient and caregiver (usually the next of kin) before

neuropsychological testing and after the procedures of the study

had been fully explained. The study procedures conformed to

Federal guidelines for the protection of human subjects and were

reviewed and approved by the UCSD Institutional Review Board.

For animal studies, all animals were handled in strict accordance

with good animal practice and all procedures were completed

under the specifications set forth by the UCSD Institutional

Animal Care and Use Committee.

Cases and Neuropathological EvaluationThe study included a total of 24 cases (Table 1); of them, 6 were

non-demented controls, 12 were DLB cases and the other 6 were

AD cases. For the present study we chose to focus on DLB because

of its frequency and widespread accumulation of a-syn in

neocortical and limbic structures [46,47,48]. Autopsy material

was obtained from patients studied neurologically and psycho-

metrically at the UCSD ADRC. At autopsy, brains were divided

sagittally, and samples from the left mid temporal cortex were

fixed in 4% paraformaldehyde (PFA) and sectioned at 40 mm for

immunocytochemical analysis. Frozen samples from the right were

used for immunoblot analysis. The temporal cortex was selected

because previous studies have shown considerable pathology and

accumulation of a-syn in this region in patients with DLB

[47,48,49].

For routine neuropathological diagnosis, paraffin sections from

neocortical, limbic and subcortical regions were stained with

haematoxylin and eosin (H&E) or thioflavine-S [50,51], and Braak

stage was assessed [52]. Based on previously published clinical and

pathological findings [53], cases were subdivided into: 1) non-

demented age-matched controls, 2) AD cases, and 3) DLB cases.

All cases met the Consortium to Establish a Registry for AD

(CERAD) and National Institute of Aging (NIA) criteria for

diagnosis and displayed neuritic plaques and tangle formation in

the neocortex and limbic system [12,54]. The diagnosis of DLB

was based on the clinical presentation of dementia and the

pathological findings of LBs in the locus coeruleus, substantia

nigra (SN), or nucleus basalis of Meynert, as well as in cortical

regions. LBs were detected using H&E stain or anti-ubiquitin and

anti-a-syn antibodies as recommended by the Consortium on DLB

criteria for a pathologic diagnosis of DLB [12]. In addition to the

presence of LBs, the great majority of these cases displayed

sufficient plaques and tangles to be classified as Braak stages III–

IV. Specifically, DLB cases had abundant plaques in the neocortex

and limbic system but fewer tangles compared to AD cases.

a-Synuclein Transgenic Mice and TreatmentsFor this study, heterozygous tg mice (Line D) expressing human

wildtype a-syn under the regulatory control of the PDGFbpromoter [55] were used. These animals were selected because

they display abnormal accumulation of detergent-insoluble a-syn

in the neocortex and limbic system and develop a-syn-immuno-

reactive inclusion-like structures in the brain [56]. Although some

nuclear staining has been observed in this model, distinct

cytoplasmic inclusion-like structures have been consistently

identified by confocal and electron microscopy [19,55,56,57].

Furthermore, these animals also display neurodegenerative and

behavioral deficits that mimic certain aspects of DLB. For western

blot and immunocytochemical studies levels of components of the

autophagy pathway were compared to littermate non tg controls

and to an APP tg model (line J9M) of AD-like pathology. These

Table 1. Summary of clinico-pathological characteristics.

Diagnosis N =Mean Age(yrs)

Mean brainweight Gender M/F

Mean PMT(hrs)

MeanDuration (yrs)

Blessedscore

Frontal cortex AmyloidPlaques (0.1sq mm)

Temporal cortex Lewybodies (0.1sq mm)

Control 6 79.862 11506115 4/2 6.562 NA 0–1 0 0

AD 6 8161 1058697 3/3 861 12.861 19–33 50 0

DLB 12 82.762 10746119 4/8 7.261.5 9.86 5–32 33 1262

doi:10.1371/journal.pone.0009313.t001

mTor Activation in LBD


mice express human APP 770/751 and 695 under the PDGFbpromoter [58].

Additional experiments with the a-syn tg mice included

treatments with the autophagy activator rapamycin (Sigma-

Aldrich, St. Louis, MO). Because rapamycin poorly crosses into

the CNS, it was infused intra-cerebrally into the lateral ventricle of

9-month old mice at a concentration of 20mg/kg. Briefly as

previously described [59], mice were anesthetized and under

sterile conditions a 26 gauge stainless steel cannula was implanted

stereotaxically into the lateral ventricle using the bregma as a

reference (Franklin and Paxinos, bregma 0.5mm; 1.1mm lateral;

depth 3mm) and secured to the cranium using superglue. The

cannula was connected via a 5 mm coil of V3 Biolab vinyl to a

model 1007D osmotic minipump (Alzet, Cupertino, CA) surgically

placed subcutaneously beneath the shoulder. The solutions were

delivered at a flow rate of 0.5ul per hour for 2 weeks. The pump

was left for an additional 2 weeks and mice were euthanized one

month after the initiation of the infusions. Brains were removed

and divided sagittally. One hemibrain was postfixed in phosphate-

buffered 4% PFA, pH 7.4, at 4uC for 48 h and sectioned at 40 mm

with a Vibratome 2000 (Leica, Nussloch, Germany) and placed in

cryosolution, while the other hemibrain was snap frozen and

stored at 270uC for RNA and protein analysis.

Construction of Lentiviral Vectors and Injection ofLV-Atg7 into a-Synuclein Transgenic Mice

The mouse Atg7 cDNA (Open Biosystems, Huntsville, AL) was

PCR amplified and cloned into the third generation self-

inactivating lentivirus vector (LV) [60] with the CMV promoter

driving expression. Lentiviruses expressing a-syn, Atg7, luciferase,

shAtg7, GFP, shLuciferase, or empty vector (as controls) were

prepared by transient transfection in 293T cells [60,61]. The

empty LV contained the viral genome with the CMV promoter,

with no gene inserted downstream of the promoter.

A total of 12 a-syn tg mice from line D (9 months old) were

injected with 3 ml of the LV preparations (2.56107 TU) into the

temporal cortex and hippocampus (using a 5 ml Hamilton syringe).

Briefly, as previously described [62], mice were placed under

anesthesia on a Koft stereotaxic apparatus and coordinates

(hippocampus: AP 22.0 mm, lateral 1.5 mm, depth 1.3 mm

and cortex: AP 2.5 mm, lateral 1.5 mm, depth 1.0 mm) were

determined as per the Franklin and Paxinos Atlas. The LVs were

delivered using a Hamilton syringe connected to a hydraulic

system to inject the solution at a rate of 1 ml every 2 min. To allow

diffusion of the solution into the brain tissue, the needle was left for

an additional 5 min after the completion of the injection. Mice

received unilateral injections (right side) to allow comparisons

against the contralateral side, with either LV-Atg7 (n = 6), or LV-

control (n = 6). Additional controls were performed by injecting

non tg littermates with either LV-Atg7 (n = 6), or LV-control

(n = 6). Mice survived for 1 month after the lentiviral injection. As

an additional control for LV injection, age matched littermates

were injected with LV-luciferase. Since no differences were

observed between the LV-control and the LV-luciferase, all data

presented here are shown with the LV-control vector.

Following NIH guidelines for the humane treatment of animals,

mice were anesthetized with chloral hydrate and flush-perfused

transcardially with 0.9% saline. Brains and peripheral tissues were

removed and divided sagittally. The right hemibrain was post-

fixed in phosphate-buffered 4% PFA (pH 7.4) at 4uC for 48 hours

for neuropathological analysis, while the left hemibrain was snap-

frozen and stored at 270uC for subsequent RNA and protein

analysis.

Cell Culture and TreatmentsFor in vitro experiments we used the previously described rat

neuroblastoma cell line B103 [63]. This model was selected

because overexpression of a-syn in these cells interferes with

neuronal plasticity (reduced neurite outgrowth and adhesion) but

does not result in overt cell death [63,64]. This model mimics the

early pathogenic process of PD where cell death is preceded by

reduced neurite outgrowth and synaptic alterations. For all

experiments, cells were plated in complete media (DMEM

[Invitrogen, Carlsbad, CA] supplemented with 10% FBS (Irvine

Scientific, Santa Ana, CA) and infected with LVs expressing a-syn,

Atg7, shAtg7, or controls at a multiplicity of infection (MOI) of 40.

After infection, cells were incubated for 48 hr in a humidified 5%

CO2 atmosphere at 37uC. All experiments were conducted in

triplicate to ensure reproducibility.

To investigate whether LC3 levels are modulated by a-syn or

Atg7 over-expression or knockdown, LC3 levels were analyzed in

coverslips with LC3-GFP. B103 cells were grown as described above

and were then plated onto poly L-lysine coated glass coverslips at a

density of 56104 cells. Five hours after plating, cells were infected

with the LV-asyn and/or LV-Atg7 or LV-shAtg7 (or controls) and

incubated for 48 hours. All coverslips were also co-infected with an

LV expressing LC3-GFP at an MOI of 40. Cultures were then

washed 26with serum-free DMEM and then fed either complete

media or serum-free media for 12 hours before fixation with 4%

PFA. Briefly as previously described [34], coverslips were treated

with Prolong Gold antifading reagent with DAPI (Invitrogen) and

imaged with the LSCM to determine the number of GFP-positive

granular structures consistent with autophagolysosomes using

semiautomatic image analysis system and the ImageQuant software.

For each condition an average of 50 cells were analyzed.

AntibodiesFor western blot and immunohistochemical analysis of the

autophagy pathway, polyclonal antibodies against mTor (1:1000,

Sigma); phosphorylated-mTor (p-mTor, 1:1000, Cell Signaling

Technology, Beverly, MA); Atg5 (1:1000, Abcam, Cambridge,

MA); Atg6 or Beclin-1 (1:1000, Novus Biologicals, Littleton, CO);

Atg7 (1:500, Abcam); Atg8 or LC3 (1:1000, Abcam); Atg10 (1:500,

Abcam); Atg12 (1:1000, Abcam); Cathepsin D (1:500, Calbio-

chem, San Diego, CA). Detection of a-syn was performed with a

rabbit polyclonal (1:500, Millipore, Temecula, CA) and a mouse

monoclonal antibody (1:500, clone syn211, Sigma).

Immunohistochemistry, Image Analysis and LaserScanning Confocal Microscopy

Briefly, as previously described [65], free-floating 40 mm thick

vibratome sections were washed with Tris buffered saline (TBS,

pH 7.4), pre-treated in 3% H2O2, and blocked with 10% serum

(Vector Laboratories, Burlingame, CA), 3% bovine serum

albumin (Sigma), and 0.2% gelatin in TBS-Tween (TBS-T). For

human brains, sections from the temporal cortex were used; for

the mice sagittal sections from the complete brain were studied.

Sections were incubated at 4uC overnight with the primary

antibodies. Sections were then incubated in secondary antibody

(1:75, Vector), followed by Avidin D-horseradish peroxidase

(HRP, ABC Elite, Vector) and reacted with diaminobenzidine

(DAB, 0.2 mg/ml) in 50 mM Tris (pH 7.4) with 0.001% H2O2.

Control experiments consisted of incubation with pre-immune

rabbit serum. To investigate the effects of postmortem delay and

fixation on the levels of mTor immunoreactivity, preliminary

studies were performed in a subset of cases (n = 5) with

postmortem delay ranging from 4–48 h.



Immunostained sections were imaged with a digital Olympus

microscope and assessment of levels of mTor, Atg7, LC3 and

Cathepsin D immunoreactivity was performed utilizing the Image-

Pro Plus program (Media Cybernetics, Silver Spring, MD). For

each case a total of three sections (10 images per section) were

analyzed in order to estimate the average number of immunola-

beled cells per unit area (mm2) and the average intensity of the

immunostaining (corrected optical density).

Double-immunocytochemical analyses was performed utilizing

the Tyramide Signal AmplificationTM-Direct (Red) system (NEN

Life Sciences, Boston, MA). Specificity of this system was tested by

deleting each primary antibody. For this purpose, sections were

double-labeled with the monoclonal antibodies against a-syn

(1:20,000, Cell Signaling) detected with Tyramide Red, and either

mTor, Cathepsin D or LC3 detected with fluorescein isothiocy-

anate (FITC)-conjugated secondary antibodies (1:75, Vector).

All sections were processed simultaneously under the same

conditions and experiments were performed twice for reproduc-

ibility. Sections were imaged with a Zeiss 63X (N.A. 1.4) objective

on an Axiovert 35 microscope (Zeiss, Germany) with an attached

MRC1024 laser scanning confocal microscope (LSCM) system

(BioRad, Hercules, CA).

Analysis of NeurodegenerationTo evaluate the integrity of the dendritic system, blind-coded

40mm-thick vibratome sections from mouse brains fixed in 4%

PFA were immunolabeled with the mouse monoclonal antibody

against microtubule associated protein 2 (MAP2) (dendritic

marker, 1:40, Millipore), as previously described [66]. After an

overnight incubation with the primary antibody, sections were

incubated with FITC-conjugated horse anti-mouse IgG secondary

antibody (1:75, Vector), transferred to SuperFrost slides (Fisher

Scientific, Tustin, CA) and mounted under glass coverslips with

anti-fading media (Vector). All sections were processed under the

same standardized conditions. The immunolabeled blind-coded

sections were imaged with the LSCM (MRC1024, Bio-Rad) and

analyzed with the Image 1.43 program (NIH), as previously

described [66,67].

Western Blot AnalysisImmunoblot analysis was performed as previously described

[68]. Briefly, brain homogenates (temporal cortex for human

tissues and cortex for mice) or cultured cells were solubilized in

lysis buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES,

pH 7.4, 140 mM NaCl, 1mM EDTA, 1mM Na3VO4, 20 mM b-

glycerophosphate, and proteinase inhibitor cocktails). Brain

homogenates were separated into cytosolic and membrane

fractions by centrifugation at 100,0006g for 1 hr at 4C. Isolation

of lysosomal fractions from brain tissue was performed by

differential centrifugation in a sucrose-based buffer essentially as

previously described [69]. Briefly, tissues were minced with a razor

blade and homogenized in ice-cold 50 mM Tris/HCl buffer,

pH = 7.4, containing 0.25 M sucrose, 10 mM EDTA, 3 mM

MgCl2, with protease and phosphatase inhibitor cocktails

(Calbiochem) added fresh (sucrose/Tris buffer). After filtration

through a mesh to remove cell debris and capsular fragments, the

homogenate was centrifuged at 1000 g for 5 min to obtain the

nuclear fraction (pellet 1, P1). The supernatant was centrifuged

again at 10,000 g for 10 min to collect the mitochondrial fraction

(pellet 2, P2). The resulting supernatant was centrifuged at

20,000 g for 10 min to obtain the lysosomal fraction (pellet 3,

P3). Finally, the supernatant remaining after the removal of the

lysosomal fraction was centrifuged at 180,000 g for 30 min to

separate the microsomal fraction (pellet 4, P4) from the cytosol/

extracellular fluid fraction (supernatant 4, S4). Pellets were gently

resuspended in 0.25 mL sucrose/Tris buffer, and all fractions were

stored at 280uC until further processing.

For western blot analysis, 20 mg of each fraction was resolved by

SDS-PAGE on 4–12% Bis-Tris gels (Invitrogen) and electroblotted

onto Immobilon membranes (Millipore). The membranes were

blocked with phosphate-buffered saline (PBS) with 0.2% Tween-

20 (PBST) containing 3% skim milk or bovine serum albumin

(BSA), followed by incubation with primary antibodies in PBST

containing 5% BSA. After washing with PBS, the membranes

were incubated with rabbit anti-mouse secondary antibodies

(1:5000, American Qualex, San Clemente, CA) and visualized

with enhanced chemiluminescence (ECL, PerkinElmer, Wellesley,

MA) and analyzed with the VersaDoc gel imaging system

(BioRad).

Electron MicroscopyBriefly, as previously described [42], vibratome sections from

control, DLB brains and a-syn tg mice were post-fixed in 1%

glutaraldehyde, then treated with osmium tetraoxide and

embedded in epon araldite. Once the resin hardened, blocks were

sectioned with an ultramicrotome (Leica). Grids were analyzed

with a Zeiss OM 10 electron microscope as previously described

[70]. Micrographs from pyramidal neurons in the temporal cortex

were randomly acquired from 3 grids, and electron micrographs

were acquired at a magnification of 25,000 each.

Statistical AnalysisAll experiments were conducted in triplicate on blind-coded

samples. After the results were obtained, the code was broken and

data were analyzed with the StatView program (SAS Institute,

Inc., NC). Comparison among groups of expression levels of

components of the autophagy pathway were performed by one-

way ANOVA with post-hoc Dunnett’s or Tukey-Kramer. All

results are expressed as mean 6 SEM.

Results

Alterations in the Levels of mTor and Atg7 AreAssociated with Lysosomal Alterations the Brains of DLBPatients

Recent evidence suggests that impaired functioning of the

lysosomal pathways (eg: autophagy) involved in a-syn clearance

might play a role in the pathogenesis of DLB [23,24]. For this

reason, expression levels of members of the autophagy pathway

were analyzed in the temporal cortex of control, AD and DLB

patients. By immunoblot analysis, mTor was identified as a triplet

band at an estimated molecular weight of 280–290 kDa that was

more abundant in the membrane than in the cytosolic fraction

(Figure 1A, B). Compared to non-demented controls and AD

cases, levels of mTor and phosphorylated-mTor (p-mTor) were

elevated in the brains of DLB cases (Figure 1A, C). In both the

membrane and cytosolic fractions Atg7 was identified as a doublet

at an estimated molecular weight of 78 kDa (Figure 1A, B). In

DLB cases, levels of Atg7 were moderately reduced compared to

controls and AD cases (Figure 1A, D). No differences were

observed between controls and AD cases (Figure 1A, D). Other

components of the autophagy pathway such as Atg5 (32 kDa) and

Atg12 (20kDa) were identified as single bands that were more

abundant in the membrane than in the cytosolic fractions

(Figure 1A, B). No differences were detected among the 3 groups

in the levels of these Atg proteins (Figure 1A, D). Beclin-1 was

detected as a single band at 50 kDa that was more abundant in the

membrane than the cytosolic fraction (Figure 1A, B). Consistent



with previous studies [34], levels of Beclin-1 in the membrane

fractions were reduced in the AD cases compared to non-

demented controls and DLB, however no differences in Beclin-1

levels were detected between non-demented controls and DLB

cases (Figure 1A, C).

Immunohistochemical analysis showed moderate levels of mTor

and Atg7 immunostaining in pyramidal neurons in control and

AD cases (Figure 2A, B, D, E). In contrast, in DLB cases levels of

neuronal mTor immunoreactivity were increased (Figure 2C, G),

while the levels of neuronal Atg7 immunolabeling were reduced

(Figure 2F, G). The increased levels of mTor and reduced Atg7

levels suggest there may be altered activation of the autophagy

pathway in the brains of DLB patients. Consistent with this

possibility, in DLB cases Cathepsin D-immunoreactive lysosomes

of normal size were scant, and pyramidal cells contained enlarged

lysosomes (Figure 2H–J, N) and increased levels of LC3

immunoreactivity (Figure 2K–M, O) compared to control cases.

By immunoblot analysis, in the DLB cases there was an increase

in immunoreactive bands representing monomeric and aggregated

a-syn both in the cytosolic and membrane fractions (Figure S1A,

B). In the AD, the levels of a-syn were slightly elevated compared

to controls (Figure S1C). Consistent with the immunohistochem-

ical analysis (Figure 2), by immunoblot, levels of LC3 and

Cathepsin D in the membrane fractions were reduced in the DLB

cases compared to non-demented controls (Figure S1A, C). In

contrast, in the AD cases levels of Cathepsin D were elevated

compared to non-demented controls (Figure S1A, C).

Compared to the unimpaired neurons in the control cases, in

DLB brains, mTor and LC3 immunoreactivity were more

abundant in neurons displaying a-syn accumulation (Figure 3A–

L). In addition, in the DLB cases, LC3 immunoreactivity was

occasionally associated with LBs (Figure 3J–L). Compared to

control cases, these neurons also showed enlarged Cathepsin D-

positive lysosomes (Figure 3M–R). Consistent with these observa-

tions, electron microscopy analysis revealed the presence of

abundant and abnormal autophagosomes in these a-syn-positive

neurons that were not detected in control cases (Figure 4A–D).

Alterations in Molecular Components of the AutophagyPathway in A-Synuclein Transgenic Mice

Consistent with the studies in human brains, levels of mTor and

p-mTor were increased in the mebrane fractions from brains of a-

syn tg mice compared to non tg controls (Figure 5A–C). Moreover,

levels of a molecular initiator of autophagy, Atg7, were reduced in

a-syn tg brains compared to APP tg mice and non tg controls

(Figure 5A, D). In contrast, other components of the autophagy

pathway such as Atg5, Atg12 and Beclin-1 were not different

among the non tg and tg mouse groups (Figure 5A, C, D).

In the non tg and APP tg mice, immunohistochemical analysis

showed moderate levels of mTor and Atg7 immunostaining in

pyramidal neurons in the neocortex (Figure 6A, B, D, E, G) and

hippocampus (not shown). In contrast, in the a-syn tg mice levels

of neuronal mTor immunoreactivity were increased, while the

levels of neuronal Atg7 immunolabeling were reduced (Figure 6C,

F, G). Compared to non tg control mice, in APP and a-syn tg mice

pyramidal cells contained enlarged lysosomes (Figure 6H–J, N)

and abnormal levels of LC3 immunoreactivity (Figure 6K–M, O).

Abundant a-syn monomer and oligomers were detected in the

membrane and cytosolic fractions of a-syn tg mice (Figure S2A, B).

By immunoblot analysis, levels of LC3 in the membrane fraction

were increased and Cathepsin D was reduced in the a-syn tg mice

compared to non tg controls (Figure S2A, C). Compared to control

mice, in the APP tg mice, levels of LC3 were elevated while levels

of Cathepsin D were unchanged in the membrane fraction (Figure

S2A, C). Double-labeling studies showed that mTor and LC3 were

abundant in neurons displaying a-syn accumulation (Figure 7A–

Figure 1. Immunoblot analysis of the autophagy pathway in the brains of AD and DLB patients. Brain homogenates from the temporalcortex of non-demented controls, AD, and DLB patients were separated into membrane and cytosolic fractions, and 20 mg of each sample wassubjected to gel electrophoresis. Immunoblots were probed with antibodies against mTor, phosphorylated (p) mTor, Beclin-1, Atg5, Atg7, Atg12 andActin. (A) Representative immunoblots of membrane fractions. (B) Representative immunoblots of cytosolic fractions. (C) Semi-quantitative analysis oflevels of mTor, p-mTor, and Beclin-1 in membrane fractions from the brains of control, AD and DLB patients. Levels of mTor were significantlyincreased in DLB patients. (D) Semi-quantitative analysis of levels of Atg5, Atg7, and Atg12 in membrane fractions from the brains of control, AD andDLB patients. Levels of Atg7 were significantly reduced in the brains of DLB patients. All semi-quantitative measurements were normalized to actinlevels as a loading control. *p,0.05 compared to non-demented controls by one-way ANOVA with post-hoc Dunnett’s test.doi:10.1371/journal.pone.0009313.g001



L). These neurons also showed the presence of enlarged Cathepsin

D-positive lysosomes compared to non tg controls (Figure 7M–R).

Electron microcopy analysis confirmed the presence of abundant

and abnormal autophagosomes in these neurons (Figure 4E–H).

Effects of Intra-Cerebral Infusion of mTor Inhibitor inA-Synuclein Transgenic Mice

Since alterations in the autophagy pathway in DLB and in a-syn

tg mice might be in part related to increased levels of mTor, we

investigated whether blocking mTor with rapamycin might

promote a-syn clearance and be neuroprotective. Compared to

vehicle-infused mice, in a-syn tg mice that received rapamycin

treatment, a-syn accumulation in neuronal cell bodies and

synapses was reduced and redistributed to the axons (Figure 8A–

C, G). Moreover, compared to vehicle-treated a-syn tg mice,

rapamycin treatment resulted in increased levels of LC3

(Figure 8D–G) and Cathepsin D (Figure 8H–J, N) immunoreac-

tivity. Furthermore, this treatment ameliorated the dendritic

pathology in the neocortex as reflected by image analysis of

MAP2 immunolabeling of the neuropil (Figure 8K–N). By

immunoblot analysis, compared to tg mice infused with vehicle

alone, a-syn tg mice that received intra-cerebral infusions with

rapamycin displayed reduced levels of a-syn in the membrane

fraction with a concomitant increase in the lysosomal fraction

(Figure 8O, P). In addition, rapamycin increased the levels of LC3

and Cathepsin D immunoreactivity (Figure 8O, P).

Taken together, these results support the possibility that

alterations in the autophagy pathway and more specifically in

mTor and Atg7 are associated with accumulation of a-syn and

neurodegeneration in DLB cases and a-syn tg models, and

activation of autophagy with rapamycin can revert this effect.

Lentivirus Delivery of Atg7 Rescues a-SynucleinAccumulation and Neuronal Deficits in Transgenic Mice

Since in addition to the increase in mTor, we also observed a

reduction in Atg7 levels in DLB and a-syn tg mice, we sought to

determine whether viral-mediated delivery of Atg7 might also

promote a-syn clearance and rescue neurodegenerative deficits in

a-syn tg mice. For this purpose, we delivered the Atg7 lentivirus

via stereotaxic injection to the temporal cortex and hippocampus

of non tg and a-syn tg mice [55].

Compared to non tg and a-syn tg mice treated with the LV-

control (Figure 9A–D, G) delivery of LV-Atg7 resulted in

increased expression in Atg7 in pyramidal neurons in the

hippocampus and in areas adjacent to the injection track in the

neocortex (Figure 9E–G). Atg7 immunostaining was localized

Figure 2. Immunohistochemical analysis of the autophagy pathway in the brains of AD and DLB patients. Vibratome sections from thetemporal cortex of non-demented controls, AD, and DLB patients were immunolabeled with antibodies against mTor, Atg7, Cathepsin D, and LC3,and imaged with a digital microscope. (A–C) Representative sections from control, AD and DLB brains immunolabeled with an antibody against mTor.(D–F) Representative sections from control, AD and DLB brains immunolabeled with an antibody against Atg7. (G) Semi-quantitative image analysisreveals a significant increase in mTor levels and a reduction in Atg7 levels in DLB patients compared to controls. (H–J) Representative sections fromcontrol, AD and DLB brains immunolabeled with an antibody against Cathepsin D. Pyramidal neurons in AD and DLB cases show enlarged CathepsinD-immunoreactive lysosomes (arrows). (K–M) Representative sections from control, AD and DLB brains immunolabeled with an antibody against LC3.(N) Increased numbers of enlarged lysosomes (.1mm) in AD and DLB brains. (O) Semi-quantitative image analysis of LC3 immunoreactivity revealsincreased LC3 levels in AD and DLB brains. Scale bar in panel (C) represents 20mm in all microscopy images. *p,0.05 compared to non-dementedcontrols by one-way ANOVA with post-hoc Dunnett’s test.doi:10.1371/journal.pone.0009313.g002



Figure 3. Double-immunolabeling analysis of autophagy markers and a-syn in the brains of patients with DLB. Vibratome sectionsfrom the temporal cortex of non-demented controls and DLB patients were immunolabeled with antibodies against a-syn, and co-labeled withantibodies against mTor, LC3 or Cathepsin D, and imaged with a laser scanning confocal microscope. (A–D) Double-immunolabeling analysis showingincreased mTor immunoreactivity in neurons of DLB patients showing a-syn accumulation. (G–L) Double-immunolabeling analysis showing increasedLC3 immunoreactivity in neurons of DLB patients showing a-syn accumulation. LC3 immunoreactivity was occasionally associated with LBs (arrows).(M–R) Double-immunolabeling analysis showing enlarged Cathepsin D-immunoreactive lysosomes (arrows) in neurons of DLB patients showing a-synaccumulation. Scale bar in panel (C) represents 15mm in panels A–L and 8mm in panels M–R.doi:10.1371/journal.pone.0009313.g003



primarily to the neuronal perykaria surrounding the injection

track with some extension to the apical dendrites (Figure 9E).

Compared to non tg mice (Figure 9H, I), a-syn tg mice injected

with the LV-control contained abundant intracellular aggregates

of a-syn (Figure 9J, K); in contrast, following LV-Atg7 injection,

there was a considerable reduction in the intra-neuronal a-syn

accumulation in the areas adjacent to the injection track

(Figure 9L–N).

Figure 4. Electron microscopic analysis of abnormal autophagosomes in patients with DLB and in a-syn tg mice. (A) Representativeimage from a non-demented control case showing normal neuronal lysosomes (arrow). (B–D) Abnormal autophagosomes and accumulation ofelectrodense deposits (arrows) in intraneuronal membrane-bound structures in the brains of patients with DLB. (E) Representative image from a nontg mouse brain showing normal neuronal lysosomes (arrow). (F–H) Abnormal autophagosome morphology and accumulation of electrodensedeposits (arrows) in intraneuronal membrane-bound structures in the brains of a-syn tg mice. Scale bar in panel (C) represents 0.5mm in all panels.doi:10.1371/journal.pone.0009313.g004

Figure 5. Immunoblot analysis of the autophagy pathway in the brains of APP and a-syn tg mice. Brain homogenates from non tg, APPtg, and a-syn tg mice were separated into membrane and cytosolic fractions, and 20 mg of each sample was subjected to gel electrophoresis.Immunoblots were probed with antibodies against mTor, phosphorylated (p) mTor, Beclin-1, Atg5, Atg7, Atg12 and Actin. (A) Representativeimmunoblots of membrane fractions. (B) Representative immunoblots of cytosolic fractions. (C) Semi-quantitative analysis of levels of mTor, p-mTor,and Beclin-1 in membrane fractions from the brains of non tg, APP tg and a-syn tg mice. Levels of mTor were significantly increased in APP tg and a-syn tg brains. (D) Semi-quantitative analysis of levels of Atg5, Atg7, and Atg12 in membrane fractions from the brains of non tg, APP tg and a-syn tgmice. Levels of Atg7 were significantly reduced in the brains of a-syn tg mice. All semi-quantitative measurements were normalized to actin levels asa loading control. *p,0.05 compared to non tg controls by one-way ANOVA with post-hoc Dunnett’s test.doi:10.1371/journal.pone.0009313.g005



Concomitant with the reduction in a-syn accumulation, analysis

of the dendritic marker MAP2 showed an increase in the percent

area of the neuropil covered by dendrites in a-syn tg mice that

received the LV-Atg7 (Figure 9O–U), supporting the possibility

that the reduction of a-syn accumulation in the LV-Atg7-treated

animals ameliorated the structural damage to neurons of a-syn tg

mice. Importantly, no significant deleterious effects on neuronal or

synapto-dendritic content were observed in non tg mice that

received injections with LV-Atg7 (not shown). Together, these

data suggest that delivery of LV-Atg7 to the a-syn tg mice reduced

the accumulation of a-syn and related neuronal pathology by

inducing a physiological autophagic response.

Overexpression of Atg7 in an In Vitro Model Reducesa-Synuclein Accumulation, while Atg7 KnockdownExacerbates Autophagic Deficits and a-SynucleinAccumulation

To further confirm the effects of LV-Atg7 in activating

autophagy and promoting a-syn clearance, we utilized a neuronal

cell model to study autophagy in the presence of a-syn

accumulation [42]. For this purpose, B103 neuronal cells were

infected with lentivirus expressing an LC3-GFP fusion protein and

co-infected with LV-control or LV-asyn. The LC3-GFP is a

marker for autophagy activation, and increased LC3-GFP

fluorescence indicates increased autophagic activity. These cells

were then infected with either LV-Atg7 or a virus encoding an

shRNA against Atg7 or a control shRNA against luciferase.

Immunocytochemical analysis demonstrated that compared to

controls (Figure S3A) levels of Atg7 were higher in LV-Atg7-

infected cells (Figure S3B), while baseline levels of Atg7 were

reduced by the LV-shAtg7 (Figure S3C). Consistent with the

immunocytochemical analysis, western blot confirmed the expres-

sion and knockdown of Atg7 in the LV infected neuronal cell line

(Figure S3D).

Immunolabeling studies with an antibody against a-syn

demonstrated that compared to controls (Figure 10A, F) and

LV-Atg7 infected cells (Figure 10B, F), there was an increase in

expression of a-syn in LV-asyn infected cells (Figure 10C, F).

Infection with LV-Atg7 resulted in increased numbers of LC3-

GFP grains per cell, consistent with an activation of autophagy

(Figure 10B, E). In cells co-infected with LV-asyn, co-expression of

Atg7 also resulted in significant activation of autophagy

(Figure 10D, E) and a reduction in a-syn accumulation

(Figure 10D, F). Similar experiments with LV-bsyn and LV-

Atg7 showed no reduction in levels of b-syn, indicating that Atg7

expression specifically reduced the accumulated levels of a-syn

(data not shown).

In contrast, compared to LV-shControl (Figure 10G, K), when

neuronal cells were infected with the LV-shAtg7, levels of LC3-

Figure 6. Immunohistochemical analysis of the autophagy pathway in the brains of APP and a-syn tg mice. Vibratome sections fromthe hippocampus of non tg, APP tg and a-syn tg mice were immunolabeled with antibodies against mTor, Atg7, Cathepsin D, or LC3, and imagedwith a digital microscope. All images are from the temporal cortex. (A–C) Representative sections from non tg, APP tg and a-syn tg brainsimmunolabeled with an antibody against mTor. (D–F) Representative sections from non tg, APP tg and a-syn tg brains immunolabeled with anantibody against Atg7. (G) Semi-quantitative image analysis reveals a significant increase in mTor levels and a reduction in Atg7 levels in a-syn tgmice compared to non tg controls. (H–J) Representative sections from non tg, APP tg and a-syn tg brains immunolabeled with an antibody againstCathepsin D. Enlarged Cathepsin D-immunoreactive lysosomes (arrows) were detected in APP tg and a-syn tg mice. (K–M) Representative sectionsfrom non tg, APP tg and a-syn tg brains immunolabeled with an antibody against LC3. (N) Increased numbers of enlarged lysosomes (.1mm) in APPtg and a-syn tg mouse brains. (O) Semi-quantitative image analysis of LC3 immunoreactivity reveals increased LC3 levels in APP tg and a-syn tgbrains. Scale bar in panel (C) represents 10mm in all microscopy images. *p,0.05 compared to non tg controls by one-way ANOVA with post-hocDunnett’s test.doi:10.1371/journal.pone.0009313.g006



Figure 7. Double-immunolabeling analysis of autophagy and a-syn in the brains of a-syn tg mice. Vibratome sections from the brains ofnon tg and a-syn tg mice were immunolabeled with antibodies against a-syn, and co-labeled with antibodies against mTor, LC3 or Cathepsin D, andimaged with a laser scanning confocal microscope. All images are from the temporal cortex. (A–D) Double-immunolabeling analysis showingincreased mTor immunoreactivity in a-syn-positive neurons in a-syn tg mice. (G–L) Double-immunolabeling analysis showing increased LC3immunoreactivity in neurons of a-syn tg mice showing a-syn accumulation. (M–R) Double-immunolabeling analysis showing enlarged Cathepsin D-immunoreactive lysosomes (arrows) in neurons of a-syn tg mice showing a-syn accumulation. Scale bar in panel (C) represents 20mm in panels A–Land 10mm in panels M–R.doi:10.1371/journal.pone.0009313.g007



GFP were reduced (Figure 10H, K). Similar effects were observed

in neuronal cells co-infected with LV-shAtg7 and LV-asyn

(Figure 10I–K). Moreover, compared LV-shControl (Figure 10G,

I, L), in cells infected with LV-shAtg7, the levels of a-syn

accumulation were increased (Figure 10J, L). Taken together,

these results support the possibility that activating the autophagy

Figure 8. Immunohistochemical and immunoblot analysis of the effects of rapamycin treatment in a-syn tg mice. For panels A–M,vibratome sections from the hippocampus of non tg and a-syn tg mice were immunolabeled with antibodies against a-syn, LC3, Cathepsin D orMAP2 and imaged with a digital microscope. All images are from the temporal cortex. For panel O, brain homogenates from non tg and a-syn tg micewere separated into membrane and lysosomal fractions, and 20 mg of each sample was subjected to gel electrophoresis. (A–C) Representativesections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated a-syn tg mice immunolabeled with an antibody againsta-syn. (D–F) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated a-syn tg miceimmunolabeled with an antibody against LC3. (G) Semi-quantitative image analysis showing reduced a-syn immunoreactivity and increased LC3immunoreactivity in the hippocampus of a-syn tg mice treated with Rapamycin. (H–J) Representative sections from the brains of vehicle-treated nontg mice and vehicle- and Rapamycin-treated a-syn tg mice immunolabeled with an antibody against Cathepsin D. (K–M) Representative sections fromthe brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated a-syn tg mice immunolabeled with an antibody against MAP2. (N)Semi-quantitative image analysis showing increased Cathepsin D immunoreactivity in the hippocampus of a-syn tg mice treated with Rapamycin.Reduced levels of MAP2 in the hippocampus of vehicle-treated a-syn tg mice is rescued by Rapamycin treatment. (O) Representative immunoblotanalysis of membrane and lysosomal fractions probed with antibodies against a-syn, LC3, and Cathepsin D. (P) Semi-quantitative image analysis ofimmunoblots showing redistribution of a-syn from membrane to lysosomal fractions and an associated increase in LC3 and Cathepsin D levels. Allsemi-quantitative measurements were normalized to actin levels as a loading control. Scale bar in panel (C) represents 40mm in panels A–C, 20mm inpanels D–E and H–J, and 10mm in panels K–M. *p,0.05 compared to vehicle-treated non tg controls by one-way ANOVA with post-hoc Dunnett’stest. #p,0.05 compared to vehicle-treated a-syn tg mice by one-way ANOVA with post-hoc Tukey-Kramer test.doi:10.1371/journal.pone.0009313.g008



pathway with rapamycin or with viral delivery of Atg7 might

reduce the accumulation of a-syn and rescue the associated

neurodegenerative alterations.

Discussion

Recent evidence in cell-based models of PD-like pathology

indicate that alterations in lysosomal functioning and autophagy

might participate in the mechanisms of a-syn-mediated neurode-

generation [23,29,35,36,37,38,39]. However it was unclear which

molecular components of the autophagy pathway might be

dysregulated in the brains of patients with DLB/PD and in a-

syn tg models. For the present study we chose investigate potential

alterations in components of autophagy in DLB cases (rather than

pure PD) because after AD, these cases represent the most

common form of dementia and movement disorders in the aging

population and display widespread cortical and subcortical

pathology.

Remarkably, we found that in DLB cases and in a-syn tg mice

levels of mTor were elevated and Atg7 expression was reduced.

mTor and LC3 was co-localized with neurons displaying a-syn

accumulation and neurodegenerative changes. This is of interest

because it provides a potential alternative explanation for the

molecular alterations in autophagy in sporadic forms of LBD. mTor

and Atg7 are early initiators of the macroautophagy pathway.

Inhibition of mTor by nutrient reduction or by activation of PI3K

results in activation of the Atg kinase 1 that in turn phosphorylates

Atgs that participate in the AV formation [71,72,73].

The mechanisms through which increased mTor and reduced

Atg7 might participate in the neuropathology of DLB are not

completely clear. However, such alterations are predicted to result

in deficient initiation of the autophagy process. This in turn might

result in progressive accumulation of a-syn aggregates that further

interfere with the fusion of lysosomes and formation of

autophagosomes, as has been suggested by other studies

[23,40,41]. This may then lead to the formation of enlarged and

Figure 9. Immunohistochemical analysis of the effects of LV-Atg7 treatment in a-syn tg mice. For panels A–M, vibratome sections fromnon tg and a-syn tg mice that received LV injections into the cortex and hippocampus were immunolabeled with antibodies against Atg7 or a-synand imaged with a digital microscope. Panels A9–M9 represent higher-power images from the hippocampus of the corresponding low-power panelsin panels A–M. For panels O–T, effects of rapamycin treatment on a-syn accumulation, autophagy and neuronal integrity in the brains of a-syn tgmice. For panels A–M, vibratome sections from the hippocampus of non tg and a-syn tg mice were immunolabeled with an antibody against MAP2and imaged with a laser scanning confocal microscope, and images were obtained from the temporal cortex. (A–F) Representative sections from thebrains of non tg (A, B) and a-syn tg mice (C–F) that received injections with LV-control (A–D) or LV-Atg7 (E, F) and were immunolabeled with anantibody against Atg7. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (G) Semi-quantitative image analysis of Atg7 immunoreactivity in non tg and a-syn tg mice show increased Atg7 levels ipsilateral to the injection sites in thebrains of animals that received LV-Atg7. (H–M) Representative sections from the brains of non tg (H, I) and a-syn tg mice (J–M) that receivedinjections with LV-control (H–K) or LV-Atg7 (L, M) and were immunolabeled with an antibody against a-syn. Images show sections from thehemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (N) Semi-quantitative image analysis of a-syn immunoreactivity in non tgand a-syn tg mice show reduced a-syn levels ipsilateral to the injection sites in the brains of a-syn tg mice that received LV-Atg7 injections. (O–T)Representative sections from the brains of non tg (O, P) and a-syn tg mice (Q–T) that received injections with LV-control (O–R) or LV-Atg7 (S, T) andwere immunolabeled with an antibody against MAP2. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sitesof injection. (U) Semi-quantitative image analysis of MAP2 immunoreactivity in non tg and a-syn tg mice shows a recovery of MAP2 levels ipsilateralto the injection sites in the brains of a-syn tg mice that received LV-Atg7 injections. Scale bar in panel (F) represents 0.1mm in panels A–F and H–M,20mm in panels A9–F9 and H9–M9, and 10mm in panels O–T. *p,0.05 compared to LV-control-treated non tg controls by one-way ANOVA with post-hoc Dunnett’s test. #p,0.05 compared to LV-control-treated a-syn tg mice by one-way ANOVA with post-hoc Tukey-Kramer test.doi:10.1371/journal.pone.0009313.g009



atypical AV-like structures [42]. Supporting this possibility, the

present study also showed that the cortical neurons in DLB cases

and in a-syn tg mice contained enlarged lysosomes and

autophagosomes similar to those described in AD [33]. The

formation of such abnormal AV-like structures in DLB and a-syn

tg mice is consistent with recent reports in neuronal cells lines

overexpressing a-syn [42]. These cells show the presence of

granular a-syn aggregates that co-localize with abnormally

enlarged LC3-positive structures [42]. The accumulation of a-

syn and the neurodegenerative phenotype in neuronal cells was

reverted by activation of the autophagy pathway with a gene

therapy approach delivering Beclin-1 with a lentivirus [42] or with

rapamycin [42,74,75]. In agreement with these findings, the

present study showed in vivo that infusion of rapamycin or injection

of LV-Atg7 into the brains of tg mice reduced the accumulation of

a-syn and was neuroprotective. This is consistent with previous in

vivo studies showing that rapamycin is neuroprotective in models of

neurodegeneration [76,77], AD [78] and Huntington’s Disease

[79,80]. Moreover, a recent study showed that blocking mTor by

overexpression of the translation inhibitor Thor (4E-BP) can

reduce the pathologic features in PD models, including degener-

ation of dopaminergic neurons in Drosophila [81]. Moreover,

rapamycin activates in vivo 4E-BP and rapamycin is also capable of

ameliorating the pathology associated with mutations in other PD

associated genes such as Pink1 and parkin [81].

In familial types of parkinsonism, mutant forms of a-syn [23]

have been shown to disrupt lysosomal clearance by blocking

CMA. Further supporting a role for lysosomal dysfunction in DLB

and PD, recent studies have shown that in lysosomal storage

disorders such as Gaucher disease [43,44] and Niemann-Pick

disease [45], there is increased predisposition to develop

parkinsonism and a-syn accumulation. Other animal studies in

models of PD, such as in animals exposed to the neurotoxin 1-

methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP), have also

revealed autophagic dysfunction associated with alterations in

signal transduction pathways [82]. In addition, increased suscep-

tibility to develop PD appears to be associated with polymor-

phisms in lysosomal genes such as those associated with Gaucher

Figure 10. Immunocytochemical analysis of the effects of Atg7 over-expression or knockdown in a neuronal cell model. For Atg7overexpression, B103 neuronal cells on coverslips and infected with a lentivirus expressing LC3-GFP in combination with empty LV-control, LV-Atg7,or LV-a-syn. For Atg7 knockdown, B103 neuronal cells on coverslips were infected with a lentivirus expressing LC3-GFP in combination with emptyLV-shControl, LV-shAtg, or LV-asyn. Cells were fixed and immunolabled with an antibody against a-syn and imaged with a laser scanning confocalmicroscope. In each set of 3 panels, the upper left panel depicts GFP fluorescence, the upper right panel depicts a-syn immunolabeling, and thelower panel depicts the merged image. (A–D) Representative images showing GFP fluorescence (marker of LC3 localization) and a-synimmunoreactivity in B103 cells infected with LV-control (A), LV-Atg7 (B), LV-asyn (C) or LV-Atg7+LV-asyn (D). (E) Semi-quantitative analysis of LC3-GFPpositive punctae shows an increase in LC3 in cultures infected with LV-Atg7 or LV-a-syn alone or in combination. (F) Semi-quantitative analysis of a-syn immunoreactivity reveals a reduction in a-syn levels in cultures co-infected with LV-Atg7 and LV-asyn. (G–J) Representative images showing GFPfluorescence (marker of LC3 localization) and a-syn immunoreactivity in B103 cells infected with LV-shControl (G), LV-shAtg7 (H), LV-asyn (I) or LV-shAtg7+LV-asyn (J). (K) Semi-quantitative analysis of LC3-GFP positive punctae shows a reduction in LC3 in cultures infected with LV-shAtg7 alone orin combination with LV-asyn. (L) Semi-quantitative analysis of a-syn immunoreactivity reveals an increase in a-syn levels in cultures co-infected withLV-shAtg7 and LV-asyn. Scale bar in large panel in (D) represents 15mm in all small panels and 10mm in all large panels. *p,0.05 compared to LV-control-treated cultures by one-way ANOVA with post-hoc Dunnett’s test. #p,0.05 compared to LV-asyn-treated cultures by one-way ANOVA withpost-hoc Tukey-Kramer test.doi:10.1371/journal.pone.0009313.g010



disease and Niemann-Pick disease. Moreover, recent studies have

shown that reduced Cathepsin D expression results in a-syn

accumulation and degeneration of the dopaminergic system in

experimental models and in patients with PD [83]. Cathepsin D is

now considered one of the main lysosomal enzymes involved in a-

syn degradation [84] and overexpression of Cathepsin D reduces

the pathology associated with a-syn accumulation [85].

Selective alterations in molecular components of the autophagy

pathway might result in degeneration of specific neuronal

populations in neurological disorders. For example, previous

studies have shown that in sporadic AD there is a profound

reduction in the levels of Beclin-1 [34], while the neurodegener-

ative process in familial forms of fronto-temporal dementia and

ALS has been linked to mutations in charged multivesicular body

protein-2B (CHMP2B) [86,87,88], and in Huntington’s Disease

polyglutamate aggregates trap mTor and disrupt autophagy [80].

The mechanisms through which levels of mTor might be

upregulated and Atg7 downregulated in DLB and a-syn tg mice

are not completely clear. However, targeted reduction of

autophagy genes including Atg7 results in behavioral defects,

including abnormal limb-clasping reflexes and a reduction in

coordinated movement, and died within 28 weeks of birth.

Furthermore, Atg7 deficiency results in neurodegeneration of the

cerebral and cerebellar cortices [89].

In conclusion, this study supports the notion that selective

molecular alterations in the autophagy pathway and more

specifically in mTor and Atg7 are associated with DLB and a-

syn tg models and supports the possibility that modulators of the

autophagy pathway might have potential therapeutic effects.

Supporting Information

Figure S1 Immunoblot analysis of a-syn levels and lysosomal

markers in the brains of AD and DLB patients. Brain

homogenates from the temporal cortex of non-demented controls,

AD, and DLB patients were separated into membrane and

cytosolic fractions, and 20 mg of each sample was subjected to gel

electrophoresis. Immunoblots were probed with antibodies against

a-syn, Cathepsin D, LC3 and Actin. (A) Representative immuno-

blots of membrane fractions. (B) Representative immunoblots of

cytosolic fractions. (C) Semi-quantitative analysis of levels of a-syn,

Cathepsin D and LC3 in membrane fractions from the brains of

control, AD and DLB patients. Levels of Cathepsin D were

increased in the brains of AD patients but reduced in the brains of

DLB patients, while levels of LC3 were increased in the brains of

both AD and DLB patients. All semi-quantitative measurements

were normalized to actin levels as a loading control. *p,0.05

compared to non-demented controls by one-way ANOVA with

post-hoc Dunnett’s test.

Found at: doi:10.1371/journal.pone.0009313.s001 (0.41 MB TIF)

Figure S2 Immunoblot analysis of a-syn levels and lysosomal

markers in the brains of APP tg and a-syn tg mice. Brain

homogenates from non tg, APP tg, and a-syn tg mice were

separated into membrane and cytosolic fractions, and 20 mg of

each sample was subjected to gel electrophoresis. Immunoblots

were probed with antibodies against a-syn, Cathepsin D, LC3 and

Actin. (A) Representative immunoblots of membrane fractions. (B)

Representative immunoblots of cytosolic fractions. (C) Semi-

quantitative analysis of levels of a-syn, Cathepsin D and LC3 in

membrane fractions from the brains of non tg, APP tg and a-syn tg

mice. Levels of Cathepsin D were significantly reduced in a-syn tg

brains, while levels of LC3 were increased in the brains of APP tg

and a-syn tg mice. All semi-quantitative measurements were

normalized to actin levels as a loading control. *p,0.05 compared

to non tg controls by one-way ANOVA with post-hoc Dunnett’s

test.

Found at: doi:10.1371/journal.pone.0009313.s002 (0.33 MB TIF)

Figure S3 Immunocytochemical and immunoblot characteriza-

tion of lentivirus-mediated Atg7 over-expression and knockdown

in a neuronal cell line. B103 neuronal cells on coverslips were

infected with LV-Atg7 or LV-shAtg7, followed by fixation and

immunolabeling with an antibody against Atg7, or lysis and

immunoblot analysis with antibodies against Atg7 or Actin. (A)

Representative image of endogenous Atg7 immunoreactivity in

cells infected with empty LV-control. (B) Representative image

showing increased Atg7 immunoreactivity in cells infected with

LV-Atg7. (C) Representative image showing reduced Atg7

immunoreactivity in cells infected with LV-shAtg7. (D) Represen-

tative immunoblot showing Atg7 levels in cells infected with LV-

Atg7 or LV-shAtg7. Scale bar in panel (C) represents 40mm in all

microscopy images.

Found at: doi:10.1371/journal.pone.0009313.s003 (0.80 MB

TIF)

Author Contributions

Conceived and designed the experiments: LC BS PD ER LH DG EM.

Performed the experiments: LC BS PD CP ADP ER AA EM. Analyzed the

data: LC BS PD CP ADP ER LH AA DG EM. Contributed reagents/

materials/analysis tools: EM. Wrote the paper: LC PD EM. Critical

revision of the article: CP ADP LH DG.

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