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THE INFLUENCE OF VIRAL VECTOR DELIVERY OF SUPEROXIDE DISMUTASE AND CATALASE TO THE HIPPOCAMPUS ON SPATIAL LEARNING, MEMORY AND

NMDA RECEPTOR FUNCTION DURING AGING

By

WEI-HUA LEE

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

2012

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© 2012 Wei-Hua Lee

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To my family

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ACKNOWLEDGMENTS

First, I would like to give special thanks to my mentor Dr. Thomas Foster for his

patience, trust and constant guidance in my Ph.D. training. I appreciate my committee

members, Dr. Jeffrey Harrison, Dr. Christiaan Leeuwenburgh, Dr. Lucia Notterpek, and

Dr. Susan Semple-Rowland for their critiques, comments and advices that polish me to

be a great scientist. I also appreciate all the friends I made in Foster lab, Dr. Ashok

Kumar, Asha Rani, Olga Tchigrinova, Michael Guidi, Xiaoxia (Sylvia) Han, Linda Been,

Dr. Kristiina Aenlle, Dr. Karthik Bodhinathan, Dr. Travis Jackson, Dr. Zane Zeier, Dr. Li

Cui, Jose Herrera, and Katrina Velez. You made my life in the lab wonderful. I would

like to thank my collaborators, Dr. Jinze Xu, Dr. Shinichi Someya, Dr. Eduardo

Candelario-Jalil, Dr. Alfred Lewin, Dr. Nicolas Muzyczka and Marvin Servanez for

helping me with my studies. I thank Ms. Betty J. Streetman at the Neuroscience office

for taking care of me all the time, and Mr. Mark Potter at vector core laboratory for AAV

supply. Last but not least, I thank my parents for being my foundation and for their

prayers. I thank my sister for always be on my side, I thank my husband, Che, and my

daughter, Shin-Ning (Julia) for their love and support along this journey.

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TABLE OF CONTENTS page

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

LIST OF TABLES............................................................................................................ 8

LIST OF FIGURES.......................................................................................................... 9

LIST OF ABBREVIATIONS........................................................................................... 11

ABSTRACT ................................................................................................................... 13

CHAPTER

1 LITERATURE REVIEWS........................................................................................ 15

Aging Effects on Cognitive Function ....................................................................... 15 Animal Models Used in Studying Aging .................................................................. 16 Cognitive Decline during Aging............................................................................... 20 Methods for Detecting Age-Related Changes in Spatial Memory in Rodents ......... 24 Mechanism of Brain Aging ...................................................................................... 30

ROS and Aging ................................................................................................ 30 ROS Markers.................................................................................................... 37

Total ROS .................................................................................................. 37 Superoxide................................................................................................. 37 Hydrogen peroxide..................................................................................... 38 Redox state................................................................................................ 39 Oxidative damages .................................................................................... 39 Antioxidant enzymes.................................................................................. 41

Antioxidant Treatments .................................................................................... 43 Viral Vectors Delivery in Central Nervous System (CNS) ....................................... 45

2 MATERIALS & METHODS ..................................................................................... 51

Animals ................................................................................................................... 51 AAV Viral Vector ..................................................................................................... 52 Construction of Lentiviral Vector and Vector Packaging ......................................... 52 SOD1 Lentiviral Vector Packaging, Concentration and Titration............................. 53 Behavior Testing..................................................................................................... 54 Hippocampal Tissue Dissection.............................................................................. 55 Western Immunoblotting ......................................................................................... 55

Preparation of Lysate from Hippocampal Tissue.............................................. 55 Determination of Protein Concentration ........................................................... 56 Electrophoresis................................................................................................. 56 Transfer of Protein............................................................................................ 56 Blotting ............................................................................................................. 56

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Stripping ........................................................................................................... 57 Immunofluorescence .............................................................................................. 58 Slice Preparation .................................................................................................... 58 Biochemical Assays................................................................................................ 60

SOD Activity ..................................................................................................... 60 Protein Carbonyls............................................................................................. 60 8-oxodGuo........................................................................................................ 60 Determination of GSH and GSSG .................................................................... 61 Determination of Glutathione Peroxidase Activity............................................. 62 Determination of Glutathione Reductase Activity ............................................. 63

Statistics ................................................................................................................. 63

3 INFLUENCE OF VIRAL VECTOR-MEDIATED DELIVERY OF SUPEROXIDE DISMUTASE AND CATALASE TO THE HIPPOCAMPUS ON SPATIAL LEARNING, MEMORY DURING AGING................................................................ 71

Results.................................................................................................................... 72 Efficiency-Specificity of The Viral Vectors ........................................................ 72 Age-Dependent Influence of Enzyme Overexpression on Spatial Learning ..... 74 Overexpression of SOD1+CAT for Four Months Improves Spatial Learning.... 76

Discussion .............................................................................................................. 79

4 THE INFLUENCE OF AAV DELIVERED SUPEROXIDE DISMUTASE 1 AND CATALASE ON HIPPOCAMPAL SYNAPTIC PLASTICITY AND NMDA RECEPTOR FUNCTION ........................................................................................ 96

Results.................................................................................................................... 99 AAV Delivery of SOD1, CAT and GFP in the Hippocampi of Rats ................... 99 SOD1 Overexpression Impairs Spatial Learning while SOD1+CAT

Overexpression Improves Spatial Learning in Aged Rats. .......................... 100 NMDAR-Mediated Synaptic Potentials are Reduced in Rats with SOD1

Overexpression ........................................................................................... 104 Effect of Overexpression of SOD1 and CAT on Glutathione Redox State ..... 105 Effect of Overexpression of SOD1 and CAT on Activities of GSH

Peroxidase and GSH Reductase ................................................................ 106 Discussion ............................................................................................................ 106

5 GENERAL DISCUSSION ..................................................................................... 122

Summary .............................................................................................................. 122 Discussion ............................................................................................................ 124

H2O2 Produced by Overexpression of SOD1 Affects Synaptic Plasticity and Memory in an Age-Dependent Manner ....................................................... 126

Co-Overexpression of SOD1 and CAT in the Hippocampi is More Beneficial Than Overexpression of SOD1 or CAT Alone............................................. 131

Mitochondrial ROS Has Minimal Effect on Synaptic Plasticity and Memory... 132

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Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) as Signal Molecules in Synaptic Plasticity ....................................................... 133

LTP During Aging ........................................................................................... 134 Global Versus Local Redox State................................................................... 135

APPENDIX: ADDITIONAL FIGURES......................................................................... 139

LIST OF REFERENCES ............................................................................................. 143

BIOGRAPHICAL SKETCH.......................................................................................... 172

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LIST OF TABLES

Table page 1-1 Studies using knock out or overexpression of antioxidant enzymes................... 50

2-1 A list of all primary antibodies used in Chapter 3 and Chapter 4 western blot and immune-fluorescence. ................................................................................. 70

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LIST OF FIGURES

Figure page 1-1 Mitochondrial respiratory chain and superoxide production................................ 49

2-1 Map of sc-trs-smCBA-Human SOD1-myc (SOD1) ............................................. 65

2-2 Map of pTR-UF11-Human SOD2 (SOD2) .......................................................... 66

2-3 Map of pTR-UF11-Human CAT (CAT) ............................................................... 67

2-4 Cue discrimination .............................................................................................. 68

2-5 Spatial and probe discrimination......................................................................... 69

3-1 Neurons are the primary cell type transduced by the AAV vectors..................... 83

3-2 Overexpression of antioxidant enzymes in the hippocampus reduces markers of oxidative stress............................................................................................... 84

3-3 Overexpression of antioxidant enzymes did not affect cue discrimination in water maze. ........................................................................................................ 86

3-4 Overexpression of SOD1 impaired spatial learning in aged rats. ....................... 87

3-5 Overexpression of SOD1 impaired acquisition of a spatial search strategy in aged rats. ........................................................................................................... 88

3-6 Long-term overexpression of SOD1+CAT improved learning in spatial trials in aged rats......................................................................................................... 90

3-7 Overexpression of SOD1+CAT for four months improves spatial learning in probe test.. ......................................................................................................... 91

3-8 Antioxidant enzymes and oxidative stress markers in hippocampus with 5-month SOD1 and SOD1+CAT overexpression................................................... 93

3-9 DNA oxidative damage is reduced by overexpression of SOD1......................... 95

4-1 Co-overexpression of SOD1+GFP and SOD1+CAT. ....................................... 112

4-2 Antioxidant enzymes and oxidative stress markers in hippocampus with 1-month SOD1+GFP and SOD1+CAT overexpression. ...................................... 113

4-3 Overexpression of SOD1+GFP or SOD1+CAT enhanced cue discrimination in the water maze compared to GFP control.. .................................................. 114

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4-4 Overexpression of SOD1+CAT improved spatial learning while overexpression of SOD1+GFP impaired spatial learning in aged rats............. 115

4-5 Overexpression of SOD1+CAT improves spatial learning in probe tests.......... 116

4-6 NMDAR-mediated synaptic potentials are reduced in rats with SOD1 overexpression. ................................................................................................ 118

4-7 Decreased level of reduced GSH (GSH), total GSH and oxidized GSH (GSSG) are observed in rats with overexpression of SOD1+GFP. .................. 120

4-8 Decreased glutathione peroxidase (GPx) and glutathione reductase (GR) activities are observed in rats with overexpression of SOD1+GFP .................. 121

5-1 A model for altered redox state in aged rats with overexpression of SOD1...... 138

A-1 Control images for hippocampal immunofluorescence. .................................... 139

A-2 SOD1 and myc expression are co-localized in the hippocampus of a rat with overexpression of SOD1. ................................................................................. 141

A-3 Expression of corresponding antioxidant enzymes are higher in the hippocampus of rats with injection of AAV-SOD1, AAV-SOD2, or AAV-CAT. .. 142

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LIST OF ABBREVIATIONS

°C degree celsius (unit for expressing temperature)

8-oxodGuo 8-oxo-7,8-dihydro-2-deoxyguanosine

AAV adeno-associated virus

ANOVAs analyses of variance

Ca+ Calcium (ionic form)

CA1 cornu ammonis area 1

cAMP cyclic adenosine monophosphate

CAT catalase

COX OxPhos complex IV subunit I

CREB cAMP response element binding protein

ERK extracellular signal-regulated kinase

Fisher’s PLSD Fisher’s protected least significant difference

GAPDH glyceraldehyde 3-phosphate dehydrogenase

GFAP glial fibrillary acidic protein

GFP green fluorescence protein

GPx glutathione peroxidase

GSH glutathione

HNE 4-hydroxy-2-nonenal

H2O2 hydrogen peroxide

LTP long-term potentiation

MAP2 microtubule-associated protein 2

NeuN neuronal nuclei

NHPs non-human primates

NMDA N-Methyl-D-aspartate

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NO nitric oxide

μg micro grams (1/1000000 of a gram; unit of mass)

μL micro liters (1/1000000 of a liter; unit of volume)

μm micro meter (1/1000000 of a meter; unit of length)

O2- superoxide anion

OH hydroxyl radical

P-GSH GSH antibody binds to GSH-protein conjugate

PP1 protein phosphatase 1

PKA protein kinase A

PKC protein kinase C

post hoc post hoc ergo propter hoc (Latin for “after this”)

ROS reactive oxygen species

S.E.M. standard error of the mean

SOD superoxide dismutase

tg-SOD SOD transgenic mice

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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

THE INFLUENCE OF VIRAL VECTOR DELIVERY OF SUPEROXIDE DISMUTASE

AND CATALASE TO THE HIPPOCAMPUS ON SPATIAL LEARNING, MEMORY AND NMDA RECEPTOR FUNCTION DURING AGING

By

Wei-Hua Lee

August 2012

Chair: Thomas C Foster Major: Medical Science - Neuroscience

Studies employing transgenic mice indicate overexpression of superoxide

dismutase 1 (SOD1) improves memory during aging. It is unclear whether the

improvement is due to a lifetime of overexpression, decreasing the accumulation of

oxidized molecules, or if increasing antioxidant enzymes in older animals could reduce

oxidative damage and improve cognitive function. The first study tested the hypothesis

that overexpression of antioxidant enzymes in hippocampi will delay age-related

memory decline by reducing oxidative damage. This study used adeno-associated virus

(AAV) to deliver antioxidant enzymes (SOD1, SOD2, CAT and SOD1+CAT) to the

hippocampi of young (4-month) and aged (19-month) F344/BN F1 male rats and

examined memory-related behavioral performance one month and four months post

injection. The second study examined the hypothesis that overexpression of antioxidant

enzymes affects N-Methyl-D-aspartate (NMDA) receptor function via regulating redox

state. AAV was used to deliver SOD1+GFP and SOD1+CAT to the hippocampi of aged

(17-month) F344 rats and examined memory-related behavioral performance one-

month post injection. Following behavioral characterization, hippocampi were removed

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and used to examine NMDA receptor function, determine antioxidant enzyme

expression, redox state-related antioxidants and enzyme activities.

The first study indicated that overexpression of antioxidant enzymes reduced

oxidative damage; however, memory function was not related to the level of oxidative

damage. Increased expression of SOD1, initiated in advanced age, impaired learning.

Increased expression of SOD1+CAT provided protection from impairments associated

with overexpression of SOD1 alone and appears to guard against cognitive impairments

in advanced age. The second study indicated that SOD1 overexpression was

associated with a decrease in the NMDAR-mediated fEPSP. The decrease in NMDAR

function in SOD1 rats could explain the impaired learning. Biochemical assays indicated

that free glutathione, oxidized glutathione and total glutathione decreased in SOD1 rats.

The reduced level of glutathione could contribute to the decreased NMDAR function.

Glutathione peroxidase and glutathione reductase activities decreased in SOD1 rats,

which might be due to altered redox state of the enzymes. In conclusion, my studies

provide support for the idea that altered redox sensitive signaling rather than the

accumulation of damage may be of greater significance in the emergence of impaired

learning and memory.

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CHAPTER 1 LITERATURE REVIEWS

Aging Effects on Cognitive Function

The lifespan of humans has increased dramatically over the last century due to the

improvement of sanitation, hygiene, health care, medicine, nutrition, and technology

(Wykle et al., 2005). A longer life has uncovered age-associated deficits in cognitive

function, challenging both personal and public health internationally. Aging is the

greatest risk factor for cognitive decline and neurodegenerative diseases (Bishop et al.,

2010). Understanding the mechanisms of aging provides one perspective or avenue of

research that can potentially prevent or delay the onset of late-life dementias.

Historically, it was once thought that the major contributor to age-related cognitive

decline was massive neuronal loss and deterioration of dendritic branching (Ball, 1977;

Brody, 1955; Coleman and Flood, 1987; Scheibel, 1979; Scheibel et al., 1976).

However, mounting research indicates that changes occurring during normal aging are

more subtle and selective than was once believed (Foster, 2012; Morrison and Hof,

1997).

Due to the limitations of approaches to examine the brains of humans, the work of

solving the mysteries of brain aging still heavily relies on studies using various animal

models. The first part of this thesis will review the use of animal models to study aging

and age-related cognitive decline. This section will focus on rodents as well as the

behavioral tasks employed to test cognitive function in rats. In addition, I will provide the

background for the oxidative stress theory of aging, and how this hypothesized

biological mechanism of aging could explain age-related memory loss. Finally, I will

layout the gaps in our knowledge that my work attempts to address.

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Animal Models Used in Studying Aging

There are inherent advantages and disadvantages with each animal model used

to study aging. The commonly used animal models of aging include nematode worms

(Caenorhadbitis elegans), fruit flies (Drosophila melanogaster), mice, rats, monkeys and

chimpanzees. C. elegans and Drosophila have the advantage of a short lifespan and

excellent genetic tools for studying how a single gene may affect aging (Olsen et al.,

2006); however, these models are evolutionarily far from humans. Thus, some

important aging genes in the invertebrates species have no mammalian homologs

(Rikke et al., 2000), and many physiologically important systems in mammals are not

found in invertebrates, including the immune system (Johnson, 2003). By contrast, 90%

of the DNA from Rhesus monkeys and almost 99% of the DNA from chimpanzees are

identical to humans; therefore, it may not be surprising that many aspects of aging,

including life course, are very similar across primates. Behaviorally, the testing protocols

used in non-human primates (NHPs) can be adapted for use in humans. With this

similarity to humans in terms of molecular and cognitive measures, the NHP studies

have a further advantage in that experiments can be conducted under tightly controlled

laboratory conditions, such as long-term regulation of diet or environmental constraints

(Colman and Anderson, 2011; Ingram et al., 1990). Unfortunately, the high cost and low

availability of aged NHPs limits the utility of these animals in aging research (Lane,

2000).

Laboratory rodents are commonly used in aging studies because of their smaller

size, ease of maintenance, and prolific reproduction. Moreover, most rodents are

relatively short lived, with the longest recorded lifespan being 4 and 5 years for mice

and rats, respectively. Mice and rats share remarkable homology with humans in terms

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of the underlying neural basis for a number of complex behaviors (Foster, 2012; LeDoux,

2000). However, before delving deeper into a discussion of the advantages of mice and

rats in aging studies, it should be pointed out that there are also important limitations for

laboratory mice and rats. A few rodent species, such as naked mole rats, beavers,

porcupines and several species of squirrels are long-lived, and can maintain a lifespan

of up to 20 years. Thus, studies limited to laboratory mice and rats with the fastest aging

process, may not provide all the desired clues for aging, particularly when attempting to

model humans, one of the slowest aging mammals (Austad, 2005; Buffenstein and

Jarvis, 2002; Jones, 1992). Indeed, some researchers have suggested that including

slow-aging rodents in studies on mechanisms of aging may provide important insights

(Gorbunova et al., 2008). A similar argument has been made for examining the

mechanisms of successful aging in humans (Murabito et al., 2012; Newman et al., 2011;

Rozing et al., 2010).

Mice are now widely used in aging studies due in part to a well described genome

and the relative ease in generating knock out or transgenic animal models (Capecchi,

2005). For example, mice with genetic changes in a single antioxidant gene have

revolutionized the study of free radicals and oxidative damage in aging and diseases

(Bartke and Brown-Borg, 2004; Hasty et al., 2003; Hasty and Vijg, 2004; Li et al., 1995;

Reaume et al., 1996). Rats, on the other hand, have a long history of being used by

experimental psychologists and behavioral neuroscientists, yielding a great body of

knowledge in cognitive and behavioral neuroscience, laying a solid foundation for the

study of cognitive decline in aging.

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Rat Strains. Genetic background is an important consideration when choosing a

rat model for aging research. Outbred strains (with breeding schemes that avoid

crosses between closely related individuals in order to maintain the maximal level of

heterozygosity in all offspring) such as Wistar, Sprague Dawley and Long-Evans rats

are genetically diverse and more accurately reflect the genetic diversity of the humans.

However, there are several disadvantages to the use of outbred rats (Lipman, 1997).

The characteristics of outbred rats can vary from one another, especially if they derived

from a small breeding population (Nadon, 2006). As a result, the sample size must be

larger in order to adjust to the genetic diversity. Inbred rats that require the rat strain

being mated brother x sister for 20 or more consecutive generation are considered

genetically identical. The genetic identity allows the experiments to be more easily

replicated in different laboratories (Phelan, 1992). However, the high homozygosity of

inbred rats also increases the chances of the population being affected by recessive or

deleterious traits, resulting in strain-specific lesions. As a result, the F1 hybrids of two

inbred strains can be the solution for both outbred and inbred rats. F1 hybrids are

genetically uniform and heterozygous for all of the genes for which the two parental

strains differ, facilitating comparison between individuals. The increased heterozygosity

in F1 hybrids protects them from the high incidence of inbred strain-specific disorders.

The National Institute on Aging (NIA) offers three strains of rats for biological aging

studies, allowing researchers to easily obtain the rats at advanced ages. The inbred

Fischer 344 (F344) strain which has been available through the NIA since the mid-

1970s is the most commonly used rat strain in aging research due to its moderate body

weight throughout life and the absence of genetic variability (Markowska and

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Savonenko, 2002; Sprott, 1991; Sprott and Ramirez, 1997). Median survival of F344

rats is approximately 24 months for males and 26 months for females (Turturro et al.,

1999). Due to the nature of this inbred strain, F344 rats have a high incidence of certain

diseases, including glomerulonephropathy and leukemia at advanced ages (Lipman et

al., 1999). The inbred Brown Norway (BN) rat lives longer than the F344 rat, with a

median life expectancy of 32 months for both males and females, and has far fewer

strain specific lesions with age (Turturro et al., 1999). The BN strain is a docile rat and

its behavioral performance has been noted to decline more slowly with age than F344

rats (Spangler et al., 1994). However, one report indicated that adult BN rats have poor

performance in many learning tasks, although their performance on reference memory

tasks was similar to other strains (Van Luijtelaar and Coenen, 1988). The F344 × BN

hybrid (F344BNF1) rat strain expresses the expected hybrid vigor and has lower levels

of the strain-specific pathologies seen in the parent populations. Thus, F344BNF1 rats

are more susceptible to environmental factors such as diet restriction (Lipman, 1997;

Lipman et al., 1999; Markowska and Savonenko, 2002; Sprott and Ramirez, 1997).

Further, adult F344BNF1 rats have been shown to exhibit good performance in various

cognitive tasks including water maze, inhibitory and passive avoidance tasks, delayed

nonmatching to position, and active avoidance (Lipman et al., 1996; Sprott and Ramirez,

1997; van der Staay and Blokland, 1996). They also live longer than F344 with the

median life expectancy for males of 34 months and 29 months for females (Turturro et

al., 1999). The hybrid vigor, ability to perform in most of the cognitive tasks, and longer

lifespan make F344BNF1 a good model to examine age-related cognitive decline in the

presence of maintained physical function (Nadon, 2006).

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For the first study of the dissertation, F344BNF1 rats were employed. We

employed F344BNF1 male rats as our animal model because of all the benefits for

aging studies as mentioned above. In addition, we chose 18-month old (in the middle of

their life span) as the aged group, because cognitive decline measured in tasks for

spatial reference memory and spatial working memory begin to emerge at this age

(Markowska and Savonenko, 2002). Our second study focused on electrophysiological

markers of aging, therefore, we chose male F344 rats in order to better compare the

results to our previous electrophysiological findings using the same rat strain. In addition,

we started treatment at the age of 17 months, because a significant drop in the level of

performance in spatial reference memory tasks was observed around this age in F344

male rats (Markowska, 1999).

Cognitive Decline during Aging

Memory and the Hippocampus. Senescence is associated with a decline in

memory that depends on the hippocampus, sometimes referred to as explicit memory,

episodic memory, or declarative memory (Singer et al., 2003). Before detailing the type

or form of memory that is processed by the hippocampus, it is important to take a

historical perspective on research focused on the neural systems involved in memory.

From a historical perspective, the most famous case is the patient, H. M., who had

bilateral medial temporal lobe resection because of his severe symptoms of epilepsy.

The surgery involved the removal of large portions of the temporal lobe from both

hemispheres of the brain, including amygdala, the entorhinal and perirhinal cortices,

and about two-thirds of the hippocampus. After the surgery, H. M. had much milder

epilepsy symptoms, but he lost the ability to form long lasting memories of the events in

his life that happened since the surgery. In other words, he could not acquire new

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factual knowledge about the world around him. He also had difficulty remembering

information acquired several years prior to his surgery. Yet, some aspects of H. M.’s

memory did remain intact, such as language, short-term memory, and learning simple

sensorimotor skills. Psychologist Professor Brenda Milner has studied H. M. more

extensively than any other investigator. Milner’s early work in studying H. M. changed

how people thought about memory. Prior to H. M., one of the dominant theories on the

biological basis of memory was based on the work of Karl Lashley, who trained rats in a

maze and then made incisions in their cerebral cortices to determine whether the site of

the incision affected the animals performance when they were reintroduced to the maze.

Lashley observed that memory for the maze degraded as a function of the amount of

damage to the cortex. However, the site of the damage did not affect memory. These

findings led to Lashley's notions of equipotentiality, the idea that the locus is not

important, and mass action, that the amount or size of damage is the critical factor. It is

important to point out that Lashley only looked at the cerebral cortex, whereas the

hippocampus and the amygdale are deep structures. The case of H. M. conflicted with

Lashley’s idea, emphasizing the importance of hippocampal formation for specific types

of memory. In addition, H. M.’s case appeared to support the dual memory model, as

proposed in 1968 by Richard Atkinson and Richard Shiffrin. According to the dual

memory model, new information needs to be transferred from short-term memory to

long-term memory in order to be remembered at a later time. H. M.’s ability to follow

conversations and retain small amounts of verbal information for ~15 seconds

suggested that his short-term memory was still intact, but he could not transfer the

short-term memory into long-term store. In addition, he maintained memories from

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before the surgery, indicating that much of long-term memory was intact. In conclusion,

H. M. and other patients with hippocampal lesions were impaired in acquiring or storing

new, consciously accessible memories, whereas procedural learning and some remote

memories that were acquired well before the lesion remained intact (Milner, 1972;

Milner et al., 1998; Rempel-Clower et al., 1996; Scoville and Milner, 1957; Zola-Morgan

et al., 1986).

With respect to the current set of studies, an important aspect of the memory

deficits associated with hippocampal damage is impaired spatial memory. The interest

in spatial processing by the hippocampus was driven to a large extent by the discovery

of ‘place cells’ in the rat hippocampus (O'Keefe and Dostrovsky, 1971). Recordings of

cell discharge activity in freely moving animals demonstrated cells that fire when an

animal was in specific locations in an environment. The ensemble of cells provides a

stable representation of the animal’s location, independent of its orientation. Place cells

are believed to be pyramidal cells in the CA1 and CA3 hippocampal subfields, or

granule cells in the dentate gyrus (Moser et al., 2008). Based on the discovery of place

cells, the hippocampus was proposed to have a primary function in forming cognitive

maps of the environment (O’Keefe & Nadel, 1978). In conclusion, the studies of humans

and animals with hippocampal damage, as well as the discovery of place cells indicate

that the hippocampus is essential in acquiring new information, consolidating short-term

memory into long-term memory, and plays an important role in spatial learning and

memory.

The deficits observed during aging are consistent with impaired hippocampal

functions. Elderly humans exhibit intact remote memories and are able to learn and

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remember new skills (i.e. procedural learning) to a similar level as that observed in

young adults. Older individuals appear to acquire new information; however, their

learning may be slower and they exhibit increased forgetting as retention delays

increase (Davis et al., 2003; Hogge et al., 2008; Huppert and Kopelman, 1989; Kral,

1962; Macdonald et al., 2006; Mitchell et al., 1990; Park et al., 1988). Interestingly,

spatial memory decline is one of the most common complaints expressed by older

people; for example, they are unable to remember the location of household objects and

frequently suffer from spatial disorientation (Jonker et al., 1996). In addition, it is quite

common for the elderly to have difficulty memorizing maps or recalling the temporo-

spatial context of a memory (Wilkniss et al., 1997).

Despite the similarity in memory deficits, impaired spatial learning and impaired

retention with increasing delays, it is important to point out that the memory deficits

observed during aging in humans are not as severe as that observed following

hippocampal damage. Similar distinctions can be made for animal models of age-

related cognitive decline. Animals with hippocampal lesions are impaired in acquiring

spatial information (Martin and Clark, 2007; Morris et al., 1990). In contrast to

hippocampal lesions, most aged animals can acquire new information with only a mild

impairment in acquisition of spatial discrimination (e.g. slower learning rate). In many

cases, with extensive training, aged animals can acquire a spatial reference memory

(memory for a location that is the same from trial to trial, i.e. trial independent) to the

same extent as young animals. In contrast, age-related deficits become more obvious

for tests that focus on acquisition of trial-dependent (locations that change from trial to

trial) spatial information and memory impairments increase with an increase in the

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retention delay (Bizon et al., 2009; Driscoll et al., 2006; Foster, 1999; Foster et al., 2003;

Frick et al., 1995). Combined with the fact that age-related memory deficits are not

associated with a loss of neurons (Gazzaley et al., 1997; Keuker et al., 2003; Merrill et

al., 2001; Merrill et al., 2000; Pakkenberg and Gundersen, 1997; Peters et al., 1994;

Rapp and Gallagher, 1996; Rasmussen et al., 1996; West et al., 1994), the differences

suggest that memory deficits during aging are not due to lesions and probably result

from more subtle changes in mechanisms for storing and maintaining information

(Foster, 1999). In addition, the results suggest that the training protocols influence the

sensitivity of each task for detecting cognitive changes during aging. The next section

will review techniques/protocols for using the water maze to access spatial memory

impairment in aged rats.

Methods for Detecting Age-Related Changes in Spatial Memory in Rodents

The Water Maze. The insight that aging affects hippocampus-dependent spatial

memory has received broad support across a range of species using a variety of spatial

tasks. Spatial mazes have been employed since the beginning of 20th century (Vincent,

1915; Watson, 1907) and are commonly used today to evaluate spatial cognition in

animals (Fouquet et al., 2011; Ingram, 1988; Kametani et al., 1989). Conventional maze

learning tasks such as the T-Maze, Barnes circular platform maze and Corridor mazes

(Barnes, 1979) have been frequently used to investigate spatial learning in rodents

(McLay et al., 1999). Generally, animals are food or water deprived and the access to

food or water is used as behavioral reinforcement or the motivation for learning the

maze. These tasks require animals to remember a route (e.g. a series of left and right

turns) or learn the spatial layout (e.g. cognitive map) in order to obtain a water or food

reward. Thus, one important factor to take into account is that animals can use different

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strategies to try and solve spatial problems. In most cases the behavior is defined in

terms of egocentric and allocentric strategies (Burgess, 2006). An egocentric strategy is

based on the information provided by bodily cues, and therefore it is independent of

spatial cues. For this strategy, the animal functions as its own central point of reference,

and so, all other object positions are defined in relation to the animal’s position in space

(O’Keefe and Nadel, 1978; Klatzky, 1998). An allocentric strategy depends on spatial

cues and their relation to each other. When using this strategy the animal memorizes

the target location in relation to the spatial position of the environmental reference

landmarks (Benhamou and Poucet, 1995).

The water maze is usually considered superior to other conventional mazes for

several reasons. First, learning on the water maze is relatively rapid such that animals

can acquire a spatial search strategy in a single training session. Second, the water

maze does not allow the animals to use aromatic cues to orient themselves in the

escape search, which may happen in the dry mazes. Third, the water maze does not

require water or food deprivation. For food and water deprivation, it may be hard to

determine if the motivation is similar across different age groups. In the case of the

water maze, the animal is motivated to learn the spatial layout of the maze in order to

escape from the water and young and old animals appear to be equally motivated. In

addition, the water maze is more suitable for examining the effects of calorie-restricted

animals. Finally, the water maze can be designed to detect impaired sensory-motor or

motivational differences, and the use of egocentric and allocentric strategies.

The water maze task was first introduced in 1982 by R. Morris’s paper in which he

developed this task for studying the role of the hippocampus in spatial learning in rats

26

(Morris, 1984; Morris et al., 1982). In the task, the rats must use distal spatial cues to

locate a hidden escape platform just under surface of the water. The original protocol

consisted of 8 days of training, one trial on the first day and 4 trials a day during the next

seven days. The rat is released from a different start location along the wall of the tank

or pool during each training trial, so the rat must use environmental cues, rather than a

series of motor responses, to navigate to the hidden escape platform. The spatial

learning is reflected by faster escape latency and shorter distance to reach the platform

over the course of training trials. Importantly, Morris tested three groups of rats in the

water maze, including a group with hippocampal lesions, a group with cortical lesions,

and a control group with intact brain structures. The rats with hippocampal lesions

exhibited a significantly longer path length to reach the platform indicating that acquiring

spatial information in water maze with a hidden platform is hippocampus-dependent.

The rats were also given a probe trial with the platform removed. During the probe trial,

the rat is released from the quadrant opposite from the quadrant that had the platform.

The animal is allowed to swim for 60 seconds in the pool and the time spending

searching each quadrant, total distance from the platform location, and number of times

the animal crosses the location that held the escape platform are recorded. The probe

trials provide a more sensitive measure of the use of a spatial search strategy that

latency or distance to reach the platform during training trials. A decrease in latency and

distance could be observed for animals using a cue-response strategy, e.g., swim in a

certain distance from the wall until they bump into the platform. In the Morris study, the

group with hippocampal lesions performed significantly worse than other two groups in

terms of spending significant less time searching in the location which had previously

27

held the platform, supporting the idea that hippocampus is involved in spatial learning.

In contrast, the rats with hippocampal lesions were not impaired in the cue

discrimination version of water maze task. For the cue discrimination task, a visible

escape platform is used as a local visual cue. This visible escape platform can be

located in different spatial locations across training trials. In addition, the area

surrounding the pool is usually separated from other part of the room by a curtain to

minimize distal spatial cues. Animals use the same sensory-motor and motivational

components as in spatial/hidden platform training; however, the most efficient way to

escape the pool is to employ a cue response strategy by simply locating the visible

platform and swimming to it. One study that examined the effect of brain lesions on the

acquisition of the cue discrimination task found that damage to the basal ganglia, but

not the hippocampus, disrupted acquisition on this task (Packard and McGaugh, 1992).

In summary, the evidence indicates that the hippocampus and basal ganglia are parts of

systems that differ in the type of memory they mediate. Furthermore, performance on

the spatial discrimination version of the task is more sensitive to impairment associated

with aging.

For the current studies, we employed the cue task prior to spatial training. This

protocol consists of cue/visual platform training in which a visibly prominent, moveable

platform with a flag on the top is used. Cue/visual training consisted of 5 blocks of three

trials, with all training delivered in one day. For cue training, the visible platform was

placed in a novel position for each trial. The cue task was used to identify sensory-

motor or motivated deficits, which would affect acquisition of a spatial search strategy.

In addition, animals learn the procedural aspect of the task, including how to swim and

28

learning that the pool wall is not a route of escape (Vorhees and Williams, 2006). When

animals are trained in the cue task first, they may perform better in the spatial task due

to the acquisition of the procedural aspects of the task (Foster, 2012). Similarly, animals

trained initially in the spatial version of water maze may perform relatively poorly, but

exhibit superior performance in subsequent cue training (Gerlai, 2001). As a result, the

initial poorer performance in spatial training may be due to deficits related to procedural

learning or to deficits in spatial learning, or a combination.

For studies presented in this dissertation, spatial training was initiated three days

following completion of the cue discrimination task. Recently it has been emphasized

that training schedules are important in determining the sensitivity of the Morris escape

task for the early detection of age-related deficits in the acquisition and retention of

spatial information in rodents (Foster, 2012). A common spatial training schedule

involves distributed training over days, in which the animals are trained 3~4 trials a day

for several days. An alternative method is to provide all the training in a single episode,

usually by massing all the training trials into a single session. Historically, much of the

research on aging has employed distributed training to examine cognitive decline during

aging. However, distributed training is not as sensitive to the earliest signs of an age-

related decline in hippocampal function. In many cases, the older animals can acquire a

spatial reference memory if provided training over several days. Thus, age differences

in acquisition, which are evident in the first couple days of training, may disappear after

several days of repetitive training. Similarly, studies in mice suggest that distributed

training procedures can mask memory deficits in mice with mutations of memory related

genes, e.g. CREB, PKA, PP1 (Genoux et al., 2002; Gulinello et al., 2009; Jones et al.,

29

2001; Kogan et al., 1997; Malleret et al., 2010). On contrast, due to the fact that the

learning or/and memory deficits are more pronounced in the initial training trials, many

laboratories have massed all the trainings into a single section in order to increase the

sensitivity of the task (Commins et al., 2003; Dash et al., 2002; Lal et al., 1973; Spreng

et al., 2002; Vasquez et al., 1983). Studies using F344BNF1 rats, the same rat strain

employed in the first study (see Chapter 3), also show that distributed testing across

several days is insensitive to cognitive decline between adult (6-month-old) and aged

(21-month-old) F344BNF1 rats (Wu et al., 2004a). In contrast, using a massed training

procedure, age-related cognitive deficits were observed between middle-age (12-15

months), aged (25-27 months), and very old (35-38 months) F344BNF1 rats (Carter et

al., 2009). Due to the sensitivity of the massed training version of the task, particularly

for animals within the age range employed in the studies presented in this dissertation,

we used a spatial training protocol consisting of five blocks of three trials per block with

training massed into a single day, for examining the effect of overexpression of

antioxidant enzymes in the hippocampus on spatial learning and memory in young and

aged F344BNF1 rats.

In the current set of studies, we employed three probe trials with the platform

removed to examine acquisition and retention of spatial information. The first probe trial

(probe 1) was given after the 5th block of spatial training, in which the rat was released

from the opposite quadrant and allowed to swim for up to 60s. This probe trial is used to

examine the acquisition of a spatial search strategy. A refresher-training block is given

immediately following probe 1. The refresher-training block is used to refresh rat’s

memory of platform location and insure that the probe trial does not result in the

30

extinction of spatial memory (Lattal et al., 2003). An hour later, a retention probe trial

(probe 2) was delivered followed by another refresher-training block. This second probe

trial is used to examine the one-hour retention of newly learned spatial search strategy.

Twenty-four hours later, a block of spatial training was given followed by a probed trial

(probe 3). The rats are tested in this third probe trial for their ability to relearn the

platform location. Note that the rats are sent back to their home cages with water and

food during the one-hour and twenty-four time periods between testing.

A camera on the roof tracks the rats’ performance on every trial, and different

measurements can be made using EthoVision software. To assess the performance

during training, latency to find the platform and path length are both used in cue and

spatial training. Using latency to find the platform, as the only measure in cue and

spatial training, may be confounded by an age-related decline in swim speed. Thus, a

longer latency may reflect slower swim speed rather than impaired learning. Similarly,

measuring only path length may ignore the fact that some animals initially tend to float

in the water giving artificially short path lengths but longer latency to find the platform

during the first block of trials. With the same consideration, we also apply multiple

measures in the probe trials, including latency to first reach the platform location,

percentage time in the goal quadrant, discrimination index: (time in goal quadrant-time

in opposite quadrant)/(time in goal quadrant +time in opposite quadrant), and number of

platform crossings.

Mechanism of Brain Aging

ROS and Aging

From the preceding review it is clear that aging is associated with selective

changes in specific cognitive functions. Furthermore, the deficits appear to be selective

31

for a particular neural system involving the hippocampus. This raises the question as to

what might cause the age-related cognitive decline. One possibility is that the age-

related memory loss is due to cellular aging. A prominent theory of cellular aging, the

free-radical theory of aging, indicates that the overproduction of endogenous reactive

oxygen species (ROS), including superoxide, hydrogen peroxide, and hydroxyl radicals

result in a pattern of cumulative damage due to modification of proteins, nucleic acids,

and polyunsaturated fatty acids of the lipid membrane (Oliver et al. 1987; Smith et al.

1991; Leutner et al. 2001; Liu et al. 2002). Normally, the production of ROS can act as a

signaling mechanism in the brain (Bokoch and Knaus, 2003; Infanger et al., 2006).

However, many studies have provided evidence for increased oxidative damage during

normal aging (Halliwell, 1992; Harman, 1992), and in relation to neurodegenerative

diseases of aging, including Alzheimer’s and Parkinson’s disease (Olanow and

Arendash, 1992). Thus, aging can be viewed as the process of accumulation of

oxidative damage.

ROS are produced by a number enzymatic reactions including cytochromes p450

(CYTP450), xanthine oxidase (XOD), phospholipase A2 (PLA2), lipoxygenase (LOX)

and ribonucleotide reductase, and nicotinamide adenine dinucleotide phosphate

(NADPH) oxidase (Noxs) (Muller et al., 2007). For example, NADPH oxidase represents

a family of proteins with oxidase activity, that contain six transmembrane domains. Nox

is found primarily in neutrophils and microglia, playing a protective role in fighting

infection by releasing superoxide (Block et al., 2004; Lambeth, 2004). Superoxide, the

primary product of the Nox protein, forms when NADPH binds to Nox on the protein’s

cytosolic side, where NADPH transfers electrons to flavin adenine dinucleotide (FAD)

32

and the heme centers and finally the oxygen on the outer membrane surface (Brown

and Griendling, 2009).

In addition, a common source of superoxide is through the mitochondria as by-

products of respiration. Superoxide is generated through oxidative phosphorylation; and

forms when an electron leaks from electron transport chain and reacts with oxygen.

During normal respiration, as much as 0.4 - 4% of total oxygen consumed by the

mitochondria can be converted to the free radical superoxide (Boveris, 1984; Boveris

and Chance, 1973; Turrens and Boveris, 1980). The majority of mitochondrial

superoxide is generated from two respiratory chain complexes, complex I (NADH

dehydrogenase) and complex III (ubiquinone-cytochrome c reductase) (Raha et al.,

2000). Complex I produces superoxide in the mitochondrial matrix and complex III

produces superoxide in both the matrix and intermembrane space (Han et al., 2001;

Miwa et al., 2003; Muller et al., 2004) (See Fig. 1-1).

Superoxide can be dismutated to hydrogen peroxide spontaneously or

enzymatically via superoxide dismutase (SOD) (Fridovich, 1983b). The properties of

superoxide and hydrogen peroxide, suggest that they act differently as damaging or

signaling molecules. Because of the short half-life and fast dismutation rate, superoxide

must be produced very close to its target to be effective as a signaling or damaging

molecule. In addition, superoxide is not able to diffuse across biological membranes due

to its negative charge. In this regard, hydrogen peroxide is more stable than superoxide

and is also capable of crossing biological membranes.

Superoxide is also capable of reacting with nitric oxide (NO), forming highly

reactive and potentially damaging peroxynitrite (OONO−). The formation of OONO− from

33

superoxide can then lead to reversible glutathionylation of proteins via reactive

cysteines, as has been described for Na+-K+ pump regulation (Figtree et al., 2009).

Superoxide is also known to react with protein thiols such as cysteine residues.

Nevertheless, the reaction rate of SOD in converting O2- to H2O2 is much faster than

that of O2- with biothiols, which helps to diminish the potential for damage (Forman et

al., 2004). H2O2 also reacts with protein thiols, and although the reaction rate of O2- with

protein thiols is chemically faster than that of H2O2, the greater stability and diffusibility

of H2O2 increase its probability of reacting with the protein thiol groups involved in ROS

signaling. This suggests that physiological protein thiol oxidation is most likely H2O2

dependent. Indeed, although the production of O2- is the main biological function of Nox

proteins and is important in the bactericidal activity of Nox2, much of the signaling that

occurs is directly mediated by its dismutation product, H2O2.

Because of the presence of SOD in the cell, H2O2 is formed rapidly from Nox-

generated O2-, or in the case of Nox4, perhaps prior to the release of O2

- from the

enzyme (Dikalov et al., 2008). H2O2 is also tightly regulated biologically by catalase,

glutathione peroxidase, and peroxiredoxins, which convert H2O2 to water and other

metabolites. H2O2 can reversibly react with low pKa cysteine residues (Winterbourn and

Metodiewa, 1999) on proteins to initially form a disulfide bond (–SSR) and sulfenic acid

(–SOH). Sulfinic acid (–SO2H) and sulfonic acid (–SO3H) can be formed by additional

oxidation; however, these latter reactions are essentially irreversible, and not useful for

signaling (Barford, 2004; Forman et al., 2002).

The balance between the production and removal of ROS is a critical factor in

determining the extent of oxidative stress. ROS mediated oxidative stress can be

34

defined or measured as irreversible oxidative damage of critical biological molecules,

causing DNA strand breaks, destruction of ion transporters or other specific proteins,

and membranous lipid peroxidation (Halliwell, 1992). In this case, the term irreversible

indicates that repair of damaged molecules will require cellular cleaning

process/degradation. There are many oxidative lesions in endogenous mammalian DNA,

of which 8-hydroxyguanine (8-oxodG) is one of the most abundant (Ames, 1989).

Oxidative DNA damage can be repaired through base excision repair (BER) and

nucleotide excision repair (NER) mechanisms. BER can recognize specific lesions in

the DNA, and thus correct only damaged bases that can be removed by a specific

glycosylase (Friedberg, 1995; Seeberg et al., 1995). For example, 8-oxo-dG is

recognized by the DNA glycosylases, 8-oxoguanine glycosylase/AP lyase (Ogg1) (Xie

et al., 2004). An additional repair mechanism in the BER system involves removing dA

mispaired to 8-oxo-dG to prevent 8-oxo-dG from forming transverse G to T mutations.

NER employs a complex set of proteins that recognize large distortions in the shape of

the DNA double helix, and remove a short single-stranded DNA segment that includes

the lesion (Sancar, 1996; Wood, 1996).

Irreversible oxidation products of amino acids are most frequently observed as

hydroxylated and carbonylated amino acid derivatives and detection of protein-

associated carbonyls represents a common way of assessing protein oxidation after

carbonyl derivatization by dinitrophenyl hydrazine (Levine et al., 1994). Oxidized

proteins are generally less active, less thermo-stable and are likely to expose

hydrophobic amino acids at their surface. In addition, protein damage can result from

protein adduct formation with lipid peroxidation products such 4-hydroxy-2-nonenal

35

(HNE) and from oxidation of glycation products leading to the formation of glycoxidation

adducts such as pentosidine or carboxymethyllysine (Berlett and Stadtman, 1997).

These latter modifications often bring carbonyl groups and/or cross-links within the

protein. In the cytosol, oxidized proteins have been shown to be preferentially degraded

by the 20S proteasome in an ATP- and ubiquitin-independent manner (Davies, 2001;

Shringarpure et al., 2003). Moreover, upon oxidative stress chaperone-mediated

autophagy can participate, to accelerate degradation of oxidized proteins carrying a

KFERQ motif (Kiffin et al., 2004).

Lipid peroxidation refers to the oxidative degradation of lipids, a process in which

free radicals steal electrons from the lipids in cell membranes, resulting in cell damage.

Polyunsaturated fatty acids are the most common lipids affected by free radicals

because they contain multiple double bonds formed by methylene-CH2-groups. The

brain is particularly susceptible to free radical attack because the brain is enriched with

polyunsaturated fatty acids. In addition, the brain is low in antioxidant capacity and has

high utilization of molecular oxygen relative to other organs (Retter, 1995). Damaged

lipids cannot be repaired; rather, they must be removed and replaced. Thus, damage of

cell membranes may accumulate. In blood, damage to lipids such as cholesterol, will

accumulate along cardiovascular vessels, which is associated with atherosclerosis

(Pratico, 2001). The end product of lipid peroxidation, such as 4-hydroxy-2-nonenal

(HNE) and malondialdehyde (MDA) can also cause damage to DNA (Marnett, 1999)

and proteins (Kaemmerer et al., 2007). As a result, lipid peroxidation has been

suggested to lead to carcinogenesis (Hu et al., 2002), Alzheimer’s disease (Dei et al.,

36

2002; Montine et al., 2002) and Parkinson’s disease (Dexter et al., 1986; Gupta et al.,

2010; Kilinc et al., 1988).

Reducing-oxidizing (redox) state is a term that has historically been used to

describe the ratio of the interconvertible oxidized and reduced form of a specific redox

couple. For example, Sir Hans Krebs defined the redox state of NAD+/ NADH couple as

[free NAD+]/[ NADH] (Krebs, 1967; Krebs and Gascoyne, 1968; Krebs and Veech,

1969). In recent years, the term redox state has been used not only to describe the

state of a particular redox pair, but also to more generally describe the redox

environment of a cell. This more general use of the term redox state is not very well

defined and differs considerably from historical uses. Schafer and Buettner suggest that

the term redox environment should be used when a general description of a linked set

of redox couples is intended. Schafer and Buettner define this kind of redox state/redox

environment as:

The redox environment of a linked set of redox couples as found in a biological fluid, organelle, cell, or tissue is the summation of the products of the reduction potential and reducing capacity of the linked redox couples present.

There are many redox couples in a cell that work together to maintain the redox

environment. The redox state of the glutathione disulfide-glutathione couple, oxidized

form of GSH (GSSG) and reduced form of GSH (GSH) can serve as an important

indicator of redox environment because the GSSG/2GSH couple is the most abundant

redox couple in a cell (Schafer and Buettner, 2001).

From the above discussion it is apparent that measures of ROS levels, oxidative

damage, and redox state are essential for examining the free radical theory of brain

aging and the possible link between these processes and cognitive decline. The

37

following section will discuss different methods for measuring ROS, redox state,

oxidative damage, and antioxidant enzymes.

ROS Markers

Total ROS

Dihydro-compounds such as 2′,7′-dichlorodihydrofluorescein (DCFH) and its

derivatives, have traditionally been used for detecting oxidative stress in vivo (LeBel et

al., 1992). Two big drawbacks for using these probes including lack of specificity for

different ROS and photosensitivity which tends to autoxidize DCFH and causes high

background fluorescence (Afzal et al., 2003; Marchesi et al., 1999; Rota et al., 1999;

Soh, 2006).

Superoxide

With one unpaired electron, superoxide is a free radical, and is toxic biologically. A

number of methods have been developed to detect superoxide. Cytochrome c, lucigenin

and luminol are the most commonly used scavenger indicators, which react with

superoxide and produce detectable products. All three chemicals have drawbacks.

While cytochrome c has low sensitivity with superoxide, both lucigenin and luminol

generate superoxide as they react to produce light, which raise an objection for using

them to quantify superoxide. More recently, several florescent probes have been

developed to detect the overall level of superoxide or superoxide specifically localized in

mitochondria. Dihydroethidium (DHE) is one such superoxide-sensitive dye used to

localize superoxide in tissues (Al-Mehdi et al., 1997; Benov et al., 1998; Murakami et al.,

1998). MitoSOX-Red is a mitochondrial specific superoxide indicator and can be paired

with a mitochondria-specific dye such as MitoTracker-Green FM to detect mitochondria

produced superoxide (Hu et al., 2007a). The fluorescent probe bis (2,4-

38

dinitrobenzenesulfonyl) fluorescein, is the only probe that is highly selective to

superoxide over other ROS, and can be used to detect real-time production of

superoxide by living cells (Maeda et al., 2005). It should be noted that, although many

methods and probes have been developed to detect superoxide, many people still

doubt that the steady state concentration of superoxide can be measured directly

because of its very short half-life at 37C (1 x 10-6 seconds). As a result, the levels of

superoxide should be determined indirectly by measuring the rates of production and

removal and the relationship between these two processes; or by measuring the

damage caused by superoxide.

Hydrogen peroxide

The classical method for measuring H2O2 concentration is through directly

measuring the absorbance at 330nm of the H2O2 molecule or through reaction with

ferrous iron (Fe2+), monitored via a subsequent reaction with dye Xylenol Orange and

detected as an increase in absorbance in solution at 550nm. Several more sensitive

assays have been developed based on the horseradish peroxidase (HRP) mediated

reaction between H2O2 and some indicating reactant. For example, in the presence of

H2O2, HRP will oxidize luminol and produce light. The light output can be proportionally

compared to the H2O2 present. Regardless of the assay used, two possible sources of

error must be controlled. The first is that other redox active compounds can also create

a signal in the assay. These signals can still be detected after treating the samples with

catalase, which specifically remove H2O2. Thus, any signal lost after treating with

catalase can be assigned to H2O2. The second possible error interfering with the assay

often comes from sample with high peroxide scavenging compounds, which can

39

continue to be active after the sample preparation. It is therefore important to work

under the conditions for minimizing peroxide degradation, e.g. shorter sample storage

time, prepare and keep the samples and all reagents used in the assay at a lower

temperature.

Redox state

Glutathione is considered to be the major thiol-disulfide redox buffer of the cell

(Gilbert, 1990). On average, the GSH concentration in the cytosol is 1–11 mM (Smith et

al., 1996). This is far higher than most other redox active compounds. Measurements of

total GSH and/or GSSG levels have been used to estimate the redox environment of

cells. Many researchers estimate the redox state of the system by taking the ratio of

[GSH]/[GSSG]. This is convenient as the units divide out, so it is not necessary to

determine an absolute concentration. A measurement in mg/mg protein, arbitrary

fluorescence units, or the area under an HPLC peak can be entered into the ratio and a

useful estimate calculated (Schafer and Buettner, 2001). Based on the results from the

first study (Chapter 3), we hypothesized that overexpression of SOD1 created a more

oxidized redox environment. Therefore, in the second study (Chapter 4), GSH and

GSSG levels were measured in hippocampal tissue following overexpression of

SOD1+GFP, SOD1+CAT, or GFP to determine the redox state. The methods for

measuring GSH and GSSG are described in more detail in Chapter 2.

Oxidative damages

For studies examining ROS induced damage to macromolecules, the most

common measures related to damage of nucleotides, proteins, and lipids. The

measurement of each is discussed below.

40

Among all the lesions discovered thus far, one of the most abundant in DNA and

RNA is the 8-hydroxyguanine due to the high oxidation potential of this base relative

to cytosine, thymine, and adenine (Ames and Gold, 1991; Gajewski et al., 1990).

Besides its abundance, 8-hydroxydeoxyguanosine (8-oxodG) and 8-hydroxyguanosine

(8-oxoG) are identified as the most detrimental oxidation lesions due to their mutagenic

effect (Ames and Gold, 1991) in which this damaged molecule can pair with both

adenine and cytosine with the same efficiency (Shibutani et al., 1991). This mis-pairing

brings about the alteration of genetic information through the synthesis of DNA and

RNA. In RNA, oxidation levels are mainly estimated through 8-oxoG-based assays. So

far, approaches developed to directly measure 8-oxoG level include HPLC-based

analysis and assays employing monoclonal anti-8-oxoG antibody. The HPLC-based

method measures 8-oxoG with an electrochemical detector (ECD) and total G with

a UV detector (Weimann et al., 2002). The ratio that results from comparing the two

numbers provides the extent that the total G is oxidized. Monoclonal anti-8-oxoG mouse

antibody is broadly applied to directly detect this residue on either tissue sections or

membrane, offering a more visual way to study its distribution in tissues and in discrete

subsets of DNA or RNA.

Protein carbonyl content is the most general indicator for protein oxidation. Several

approaches have been taken to detect and quantify the carbonyl content in protein

preparation. The most convenient procedure is the reaction between 2,4-

dinitrophenylhydrazine (DNPH) and protein carbonyls. DNPH reacts with protein

carbonyls, forming a Schiff base to produce the corresponding hydrazine, which can be

analyzed spectrophotometrically (Reznick and Packer, 1994).

41

Two lipid peroxidation products are commonly used as markers for lipid damage,

4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA). HNE is formed by

peroxidation of linoleic acid. HNE is more stable than free radicals and it is able to

migrate to sites that are distant from its formation, resulting in greater damage. The

most damaging effect of HNE is its ability to form covalent adducts with histidine, lysine,

and cysteine residues in proteins, enabling a modification in the activity of proteins

(Butterfield et al., 1997). It has been shown that the HNE-modified proteins, along with

neurofibrillary tangles, are present in the senile plaques in aged dogs (Papaioannou et

al., 2001). Increased levels of HNE have also been found in Alzheimer’s and

Parkinson’s disease (Yoritaka et al., 1996; Zarkovic, 2003). These findings support the

idea that oxidative stress/damage is elevated in neurodegenerative diseases and lipid

peroxidation may contribute to the deterioration of central nervous system (CNS)

function. MDA is formed by peroxidation of arachidomic acid. MDA induces DNA

damage by reacting with amino acids in protein to form adducts that disrupt DNA base-

pairing. Increased levels of MDA have been found in the aged canine brain (Head et al.,

2002). In the aged human brain, increased levels of MDA have been found in inferior

temporal cortex and in the cytoplasm of neurons and astrocytes (Dei et al., 2002), as

well as in the hippocampus and cerebellum of aged rodents (Cini and Moretti, 1995;

Gemma et al., 2002). Lipid peroxidation from biological samples can be detected by

antibodies (Opalach et al., 2010) or through thiobarbituric acid reaction (Cui et al., 2009;

Dawn-Linsley et al., 2005).

Antioxidant enzymes

Antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT)

and glutathione peroxidase (GPx) (Halliwell, 1991; Brigelius-Flohe, 1999) are the

42

primary factors to protect neural tissue from toxic free radicals. Thus, a change in

antioxidant enzyme activity can be used as biomarkers for detecting a possible change

of ROS. Certainly, SOD activity is a major defense mechanism against oxygen toxicity

(McCord and Fridovich, 1969; Fridovich, 1983) by converting superoxide to the less

reactive hydrogen peroxide. Catalase (CAT) and glutathione peroxidase (GPx)

cooperate with SOD to decompose H2O2 to water and O2. Although age-related

changes in the antioxidant enzymatic system have been described, results on the SOD,

GPx, and CAT activities are contradictory. Total SOD activity was reported to be

unchanged during aging in whole brain from older Wistar rats (Sahoo and Chainy, 1997)

and in older Fisher 344 rats (Tian et al., 1998). Other studies reported that the activity of

SOD was decreased in cerebrum, hypothalamus, hippocampus, cerebellum, brain stem

and spinal cord with increasing age from Charles Foster rats (Gupta et al., 1991) and in

whole brain from Fischer 344 rats (Semsei et al., 1991). Catalase activity in the whole

brain was significantly decreased in aged Fisher 344 rats (Tian et al., 1998) and Wistar

rats (Sahoo and Chainy., 1997). As described above, catalase has very low activity in

the brain, and it seems to play a secondary role in converting H2O2 compared with other

organs. The activity of GPx was reported to be unchanged in the cortex (Cand and

Verdetti, 1989; Dogru-Abbasoglu et al., 1997), and in whole brain extracts (Sahoo and

Chainy, 1997) from Wistar rats; whole brain (Tian et al., 1998) and several brain regions

including hippocampus, striatum, substantia nigra, and three different cortical regions of

the cerebrum (frontal, parietotemporal and occipital regions) (Carrillo et al., 1992) from

Fischer 344 rats; however, a decrease activity during aging has also been reported

(Benzi et al., 1989). In addition, changes in the activity of antioxidant enzymes have

43

been reported to be markedly dependent on the sex of animals (Guo et al., 1993; Tam

et al., 2003; Banos et al., 2005; Ehrenbrink et al., 2006). Furthermore, one paper

suggest that it is the cooperation of antioxidant enzyme network, rather than their

individual levels that is crucial for the overall oxidant/antioxidant status during aging

process (Sobocanec et al., 2008). In conclusion, although the conflicting results may

arise from different factors like animal strains, conditions of animal maintenance, or

experimental procedures, the results still indicate that the activities of antioxidant

enzymes are age- and tissue-specific.

Antioxidant Treatments

Previous studies have shown that in both young and aged rats, dietary

supplements of fruit and vegetable extracts, with high antioxidant activity, retarded age-

related declines in neuronal and cognitive function (Joseph et al., 1999). Several studies

have also addressed the effects of antioxidant dietary supplementation on humans. The

most common antioxidants, for example, tocopherol (Vitamin E) and ascorbate (vitamin

C) are usually taken through supplementation or vitamin-enriched food. However,

vitamin E only operates under strong oxidative condition, i.e. Alzheimer’s disease,

whereas it can evolve into pro-oxidant under mild oxidative conditions (Bowry et al.

1992). Vitamin C, on the other hand, has little effect on improving cognition

(Mendelsohn et al., 1998; Grodstein et al., 2003; Maxwell et al., 2005). Vitamin C is

excreted rapidly after ingestion, and the hydrophilic character of vitamin C makes it

difficult for it to penetrate through blood brain barrier (BBB). Though some studies

indicated that vitamin C can reduce the amount of metal ions, which may down-regulate

the free radicals production from the fenton reaction (Stohs and Bagchi , 1995; Carr and

Frei, 1999). Thus, although adding antioxidant supplements or an antioxidant enriched

44

diet may seem like a relatively simple therapy, it may not act at the source of ROS in the

nervous system; as such it may have little effect on normal cognitive aging.

Different invertebrate animal models that overexpress antioxidant enzymes have

been created as tools for studying the relationship between oxidative stress and

longevity. The literature concerning whether increasing antioxidant enzymes can delay

aging is controversial. Overexpression of Cu/Zn SOD, MnSOD or catalase extend life

span in the short-lived strain Drosophila melanogaster but, the same phenomena was

not observed in a long-lived strain (Mockett et al., 2003; Orr et al., 2003; Orr and Sohal,

1993, 1994; Sun et al., 2002). Overexpression of catalase, targeted to mitochondria,

extends life span in mice (Schriner et al., 2005). Administration of synthetic superoxide

dismutase/ catalase mimetics have been shown to extend the life span of

Caenorhabditis elegans (Melov et al., 2000; Sampayo et al., 2003a; Sampayo et al.,

2003b), but the effect may depend on the experimental conditions (Keaney and Gems,

2003; Keaney et al., 2004).

The hypothesis that age-related cognitive decline could be reversed by increasing

antioxidant defenses has been tested in transgenic mouse models expressing different

forms of SOD. Relative to wild-type controls, the enzymatic activity in the brain of

transgenic mice represents a 6-fold increase for SOD1 (Kamsler and Segal, 2003b), a

2.3-fold increase for SOD2 (Maragos et al., 2000), and about a 10-fold increase for

SOD3 (Thiels et al., 2000). Depending on the type of SOD examined, genetic

manipulations of SOD expression can have a profound effect on age-related cognitive

impairment, and an age-dependent influence on synaptic plasticity. Young adult mice

overexpressing SOD1 or extracellular SOD exhibit impairment in long-term potentiation

45

(LTP), a form of synaptic plasticity that is thought to be involved in memory. These mice

also exhibit impaired hippocampus-dependent memory (Gahtan et al., 1998; Kamsler

and Segal, 2003b; Levin et al., 1998; Thiels et al., 2000). In contrast, aged transgenic

mice over expressing SOD1 or SOD3 exhibit improved LTP and SOD3 transgenic mice

exhibit better performance in the Morris water maze compared with wild-type littermates

(Kamsler and Segal, 2003b; Levin et al., 2002). Up regulation of SOD2, on the other

hand, has no obvious effect on hippocampal LTP (Hu et al., 2007a). Taken together,

these results indicate that overexpression of SOD1 and SOD3 affect synaptic plasticity

and memory in an age-dependent fashion (See Table 1-1 for a summary of antioxidant

enzymes studies in mice).

Viral Vectors Delivery in Central Nervous System (CNS)

Adeno-associated virus (AAV) vectors have been utilized in pre-clinical and clinical

gene delivery studies, primarily because the vectors can transduce non-dividing cells

and confer long-term stable gene expression, without associated inflammation or

toxicity (Bessis et al., 2004; Goncalves, 2005; Haberman et al., 1998). AAV is a small,

icosahedral, nonenveloped virus that belongs to Parvoviridae family. The name

“associated” came from the fact that Adenovirus is usually served as helper virus for a

productive infection of AAV to occur. The wild AAV has a linear single-stranded DNA

genome of about 4.7 kb of either plus or minus polarity. There are two inverted terminal

repeats (ITRs) flanking the two viral genes, rep (replication) and cap (capsid). The rep

gene encodes four regulatory proteins (Rep78, Rep68, Rep52 and Rep40) required for

the AAV life cycle. The cap gene encodes three structural proteins (VP1, VP2 and VP3)

to form a capsid that is approximately 22 nm. In our studies, we used recombinant AAV

(rAAV) to deliver antioxidant enzymes to the hippocampus. rAAV does not contain any

46

AAV rep and cap genes. Rather, rep and cap genes are replaced with a gene or

construct of interest, which is flanked by the ITRs containing all the cis-acting elements

necessary for replication and packaging. Use of rAAV does have some disadvantage.

The cloning capacity of the vector is relatively limited. Efficient packaging of rAAV can

be performed with constructs ranging from 4.1 kb to 4.9 kb in size. Large genes are,

therefore, not suitable for use in a standard rAAV vector.

There have been at least 11 AAV capsid serotypes described. The surface of the

AAV capsid is involved in cell binding, internalization, and trafficking within the targeted

cell; therefore, each capsid exhibits a unique tissue tropism and transduction efficiency

(Van Vliet et al., 2008). Serotype 2 (AAV2) has been the most extensively examined

(Bartlett et al., 1998; Burger et al., 2004; Fischer et al., 2003; Nicklin et al., 2001). AAV2

is used to deliver genes to cells in CNS (Flannery et al., 1997; Kaplitt et al., 1994;

McCown, 2005, 2011; McCown et al., 1996) and primarily transduces neurons (Bartlett

et al., 1998; Burger et al., 2004; Klein et al., 1998) through the interaction of AAV2

capsid proteins with heparin sulphate proteoglycan (HSPG) moieties on the neuronal

cell surface. In our current studies, we used pseudotyped rAAV2/5 (putting the genome

of rAAV2 into the capsid of rAAV5) because rAAV2/5 has been shown to transduce

hippocampus with high efficiency in CA1-CA3 and the dentate gyrus (Burger et al.,

2004). We also take the advantage of AAV that can affect various genes simultaneously

by using multiple vectors (Mandel et al., 1998; Muramatsu et al., 2002; Rendahl et al.,

1998; Shen et al., 2000). We transduced hippocampal neurons with two separate AAV

vectors to overexpress SOD1 and CAT or GFP simultaneously in the same cell.

47

Lentiviruses are a subclass of Retroviruses, developed based on the human

immunodeficiency virus (HIV). Unlike other retroviruses that can only infect dividing cells,

lentivirus has the capacity to integrate into the genome of non-dividing cells, which

makes it an attractive CNS gene transfer tool (Blomer et al., 1997; Naldini et al., 1996a).

Lentivirus has relatively large cloning capacity compared to AAV. The capacity is

generally considered to be 8-10 kb (Naldini et al., 1996b; Sinn et al., 2005).

A process known as pseudotyping can modify the host range of lentiviral vectors.

Psudotyped lentiviral vectors consist of vector particles carrying glycoproteins derived

form other enveloped viruses. Such particle has the tropism of the virus from which the

glycoproteins were obtained. For our initial studies, we chose the most widely used

glycoproteins for pseudotyping lentiviral vector, the vesicular stomatitis virus

glycoproteins (VSV-G) (Barsoum et al., 1997; Naldini et al., 1996b; Reiser et al., 1996;

Russell and Cosset, 1999). VSV-G pseudotyped lentiviral vector has been shown to

have broader transduction range (Coil and Miller, 2004), increased transduction

efficiency (Barsoum et al., 1997), and stronger viral particles to make concentration by

ultracentrifugation possible (Burns et al., 1993). However, the virion transport of VSV-G

pesudotyped lentivirus is limited to relatively shorter distance (Akli et al., 1993; Alisky et

al., 2000). Lentivirus has been demonstrated to transduce most cell types in CNS in

vivo, including neurons, astrocytes, adult neuronal stem cells, oligodendrocytes, and

glioma cells with desired cell type promoters or modified envelop proteins that bind to

the receptors only found in the desired cell type (Blomer et al., 1997; Consiglio et al.,

2004; Geraerts et al., 2006; Jakobsson et al., 2003; Miletic et al., 2004). For safety

reasons, lentivirus never carries the genes for replication (Dull et al., 1998; Zufferey et

48

al., 1998; Zufferey et al., 1997). As a result, to produce a lentivirus, several plasmids

need to be transfected into a packaging cell; including the packaging plasmids that

encode virion proteins, such as the capsid and the reverse transcriptase and the

plasmid contains genetic material (See Chapter 2 for SOD1 lentiviral vector packaging,

concentration and titration).

49

Figure 1-1. Mitochondrial respiratory chain and superoxide production. The mitochondrial respiratory chain consists of five enzyme complexes (complexes I-V) and two intermediary substrates (coenzyme Q and cytochrome c). Electrons donated from NADH and FADH donate electrons to complex I and complex II, respectively were then passed to coenzyme Q, complex III, cytochrome c and finally complex IV. In complex IV, electrons mediate the combination of hydrogen ions and molecular oxygen to form water. With the movement of electrons, hydrogen ions are pumped from the matrix to the intermembrane place in Complex I, III and IV to generate an electrochemical gradient. The complex V, also called ATP synthase then uses the electrochemical gradient to generate ATP from ADP and inorganic phosphate (Pi). Superoxide is generated through oxidative phosphorylation; and forms when an electron leaks from electron transport chain and reacts with oxygen. During normal respiration, as much as 0.4 - 4% of total oxygen consumed by the mitochondria can be converted to the free radical superoxide. The majority of mitochondrial superoxide is generated from complex I and complex III. Complex I produces superoxide in the mitochondrial matrix and complex III produces superoxide in both the matrix and intermembrane space.

50

Table 1-1. Studies using knock out or overexpression of antioxidant enzymes Gene symbol

Gene name (localization)

Cognition Life span

Main antioxidant function

Reference

Sod1-/- CuZn superoxide dismutase (C, M)

Not reported

Mutiple pathologies, ~30% shortened life span

Scavenger of O2

-

Elchuri et al., 2005

Sod2-/- Mn superoxide dismutase (M)

Not reported

Neonatal lethal Scavenger of O2

- Li et al., 1995

Sod3-/- Extracellular superoxide dismutase (E)

Not reported

“Normal,” no reduction in life span

Scavenger of O2

- Sentman et al., 2006

Cat-/- Catalase (C) Not reported

“Normal,” life span not reported

Scavenges H2O2

Ho et al. 2004

Gpx1-/- Glutathione peroxidase 1 (C)

Not reported

“Normal,” life span not reported

Scavenges H2O2

Ho et al. 1997

Gpx2-/- Glutathione peroxidase 2 (E)

Not reported

“Normal,” life span not reported

Scavenges H2O2

Esworthy et al. 2001

Gpx4-/- Phospholipid glutathione peroxidase (M, C)

Not reported

Embryonic lethal Scavenges H2O2

Yant et al., 2003

Tg-SOD1 CuZn superoxide dismutase (C, M)

Impaired in young Enhanced in old

“Normal,” does not extend life span

Scavenger of O2

- Gahtan et al., 1998; Kamsler and Segal, 2003; Kamsler et al., 2007; Huang et al., 2000

Tg-SOD2 Mn superoxide dismutase (M)

No effect Growth retardation and decreased fertility, life span not reported

Scavenger of O2

- Hu et al., 2007; Raineri et al., 2001

Tg-SOD3 Extracellular superoxide dismutase (E)

Impaired in young Enhanced in old

Not reported Scavenger of O2

- Thiels et al., 2000; Levin et al., 1998; Hu et al., 2007; Levin et al., 2002;

MCAT; Tg-CAT

Catalase (M); Catalase (Peroxision)

Not reported

Extend medium and maximal life span (MCAT) Does not extend maximal life span (PCAT)

Scavenges H2O2

Schrier et al., 2005; Chen et al., 2004

51

CHAPTER 2

MATERIALS & METHODS

Animals

All treatments were approved by the Institutional Animal Care and Use Committee

and were in accordance with guidelines established by the United States Public Health

Service Policy on Human Care and Use of Laboratory Animals. Young tg-SOD1 mice

exhibit impaired spatial learning (Gahtan et al., 1998); therefore, young rats were

included to examine age effects and specificity of gene overexpression in the first study.

In the first study, AAV was injected into the hippocampus of young (4-month) and aged

(19-month) male Fischer 344/Brown Norway F1 rats (Harlan Laboratories) in order to

express SOD1 (young: n = 14; aged: n = 13), SOD2 (young: n = 9; aged: n = 9), CAT

(young: n = 10; aged: n = 9), mix of SOD1+CAT (young: n = 11; aged: n = 12) or GFP

(young: n = 8; aged: n=8). We also included young (n = 4) and aged (n = 4) no surgery

controls. No behavioral difference was observed between GFP and no surgery controls;

therefore, these animals were combined for behavioral analyses. In the second study,

AAV was stereotactically injected bilaterally into dorsal hippocampi of 17-month old

male Fischer 344 rats (Harlan Laboratories). Rats were injected with GFP as control (n

= 10); SOD1+GFP (n = 6); or SOD1+CAT (n = 7). In the second study, AAV containing

antioxidant enzymes genes were stereotactically injected bilaterally into dorsal

hippocampi of 17-month old male Fischer 344 rats. Rats were injected with GFP as

control (n = 14); SOD1+GFP (n = 11); or SOD1+CAT (n = 12). Behavioral testing in the

water maze was initiated 1 month following virus injections.

52

AAV Viral Vector

Virus particles were produced and quantified by dot blot analysis, through the

Powell Gene Therapy Center at the University of Florida (Gainesville, FL). Human

SOD1 gene with a c-terminal myc tag (myc) was cloned into, self-complementary AAV

(scAAV), containing a cytomegalovirus/chicken β-actin promoter with a

cytomegalovirus-immediate early (CMV-IE) enhancer (Figure 2-1). Plasmids pTR-UF11-

hSOD2 and pTR-UF11-hCAT were gifts from Dr. Nicolas Muzyczka (Department of

Molecular Genetics and Microbiology, University of Florida). These plasmids express

the human SOD2 gene, which codes for Mn-superoxide dismutase, or the human CAT,

which encodes catalase, respectively (Figure 2-2 and 2-3). Plasmid UF11-GFP was

used as control vector. Expression of each gene was driven by the chicken β-actin

proximal promoter and the CMV immediate early enhancer (CBA promoter). All vectors

contain AAV2 inverted terminal repeats for packaging in pseudotyped AAV5 capsid.

Rats were anesthetized with ketamine/xylazine (90/10 mg/kg) and virus was

stereotactically injected at 2 sites bilaterally in the hippocampus using glass pipettes.

Each injection consisted of 2 μL of GFP (dot blot titer: 1.02 x 1013 vg/mL), SOD1 (dot

blot titer: 1.14 x 1013 vg/mL), SOD2 (dot blot titer: 6.99 x 1012 vg/mL), CAT (dot blot

titer: 2.53 x 1013 vg/mL), or 3 uL 2:1 mix of SOD1 and CAT.

Construction of Lentiviral Vector and Vector Packaging

pTYF-EF1-CuZnSOD-IRES-GFP. The open reading frame encoding the human

Cu/Zn SOD (SOD1) was amplified from cDNA clone using primers that introduced NheI

and ClaI sites on the 5`- and 3`-ends of the coding region ( 5’t a a g c t a g c t t t g c g t c

g t a g t c t c c t g c 3’ and 5’t a t a t a t c g a t a a g g g a a t g t t t a t t g g g c g 3’). The

resulting 567-base pair SOD1 PCR products were subcloned into TOPO TA cloning

53

vector. The human SOD1 cDNA was removed from TOPO vector and ligated into pTYF-

EF1a-IRES-eGFP linker vector by NheI and ClaI to create pTYF-EF1-CuZnSOD-IRES-

GFP (LV-SOD1/eGFP).

SOD1 Lentiviral Vector Packaging, Concentration and Titration

SOD1 transducing vectors were packaged into vesicular stomatitis virus envelop G

(VSV G)-pseudotyped vector to make lentivirus using a three plasmid packaging system.

The 293 FT cells (Invitrogen, no. R70007) were plated at a density of 3 x 107 cells in

T225 flasks and were transfected the next day (80-90 % cell confluence) with the three

plasmid vectors, pNHP, pHEF-VSVG and the SOD-1 transducing vector using

Superfect (Qiagen). Media containing virus were collected at 30 hours and 45 hours

post-transfection. The medium collected 30 hours after transfection were centrifuged at

2000 rpm for 5min at 4 °C to remove cell debris and stored at 4 °C until the next day.

The medium collected 45 hours after transfection were similarly centrifuged, mixed with

the 30 hour sample and filtered through a 0.45 um low-protein-binding durapore filter

device (Millipore). All but 26 mL of the filtrate were centrifuged at 2500 rcf for 1 h at 4 °C.

The retentate recovered from the column were mixed with the 26 mL of unconcentrated

medium, loaded into a 30 mL conical polyallomer tube (Beckman), and centrifuged at

24,000 rpm in a SW28 rotor (Beckman) for 1.5 hr at 4°C. The resulting viral pellet was

overlayed with DMEM and stored at 4 °C overnight. The next day, the viral solution was

aliquoted and stored at -80 °C until use. Particle titers were determined using a HIV p24

ELISA kit (Cell biolabs, no. VPK-108-HIV p24), according to the protocols provided.

Biological titration was determined by transfecting HeLa cells with serial diluted

lentivirus, and counting the cells expressing GFP by flow cytometry.

54

Behavior Testing

Methods for water maze testing have been published previously (Foster et al.,

2001). Rats were first trained to find a visible platform using 15 trials separated to 5

blocks. Rats that did not reach the platform within 60 seconds on all trials during the 5th

block were removed from the experiment. Standard water maze testing across several

days is insensitive to cognitive decline across this age span in Fischer 344/Brown

Norway F1 rats (Wu et al., 2004b). Therefore, the task difficulty was increased by

employing a single day massed training protocol, which is sensitive to age (Carter et al.,

2009). Spatial discrimination testing occurred three days later and consisted of 18 trials

separated into 6 blocks. A free swim probe trial was inserted between blocks 5 and 6.

For the probe trial, the number of platform crossings was counted and the time spent in

both goal and opposite quadrants was recorded.

The water maze protocol used in the second study was similar to the one used in

the first study but with some modifications. Rats were first trained to find a visible

platform using 15 trials separated to five blocks (Table 2-5). Rats that did not reach the

platform within 60 s on more than one trial during the fifth block were removed from the

experiment. Four days later, a single-day massed training protocol, which is sensitive to

age was used to test spatial discrimination (Table 2-6). The spatial training protocol

consists 15 trials separated into five blocks. A free swim probe trial with the platform

removed (probe 1) was inserted between blocks 5 and 6. For the probe trial, the number

of platform crossings was counted and the time spent in both goal and opposite

quadrants was recorded. A refresher training block was given following probe 1. An

hour later, a retention probe trial (probe 2) was delivered followed by a refresher training

block. Twenty-four hours later, the rats were tested for their ability to relearn the

55

platform location. A single block of spatial training was given followed by a probed trial

(probe 3). Note that the rats were sent back to their home cages with water and food

during the one-hour and twenty-four time periods.

Hippocampal Tissue Dissection

Starting one week after finishing behavioral testing, animals were euthanized

using isoflurane (Webster, Sterling, MA) and decapitated with a guillotine (Myneurolab,

St Louis, MO). The brains were rapidly removed and transferred to a beaker containing

ice-cold artificial cerebrospinal fluid (ACSF). For biochemical studies, the hippocampi

were removed and flash frozen in liquid nitrogen. The samples were then stored at –

80C until further processing. For the animals used in electrophysiology, one

hippocampus from each animal was flash frozen in liquid nitrogen and stored at –80C

for biochemical analysis and the other hippocampus from the same animal was

dissected out and prepared for electrophysiological recording (see slice preparation).

Western Immunoblotting

Preparation of Lysate from Hippocampal Tissue

The hippocampi were frozen in liquid nitrogen, and stored at -80°C. Hippocampal

tissues were grinded into powder by mortar and pestle with liquid nitrogen. Half of the

tissue powder was homogenized with lysis buffer [Radioimmunoprecipitation assay

buffer (RIPA buffer) supplemented with protease inhibitor, phosphatase inhibitor and

EDTA (Thermo scientific, Rockford, IL)] and incubated for 1 hour on ice with intermittent

vortexing. The remaining tissue powder was gathered in 1.5 mL tube and stored in -

80°C. The lysates were centrifuged at 20,000 g for 30 minutes at 4 °C.

56

Determination of Protein Concentration

The lysates were first diluted 1/20 in a total of 50 L of ddH2O, and were used to

measure protein content using a BCA kit (Pierce, Rockford, IL). The protein

concentration was measured on microplate reader (Bio-Rad Laboratories, Hercules, CA)

at 560 nm. The samples were diluted with lysis buffer to the same protein concentration.

The samples were then mixed with 4X LaemmLi’s SDS-sample buffer [Tris-HCL (250

mM, pH 6.8), SDS (8%), glycerol (40%), -mercaptoethonol (8%) and bromophenol

(0.02%), Boston BioProducts, Ashland, MA] and heated to 100 °C for 5 minutes .

Electrophoresis

Samples (20 g/lane) were loaded on 4-15 % gradient polyacrylamide gels (Bio-

Rad Laboratories, Hercules, CA) and ran at 90 Volts in electrophoresis buffer (25 mM

Tris, 192 mM glycine, 0.1% SDS, pH 8.3, Bio-Rad Laboratories, Hercules, CA) until the

blue indicator line reached the bottom of the gel.

Transfer of Protein

Proteins were transferred to PVDF membranes (GE Healthcare) overnight at 50

Volts, 4 °C in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol). Blots

were then stained with Ponceau S and examined for transfer quality (e.g. roughly how

much protein was transferred to the membrane, verifying no air bubbles, or other signs

of transfer failure were present).

Blotting

Blots were washed 5 min in TBST and subsequently blocked in 5% milk in TBST

for 1 hour. Primary antibodies were then applied to blots overnight at 4 °C (Table 2-1

for a list of all antibodies used), washed 3 times (10 min each time) with TBST, and

secondary antibodies applied for 1~2 hours at room temperature. Blots were again

57

washed 4 times (10 min each time) with TBST and developed using Amersham ECL

Plus Western Blot Detection Kit (GE Health Care) on biomax film (Kodak). Blots were

scanned using 6500 scanner (Bio-Rad), and densitometry determined using Image J

Stripping

Stripping of the membranes allowed us to re-probe with different antibodies, thus

avoiding inconsistent sample loading between each electrophoresis blotting, and

conserved the samples for other assays. Typically, the blot was first probed with

antibody with lowest level of signal. This blot was then stripped, blocked with 5% milk in

TBST and re-probed with another primary antibody made from different species. For

example, if the first blot was probed with mouse antibody, then the second blot would be

re-probed with antibody made from another species rather than mouse. This prevented

using of the same secondary antibody in the first and second blotting, which may result

to additional signals from the residual primary antibody after stripping. The blot was then

stripped again and re-probed with either a third antibody or with anti-GAPDH antibody

as loading control. Each membrane was stripped for up to 3 times.

A good stripping should remove the antibodies, but leave transferred proteins. The

recipe for stripping buffer varied from simple as 0.2~0.5 M NaOH or 0.2 M glycine (HCl)

in ddH2O to a more complex buffer adding SDS, Tris, Tween 20, and -mercaptoethanol.

The tripping process can be done at room temperature or at 37C for better results. The

stripping buffer used in current studies was from Thermo Scientific. The membrane was

incubated with the stripping buffer for 15 min at 37C, and 3X washed with TBST before

blocked with 5% milk in TBST.

58

Immunofluorescence

Brains were postfixed in 4% paraformaldehyde followed by 30% sucrose in PBS

(4°C). Sections (10-20 μm) were incubated with primary antibodies overnight at 4°C.

(See Table 2-1 for a list of primary antibodies used in western blot and

immunofluorescence). The brain sections were then washed and incubated in

corresponding Alexa 488 or 594 secondary antibodies (Molecular Probes, Eugene, OR)

for 1 hour at room temperature. Sections were washed and counterstained with 4'-6-

Diamidino-2-phenylindole (DAPI) solution (0.1 μg/mL in PBS) before mounting.

Expression was confirmed using fluorescent microscopy (Zeiss Axioplan 2 upright

fluorescent microscope, equipped with a QImaging Retiga 4000R Camera with RGB-

HM-5 Color Filter and QImaging QCapture Pro 6.0 software; QImaging Surrey, BC

Canada). No primary antibody control and isotype control were made to be compared

with SOD1 and c-myc staining. In no primary antibody control, the tissue was incubated

with antibody diluent (3% goat serum in PBS), without the primary antibody included.

The tissue was then incubated with secondary antibody and DAPI before mounting. In

isotype control, the tissue was incubated with antibody diluent, supplemented with a

non-immune immunoglobulin of the same isotype (for example, IgG2a for c-myc control)

and concentration as the primary antibody. The tissue was then incubated with the

secondary antibody and DAPI before mounting. The isotype control helped ensure that

what appeared to be specific staining was not caused by non-specific interactions of

immunoglobulin molecules with the sample.

Slice Preparation

For physiology experiments, one animal per day was killed, staring were one week

after completing the water maze testing. Recordings were obtained from hippocampi of

59

aged, SOD1+GFP, SOD1+CAT or GFP transduced animals. Rats were anesthetized

with isoflurane (Halocarbon Laboratories, River Edge, NJ) and the hippocampi were

collected from rats after decapitation. Transverse hippocampal slices (400 m) were cut

parallel to the alvear fibers using a tissue chopper. Slices were maintained in an

interface chamber (Harvard Apparatus, Boston, MA) at 30 °C and perfused with an

oxygenated artificial cerebrospinal fluid (ACSF) (in mM: 124.0 NaCl, 2.0 KCl, 1.25

KH2PO4, 1.5 MgSO4, 2.4 CaCl2, 26.0 NaHCO3, and 10 glucose. Slices were permitted

to recover for 120 min before recording. Extracellular field excitatory post synaptic

potentials (fEPSPs) were recorded from Schaffer collateral-CA1 synapses using glass

micropipettes (4–6 MΩ) filled with recording medium (ACSF). Two concentric bipolar

stimulating electrodes (outer pole: stainless steel, 200 m diameter; inner pole:

platinum/iridium, 25 m diameter, FHC, Bowdoinham, ME) were positioned

approximately 1 mm from either side of the recording electrode localized in the middle

of the stratum radiatum.

Baseline fEPSP responses to stimuli of a given intensity (input-output curves)

were measured to examine basal synaptic transmission at the Schaffer collateral CA3–

CA1 synapse in hippocampal slices. Stimulus intensity was from 0 V to 28 V when

examining the total fEPSP, and from 0 V to 32 V when examining the NMDAR-mediated

fEPSP in order to achieve the maximum response. To obtain the NMDAR-mediated

fEPSP, slices were incubated in ACSF containing low extracellular Mg2+, (0.5 mM),

AMPA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 30 M), and GABA

receptor antagonist, picrotoxin (PTX, 10 M).

60

For induction of LTP, the stimulus current was set to produce a response 50–60%

of the maximal fEPSP slope (from input-output curve). After stable baseline recording at

0.033 Hz for at least 20 min, high-frequency stimulation (HFS) was delivered to the

pathway with 4 episodes of 100 Hz (each episode was 1 sec with an inter episode time

of 1 sec) at the baseline stimulus current, and recorded for 60 min after HFS. A

simultaneously recorded control (non-HFS) pathway received the test stimulus but not

the HFS.

The signals were amplified, filtered between 1 Hz and 1 kHz, and stored for off-line

analysis. For analysis, two cursors were placed around the initial descending phase of

the EPSP waveform, and the maximum slope (mV/ms) of the EPSP was determined by

a computer algorithm that found the maximum change across all sets of 20

consecutively recorded points (20 kHz sampling rate) between the two cursors.

Biochemical Assays

SOD Activity

SOD activity was measured using the HT superoxide dismutase assay kit

(Trevigen Inc, Gaithersburg, MD) according to the manufacturer’s instructions.

Protein Carbonyls

Protein carbonyls were measured using a commercial ELISA (Zentech PC Test,

Protein Carbonyl Enzyme Immuno-Assay Kit, Biocell Corp, Papatoetoe, NZ) according

to the manufacturer’s instructions.

8-oxodGuo

8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodGuo) levels were determined

according to the neutral guanidine isothiocyanate (GTC) method previously described

(Cui et al., 2009; Hofer et al., 2006). Briefly, after homogenization in GTC in the

61

presence of the metal chelator deferoxamine mesylate (DFOM; Sigma), proteins and

lipids were removed using organic solvents and centrifugation. After salt/isopropanol

precipitation of nucleic acid at 80 °C and washing in 70% ethanol, nucleic acids were

dissolved in 30 μM DFOM and hydrolyzed using 4 U Nuclease P1 (MP Biomedicals,

Irvine, CA) and 5 U alkaline phosphatase (Sigma) in 30 mM sodium acetate, 20 μM

ZnCl2, pH 5.3, at 50 °C for 60 min. After filtration to remove enzymes, nucleosides were

separated using HPLC-EC-UV and analyzed for dGuo and 8-oxodGuo

electrochemically using a Coulochem III detector from ESA Inc. (Chelmsford, MA).

HPLC peaks were quantified against daily made calibration curves of standards from

Sigma and Calbiochem (San Diego, CA).

Determination of GSH and GSSG

A glutathione fluorescent detection kit (Arbor Assays LLC, Ann Arbor, MI) was

used to measure reduced/oxidized glutathione (GSH/GSSG) ratio in rat hippocampus

samples. Rat hippocampi were frozen by liquid nitrogen and stored in -80 C until use.

The tissues were homogenized in ice cold RIPA buffer supplemented with protease

inhibitor, phosphatase inhibitor and EDTA (Thermo scientific, Rockford, IL), and

centrifuged at 14,000 rpm for 10 minutes at 4°C. An aliquot of the supernatant was

removed for protein determination. A second aliquot of the supernatant containing 50 g

of protein was precipitated with an equal volume of ice cold 5% (w/v) 5-sulfosalicylic

acid (SSA) solution. After 10 minutes incubation with SSA at 4°C, the samples were

centrifuged at 14,000 rpm for 10 minutes at 4°C to remove precipitated protein. The

supernatant was collected and diluted with assay buffer to reduce the SSA

concentration to 1%. After mixing the sample or standard with a proprietary

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nonfluorescent molecule, ThioStar®, that covalently binds to the free thiol group on

GSH to yield a highly fluorescent product, the samples were incubated at room

temperature for 15 minutes, and the fluorescent product was read at 510 nm on a

fluorescent plate reader with excitation at 370-410 nm. Free glutathione, GSH, was read

first, followed by addition of a reaction mixture that converts all the oxidized glutathione,

GSSG, into free GSH, which then reacts with the excess ThioStar® to yield the signal

related to total GSH content. The concentration of GSSG was estimated by subtracting

the measured free GSH from the measured total GSH.

Determination of Glutathione Peroxidase Activity

Glutathione peroxidase (GPx) activity was measured by using a glutathione

peroxidase assay kit (Cayman, Ann Arbor, MI). Briefly, frozen rat hippocampal tissues

were homogenized in cold RIPA buffer supplemented with protease inhibitor,

phosphatase inhibitor and EDTA (Thermo scientific, Rockford, IL), and centrifuged at

10,000 g for 15 minutes at 4°C. The supernatants were removed for analyses. The 20

uL samples containing 40 μg total proteins were added to a solution containing GSH,

glutathione reductase, and nicotinamide adenine dinucleotide phosphate (NADPH). The

reaction was initiated by adding substrate cumene hydroperoxide (final concentration:

0.22 mM), and the reduction was recorded at 340 nm using a kinetic program (6

readings at 1-minute intervals). The GPx activity was determined by the rate of

decrease in absorbance at 340 nm. The GPx activity is calculated using the following

formula: GPx activity = (A340/min)/(0.00373 M-1) x 0.19 mL/0.02 mL =

nmol/min/mL/20 μg protein.

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Determination of Glutathione Reductase Activity

A glutathione reductase (GR) fluorescent activity kit (Arbor Assays LLC, Ann Arbor,

MI) was used to measure GR activity in rat hippocampus samples. Rat hippocampi

were frozen by liquid nitrogen and stored in -80 C until use. The tissues were

homogenized in ice cold RIPA buffer supplemented with protease inhibitor,

phosphatase inhibitor and EDTA (Thermo scientific, Rockford, IL), and centrifuged at

14,000 rpm for 10 minutes at 4°C. An aliquot of the supernatant was removed for

protein determination. 25 L of GR standards or 25 L of samples containing 50 g of

protein were added to the 96-well plate followed by addition of 15 L nonfluorescent

molecule, ThioStar®, that covalently bind to the free thiol group on GSH to yield a highly

fluorescent product. The plate was incubated at room temperature for 5 minutes, and

the fluorescent product was read at 510 nm in a fluorescent plate reader with excitation

at 370-410 nm. This data was used to subtract any background thiol signal in samples.

By adding the oxidized glutathione, GSSG and NADPH to the samples, the GR activity

of the sample was determined by measuring the amount of GSH generated from the

reduction of GSSG by reacting the GSH with ThioStar® to covalently bind the free thiol

group on GSH and yield a highly fluorescent product, which was used to compared to

the readings from standards.

Statistics

All statistical analyses were performed using StatView 5.0 (SAS Institute).

Analyses of variance (ANOVAs) were used to establish main effects and interactions.

Follow-up ANOVAs and/or Fisher’s protected least significant difference (PLSD) post

hoc comparisons with p < 0.05 were employed to determine specific differences.

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Student t tests were used to determine whether quadrant search behavior was different

than that expected by chance. Western blots were analyzed using Image J (National

Institutes of Health), a Java-based image processing program for densitometry; data

significant at *p<0.05. Data from western blots were analyzed using an ANOVA. Post-

hoc analysis was determined using Fisher’s PLSD test. All graphs were produced in

GraphPad Prism software (GraphPad Prism Inc).

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Figure 2-1. Map of sc-trs-smCBA-Human SOD1-myc (SOD1)

66

Figure 2-2. Map of pTR-UF11-Human SOD2 (SOD2)

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Figure 2-3. Map of pTR-UF11-Human CAT (CAT)

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Figure 2-4. Cue discrimination

69

Figure 2-5. Spatial and probe discrimination

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Table 2-1. A list of all primary antibodies used in Chapter 3 and Chapter 4 western blot and immune-fluorescence.

Antibody Concentration Host Clonality Company

CAT 1:700 Mouse Monoclonal Abnova

CAT (Immuno-Fluo) 1:500 Rabbit Polyclonal abcam

COX (Immuno-Fluo) 1:300 Mouse Monoclonal Invitrogen

GAPDH 1:6000 Mouse Monoclonal EnCor Biotechnology

GFAP (Immuno-Fluo) 1:500 Rabbit Polyclonal DakoCytomation

GFP 1:2000 Rabbit Polyclonal Invitrogen

GPx1 1:600 Rabbit Polyclonal Abcam

GPx4 1:1000 Rabbit Polyclonal Abcam

HNE 1:100 Mouse Monoclonal Abcam

Iba1 (Immuno-Fluo) 1-2 g/mL Rabbit Polyclonal Wako

MAP2 (Immuno-Fluo) 1:10,000 Chicken Polyclonal Abcam

Myc-tag 1:1000 Mouse Monoclonal Cell Signaling

Myc-tag (Immuno-Fluo) 1:2000 Mouse Monoclonal Cell Signaling

NeuN (Immuno-Fluo) 1:1000 Mouse Monoclonal Chemicon/Millipore

P-GSH 1:1000 Mouse Monoclonal Arbor Assays

SOD1 1:5000 Rabbit Polyclonal Abcam

SOD1 (Immuno-Fluo) 1:1000 Rabbit Polyclonal Abcam

SOD2 1:6000 Rabbit Polyclonal Abcam

SOD2 (Immuno-Fluo) 1:500 Rabbit Polyclonal Abcam

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CHAPTER 3 INFLUENCE OF VIRAL VECTOR-MEDIATED DELIVERY OF SUPEROXIDE

DISMUTASE AND CATALASE TO THE HIPPOCAMPUS ON SPATIAL LEARNING, MEMORY DURING AGING

The brain is highly sensitive to oxidative stress (Halliwell, 1992), and the

accumulation of damaged molecules may contribute to age-related memory

impairments (Butterfield and Sultana, 2007; Forster et al., 1996; Knoferle et al., 2010;

Navarro et al., 2002; Nicolle et al., 2001). Antioxidant molecules and enzymes balance

the biological activity of reactive oxygen species (ROS), superoxide (O2·-) and hydrogen

peroxide (H2O2). Superoxide dismutase (SOD) catalyzes O2·- into H2O2 and catalase

(CAT) or glutathione peroxidase (GPx) converts H2O2 to water and oxygen. SODs are

classified according to their metal cofactors and cellular localization. The Cu/Zn-SOD1

(SOD1) is distributed throughout the cytoplasm, nucleus, and inner membrane space of

mitochondria, Cu/Zn-SOD3 (SOD3) is located in the extracellular space, and Mn-SOD

(SOD2) is restricted to the mitochondrial matrix (Chang et al., 1988; Fridovich, 1983a;

Keller et al., 1991).

SOD1 overexpression differentially influences brain function over the course of

aging (Hu et al., 2007b). Enhanced long-term potentiation (LTP) and spatial memory are

observed in aged transgenic SOD1 (tg-SOD1) mice (Kamsler et al., 2007; Kamsler and

Segal, 2003b). In contrast, tg-SOD2 mice do not exhibit altered synaptic plasticity or

memory (Hu et al., 2007b). It is unclear if increased SOD1 activity is required over the

lifespan in order to prevent the accumulation of oxidative damage; or whether enhanced

SOD1 activity, initiated in aged animals, might be therapeutic. To test this hypothesis,

we used adeno-associated virus (AAV) to deliver antioxidant enzymes (SOD1, SOD2,

CAT, or SOD1+CAT) to the hippocampus of young and aged rats. The results

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demonstrate a dissociation of learning from measures of oxidative stress and suggest

that changes in redox sensitive signaling may mediate cognitive impairment.

Results

Efficiency-Specificity of The Viral Vectors

Following behavioral testing (see below) a subset of animals was killed for

examination of vector expression. Figure 3-1 illustrates the pattern of AAV-mediated

transduction. Strong expression was observed throughout the dorsal hippocampus,

extending > 3,000 μm along the anterior-posterior axis and included all major cell layers

(Fig. 3-1A). Transduction was limited to the hippocampus in confirmation of our previous

work (Foster et al., 2008) and mainly observed in neurons. Immunostaining for the myc

tag of SOD1-myc revealed transduction in neurons identified by NeuN (Fig. 3-1B) or

MAP2 (Fig. 3-1C) staining. Myc immunofluorescence was not localized to astrocytes or

microglia, assessed by immunostaining for GFAP (Fig. 3-1D) and Iba1 (Fig. 3-1E).

Injections of SOD1+CAT resulted in co-localization within the soma and dendrites of

neurons (Fig. 3-1F). SOD2 was observed in the soma, but not the nucleus and co-

localized with COX consistent with mitochondrial localization (Fig. 3-1G).

Figure 3-2 shows the increase in enzyme expression and decrease in oxidative

damage associated with viral vector treatment. For quantification, band intensities were

normalized to GAPDH, and then normalized to the mean value for young animals. Two

SOD1 bands were observed for young and aged SOD1-myc injected rats (Fig. 3-2A).

The band representing endogenous SOD1 was observed at 19 kDa and a larger band

representing the myc tagged human SOD1 was located at 23 kDa. Densitometry

indicated that rats injected with SOD1-myc exhibited a fourfold increase in total SOD1

[F(1,16)=45.59, p<0.0001) (Fig. 3-2E). Measurement of the 19 kDa band indicated that

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SOD1-myc expression did not modify the level of endogenous SOD1 (data not shown).

An increase in the relevant vector was observed for SOD2 [F(1,11)=7.9, p<0.05] (Figs.

3-2B, 3-2F) and CAT [F(1,8)=7.14, p<0.05] (Figs. 3-2C, 3-2G). Finally, injection of

SOD1+CAT increased the expression of SOD1 [F(1,10)=14.54, p<0.01] and CAT

[F(1,12)=7.83, p<0.05] (Figs. 3-2D, 3-2H). It should be noted that the level of expression

for SOD1 under this condition was reduced relative to injection of SOD1-myc (i.e. 2

versus 4 fold).

As shown in Figure 3-2, SOD1, SOD2, CAT, or SOD1+CAT overexpression was

associated with reduced HNE-staining. Quantification confirmed a decrease in HNE-

reactive protein for SOD1 [F(1,8)=9.26, p<0.05], SOD2 [F(1,12)=11.45, p<0.01, CAT

[F(1,8)=18.53, p>0.01], and SOD1+CAT [F(1,8)=16.53, p<0.01]. A decrease in protein

S-glutathionylation was observed for rats overexpressing SOD1 [F(1,12)=15.17,

p<0.005], SOD2 [F(1,12)=7.83, p< 0.05], CAT [F(1,8)=22.54, p<0.01], and SOD1+CAT

[F(1,8)=7.3, p<0.05]. Examination of SOD activity indicated that SOD1-myc rats

exhibited a two-fold increase in SOD activity compared to GFP-injected rats (p<0.05; n

= 4/group, data not shown).

SOD overexpression can increase the level of H2O2 and the expression of

downstream antioxidant enzymes (de Haan et al., 1996; Fullerton et al., 1998; Kelner et

al., 1995; Liochev and Fridovich, 2007). Western blots indicate that GPx4 and CAT

levels did not differ among groups; however, there was a tendency for a treatment effect

for GPx1 [F(1,15)=3.46, p=0.08] and post hoc comparisons indicated GPx1 expression

significantly increased in aged SOD1 rats (Fig. 3-2E).

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Age-Dependent Influence of Enzyme Overexpression on Spatial Learning

Spatial learning in the water maze was tested one month following virus injections.

Figure 3-3 shows the mean escape latency and escape path length across the five

training blocks during cue discrimination training. All groups exhibited a decrease in

escape latency and path length during training, and young animals exhibited superior

latency and path length relative to aged rats. An ANOVA on latency indicated an effect

of training [F(4,404 =107.21, p<0.0001] and an age effect [F(1,404)=34.71, p<0.0001] in

the absence of a treatment effect (Figs. 3-3A, 3-3B). Similarly, an ANOVA on path

length indicated an effect of training [F(4,404)=71.23, p<0.0001] and age

[F(1,404)=40.084, p<0.0001] in the absence of a treatment effect (Figs. 3-3C, 3-3D).

For spatial discrimination, an ANOVA on escape latency indicated significant main

effects of training [F(5,505)=68.03, p<0.0001] and age [F(1,505)=37.35, p<0.0001], with

a tendency for a treatment effect [F(4,505)=2.10, p=0.08] (Figs. 3-4A, 3-4B). Analysis of

escape path length indicated effects of training [F(5, 505)=81.19, p<0.0001] and age

[F(1,505)=28.83, p<0.0001], with a tendency for a treatment effect [F(4,505)=2.27,

p=0.06] (Figs. 3-4C, 3-4D). To localize treatment effects, the data for the last two

training blocks were averaged and an ANOVA was run within each age group. No

treatment effects were found in young rats. A tendency [F(4,50)=1.92, p=0.12] for a

treatment effect on latency was observed for aged rats, and post hoc analyses indicated

that aged SOD1+CAT rats exhibited significantly less time to reach platform compared

to aged rats expressing SOD2 or SOD1 alone (Fig. 3-4E). The results were confirmed

for distance (Fig. 3-4F). An ANOVA on the mean for the last two blocks revealed

tendency for a treatment effect in aged rats [F(4,50)=2.5, p=0.05] and post hoc analysis

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indicated that aged SOD1+CAT rats had a shorter path length compared to aged SOD2

or SOD1 rats.

A 60 sec probe trial was delivered between training blocks 5 and 6. Analyses were

performed on two consecutive 30 sec segments. An ANOVA on the percent time in the

goal quadrant during the first 30 seconds indicated young spent more time in the goal

quadrant than aged rats [F(1,100)=4.82, p<0.05] and there was a tendency for a

treatment effect [F(4,100)=2.07, p=0.08] (Figs. 3-5A, 3-5B). ANOVAs within each age

group revealed a treatment effect for aged animals [F(4,50)=3.07, p<0.05] and post hoc

comparisons indicated that aged control rats spent more time in the goal quadrant

compared to aged rats with SOD1 or CAT overexpression. Aged SOD1+CAT rats spent

significantly more time in the goal quadrant relative to aged SOD1 rats. The percent

time in the goal quadrant was compared to that expected by chance (i.e. 25%). For the

first 30 seconds of the probe trial, all groups except for aged SOD1 or CAT performed

above chance. Examination of time spent searching the goal quadrant for the last 30

sec of the probe trial indicated a tendency for an age effect [F(1,100)=5.06, p=0.08] in

the absence of a treatment effect (Figs. 3-5C, 3-5D). An examination of the percent time

in the goal quadrant indicated that all groups except aged controls and aged SOD1 rats

spent significantly greater time in the goal quadrant relative to chance (Figs. 3-5C, 3-

5D). The fact that aged SOD1 rats did not spend a significant portion of their search

behavior in the goal quadrant for either segment of the probe trial indicates that these

animals did not acquire a spatial search strategy.

The aged SOD1 impairment was confirmed by measuring the latency to the first

platform crossing (Figs. 3-5E, 3-5F). An ANOVA indicated an age [F(1,100)=10.95,

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p<0.01] and treatment [F(4,100)=2.52, p<0.05] effect. ANOVAs within each age group

indicated a tendency for a treatment effect in aged animals [F(4,49)=2.38, p=0.06] and

post hoc comparisons indicated that aged SOD1 rats required a longer time to cross the

platform location compared to age matched controls, CAT, and SOD1+CAT expressing

groups. There was a tendency (p=0.08) for a difference between SOD1 and SOD2

groups. An ANOVA on the total number of platform crossing indicated an age difference

[F(1,100)=13.15, p<0.001] (Figs. 3-5G, 3-5H), in the absence of a treatment effect. In

sum, the results indicate that SOD1 overexpression for one month was associated with

impaired spatial learning in aged rats. The fact that the SOD1+CAT exhibited superior

performance relative to SOD1 group indicates that the impairment may be rescued to

co-overexpressing CAT.

Overexpression of SOD1+CAT for Four Months Improves Spatial Learning

Our results contrast with studies in tg-SOD1 mice, which indicate that young tg-

SOD1 mice exhibit impaired spatial learning while aged tg-SOD1 mice show enhanced

spatial memory (Gahtan et al., 1998; Kamsler et al., 2007). The difference may result

from an interaction of age and the duration of overexpression. To examine this

possibility, a subset of animals tested at one month was retested at four months post-

injection. The experimental groups included young rats (8 mo) injected with SOD1 (n =

6), SOD1+CAT (n = 10), GFP (n = 2), no surgery controls (n = 4), and aged rats (23 mo)

injected with SOD1 (n = 6), SOD1+CAT (n = 10), GFP (n = 2) or no surgery controls (n

= 4).

For examination of spatial learning 4 months after viral vector injections, the

platform location in the maze was shifted to a new quadrant. An ANOVA on escape

latency across training blocks indicated main effects of training [F(5,185)=40.53,

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p<0.0001] and age [F(1,185) =5.19, p<0.05], with a tendency for a treatment effect

[F(2,185)=5.19, p=0.07] (Figs. 6A, 6B). Analysis of escape path length indicated effects

of training [F(5, 185)=36.12, p<0.0001] and age [F(1,185)=5.20, p<0.05] (Figs. 6C, 6D).

To localize treatment effects, the data for the last two training blocks were averaged and

an ANOVA for treatment effects was run within each age group. An ANOVA for young

animals indicated a tendency for a treatment effect [F(2,18)=7.12, p=0.14], however;

post hoc comparisons did not reach significance; although there was a trend for young

SOD1 rats to exhibit an increase in the escape latency relative to young control rats

(p=0.05). An ANOVA for aged animals indicated a tendency for a treatment effect

[F(2,19)=3.24, p=0.06] and post hoc comparisons indicated that aged SOD1+CAT rats

required less time to reach platform compared to aged SOD1 rats (Fig. 3-6B). Analysis

of the mean distance for the last two training blocks also indicated a tendency for a

treatment effect in aged rats [F(2,19)=2.67, p=0.09) (Figs. 3-6C, 3-6D) and post hoc

comparisons confirmed that aged SOD1+CAT rats had a shorter path length to escape

compared to aged SOD1 rats (Fig. 3-6D).

Examination of the first 30 seconds of the probe trial delivered between blocks 5

and 6, indicated a tendency for young rats to spend more time in the goal quadrant

relative to aged rats [F(1,37)=3.33, p=0.07] in the absence of a treatment effect (Figs. 3-

7A, 3-7B). The percent time searching the goal quadrant was significantly above chance

for all groups, indicating that all groups had acquired a spatial search strategy. A

tendency for a treatment effect [F(2, 37)=2.47, p=0.09] in the absence of an age

difference was observed for the second half-minute of the probe trial (Figs. 3-7C, 3-7D).

Again, all groups spent a significantly longer time in goal quadrant than expected by

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chance (Figs. 3-7C, 3-7D). An ANOVA on the latency to the first platform crossing

indicated a significant age effect [F(1,37)=12.73, p<0.01], and a tendency for a

treatment effect [F(2,37)=2.24, p=0.1]. An ANOVA within each age group indicated a

tendency for a treatment effect only in the aged group [F(2,19)=3.29, p=0.05]. Post hoc

comparison indicated that aged SOD1+CAT rats required less time for the first platform

crossing compared to aged SOD1 rats and there was a tendency (p=0.08) for

SOD1+CAT rats to cross quicker than aged controls. Finally, an ANOVA on total

platform crossing indicated an age effect [F(1,37)=12.67, p<0.005] and a tendency for a

treatment effect [F(2,37)=2.54, p=0.09]. An ANOVA within each age group indicated a

tendency for a treatment effect in aged rats [F(2,19)=3.45, p=0.05] and post hoc

comparisons indicated that aged SOD1+CAT rats exhibited significantly more platform

crossings than aged controls (Fig. 3-7H). In fact, aged SOD1+CAT rats exhibited

performance similar to young animals.

Figure 3-8 shows western blot data on enzyme expression and lipid peroxidation

in the hippocampus for animals that overexpressed viral vectors for ~5 months. As

shown in Figure 8A, the two SOD1 bands (19 and 23 kDa) were observed for SOD1-

myc injected rats. Densitometry confirmed that rats injected with SOD1-myc exhibited

~3 fold increase in total SOD1 [F(1,12)=133.08, p<0.0001) (Figs. 3-8A, 3-8C). Again,

the 19 kDa band was not influenced by SOD1-myc expression (data not shown).

Injection of SOD1+CAT increased the expression of SOD1 [F(1,12)=31.31, p<0.001]

and CAT [F(1,8)=10.40, p<0.05] (Figs. 3-8B, 3-8D). Similar to 2-month overexpression,

the level of expression for SOD1 under this condition was reduced relative to injection of

SOD1-myc alone (i.e. 2 versus 2.5 fold).

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A decrease in HNE-reactive protein was observed for rats that overexpress SOD1

[F(1,12)=24.23, p<0.001] or SOD1+CAT [F(1,12)=7.63, p<0.05] (Fig. 3-8). For SOD1

rats, there was an age effect on CAT expression [F(1,12)=6.58, p<0.05] in the absence

of a treatment effect. Post hoc tests within each treatment group indicated an age-

related decrease in expression in animals overexpressing SOD1 (Fig. 3-8C).

Examination of GPx1 indicated no effect of age or treatment for SOD1 animals and

SOD1+CAT animals exhibited an effect of age [F(1,16)=15.00, p<0.005] and treatment

[F(1,16)=4.27, p<0.05]. Post hoc analyses indicated that GPx1 expression was reduced

in aged rats that overexpressed SOD1+CAT [F(1,8)=7.9, p<0.05] compared to aged

controls (Figs. 3-8B, 3-8D). Levels of protein carbonyls were measured; however, the

results did not reveal any information concerning the effect of SOD1 overexpression,

possibly due to the lack of specificity of carbonyl measures (Adams et al., 2001) or the

fact that H2O2 is a poor mediator of stable oxidative damage for protein carbonyls

(Shacter, 2000b). Furthermore, previous research indicates that lipid peroxidation may

be more sensitive since SOD overexpression in mice markedly reduces lipid

peroxidation in the absence of a significant effect on protein carbonyls (Jang et al.,

2009). Consistent with the lipid peroxidation results, overexpression of SOD1 resulted in

a decrease in Oxo8dG [F(1,21)=16.58, p<0.0005] in the absence of an age difference

(Fig 3-9), confirming that SOD1 overexpression decreased oxidative damage.

Discussion

While it is clear that oxidative stress is a likely component of aging, it is unclear

whether increased production of ROS or the accumulation of oxidative damage is the

primary cause of functional decline. Consistent with previous work in tg-mice,

overexpression of SOD1, SOD2, and catalase reduced markers of oxidative stress

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(Perez et al., 2009a). However, decreased oxidative damage was not associated with

improved cognitive function. Moreover, SOD1 overexpression impaired spatial learning

in aged rats. The impairment was specific to SOD1 and spatial learning in aged animals.

The results indicate that accumulation of oxidative damage is not the only mechanism

for cognitive decline. Other processes influenced by oxidative stress may be more

relevant to age-related cognitive decline, including mitochondrial function, redox

signaling, and inflammation.

SOD1 can be toxic to motor neurons (Bosco et al., 2010). However, in the current

study, the deficit was limited to aged SOD1 animals, indicating that it was not simply

due to SOD1 overexpression. Further, one would expect that toxicity should increase

over time. On the contrary, we observed that the spatial learning deficit decreased

following four months of SOD1 overexpression in our oldest rats. Although, there was a

tendency for the emergence of impairment as young SOD1 rats approached middle-age

(Fig. 3-6). SOD1 catalyzes O2·- into H2O2 and the levels of O2

·- and H2O2 in brains of Tg-

SOD1 mice are decreased and increased, respectively (Malinska et al., 2009) and tg-

SOD1 overexpressing mice exhibit age-dependent effects on cognition and

hippocampal synaptic plasticity which are thought to result from elevation of ambient

H2O2 levels (Kamsler and Segal, 2003a). Thus, the specificity may be due to ambient

H2O2 levels. One indication that H2O2 was increased in the current study is that aged

SOD1 animals exhibited increased GPx1 expression (Fig. 3-2A). In other tissues, H2O2

can provoke an increase in GPx activity (Noack et al., 1998; Rohrdanz et al., 2001;

Wijeratne et al., 2005). Finally, aged SOD1+CAT rats did not exhibit cognitive

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impairments, suggesting improved cognition resulted from better homeostatic regulation

of redox signaling due to CAT processing of excess H2O2.

How can SOD1 overexpression reduce oxidative damage if H2O2 levels increase?

H2O2 is a relatively mild oxidizing agent. Irreversible oxidative damage results from O2·-;

SOD1 removes O2·-and overexpression of SOD1 reduces the level of O2

·- in the brain

(Malinska et al., 2009). If free iron is available, H2O2 can produce hydroxyl radicals (OH.)

through Fenton-type reactions. However, a large body of evidence indicates that H2O2

per se may not induce irreversible oxidative damage (Catala, 2010a; Leutner et al.,

2001b; Linden et al., 2008a; Shacter, 2000b). In contrast, H2O2 can readily and

reversibly influence redox sensitive signaling cascades. Age-related changes in redox

state disrupt calcium homeostasis, increasing the release of calcium from internal stores,

decreasing CaMKII activity, and impairing LTP (Bodhinathan et al., 2010a, b; Foster,

2007). H2O2 application to hippocampal slices mimics age-related changes, promoting

calcium release from internal stores (Gonzalez et al., 2006), decreasing synaptic

CaMKII activity (Shetty et al., 2008), and impairing LTP (Auerbach and Segal, 1997;

Kamsler and Segal, 2003a; Pellmar et al., 1991). The LTP impairment in tg-SOD1 mice

is attenuated by catalase suggesting the involvement of elevated H2O2 (Gahtan et al.,

1998). Together the results suggest that SOD1 overexpression could impair learning

through increased H2O2 acting on redox signaling pathways involved in memory

formation.

Adaptation to Altered Redox State: The effect of SOD overexpression on

memory changes with age (Hu et al., 2007b). SOD1 overexpression impaired or had no

effect on learning in young mice and enhanced spatial memory in older mice (Kamsler

82

et al., 2007; Kamsler and Segal, 2003b). The authors suggest that aged mice adapt to

elevated H2O2. Transcription of genes regulating redox homeostasis increase during

middle-age (Aenlle et al., 2009; Blalock et al., 2003; Zeier et al., 2011). Thus, the

increase in GPx1 expression in aged SOD1 mice may represent an adaptive response.

Other adaptive changes may underlie the improvement in behavior following four-

months of SOD1 overexpression. Finally, aged SOD1+CAT rats exhibited superior

water maze performance, indicating that treatment to reduce O2·- and H2O2 may be

more beneficial than simply enhancing SOD1. Indeed, administration of a superoxide

dismutase/catalase mimetic improved memory in aging mice (Liu et al., 2003). Future

studies should employ redox proteomics to identify specific changes related to cognitive

decline.

In summary, memory function was not related to the level of oxidative damage.

Increased SOD1 expression impaired learning in older rats. In contrast, increased

expression of SOD1+CAT provided protection from age-related cognitive impairments.

The results extend a growing body of evidence indicating the importance of H2O2 and

the redox signaling for regulating cognitive function during aging.

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Figure 3-1. Neurons are the primary cell type transduced by the AAV vectors. (A) Panels show that transduction of myc (green) for SOD1-myc extended at least 3,000 μm though the hippocampus, was observed in all three cell layers, and was limited to the hippocampus. The distance is calculated relative to bregma. (B) Merged images showing co-localization (yellow) of myc (green) for SOD1-myc and NeuN (red) in neuron cell bodies, and (C) in dendrites determined by MAP2 (red). Myc did not co-localize with (D) GFAP (red) or (E) Iba1 (red). (F) Merged figure for the hippocampus of a rat injected with SOD1+CAT and immunostained for myc (green) for SOD1-myc and CAT (red) and co-localization (yellow). (G) Merged figure of a cell in the CA1 pyramidal cell layer of a rat injected with the SOD2 vector showing co-localization of SOD2 (green) with the OxPhos Complex IV subunit I (COX) (red). Note the expression is not observed in the nucleus (N) consistent with mitochondrial localization. Cell nuclei in A, D and E were stained with DAPI (blue). Calibration bars represent 500 µm (A), 100 µm (B-F) and 10 µm (G).

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Figure 3-2. Overexpression of antioxidant enzymes in the hippocampus reduces

markers of oxidative stress. Western blot analysis of hippocampal lysates from young and aged rats injected with viral vectors to express (A, E) SOD1-myc, (B, F) SOD2, (C, G) CAT, or (D, H) SOD1+CAT. For quantification, band intensities were normalized to expression of GAPDH and this value was

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normalized to the mean value of young controls from the same blot. In each case, transduction resulted in an increase in the expression of the antioxidant enzyme. For SOD1-myc (A, D), two bands were observed representing endogenous rat SOD1 (rSOD1) and the human myc tagged SOD1 (hSOD1). Lipid peroxidation (HNE) and S-glutathionylated proteins (GSH) were decreased by enzyme overexpression. To determine whether overexpression of antioxidant enzymes would influence expression of downstream antioxidant enzymes, immunostaining was performed for glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), and CAT. Overexpression of SOD1-myc was associated with an increase in GPx1 (E). Each bar represents the normalized mean + SEM (n = 3-6). Asterisks indicate significant (p < 0.05) differences determined by post hoc Fisher PLSD.

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Figure 3-3. Overexpression of antioxidant enzymes did not affect cue discrimination in

water maze. All groups learned to reach the visible platform, as indicated by a significant overall decrease in latency (A, B) and distance (C, D) across blocks. Y = 5-month and A = 20-month old rats injected with SOD1, SOD2, CAT, or SOD1+CAT. Age matched controls (Cont) included rats injected with GFP and no surgery controls.

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Figure 3-4. Overexpression of SOD1 impaired spatial learning in aged rats. Behavioral

measures for young and aged rats during training on the spatial version of the water escape task. Mean latency (A, B) and mean path length (C, D) to escape during spatial discrimination training. No treatment effects were found for young rats. Examination of the mean latency (E) and mean path length (F) averaged across the last two training blocks in aged animals indicated an effect of treatment. Due to better performance by the SOD1+CAT group relative to the SOD1 and SOD2 groups. Asterisk indicates a significant (p < 0.05) difference.

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Figure 3-5. Overexpression of SOD1 impaired acquisition of a spatial search strategy in

aged rats. A single probe trial was given between block 5 and block 6 of spatial training. Percent time spent searching the goal quadrant during the first 30 seconds of for (A) young and (B) aged rats. The search behavior of aged rats with overexpression of SOD1 or CAT was not different from chance

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levels (25% dashed line). Aged SOD1 rats spent significant less time compared to aged control and aged rats with overexpression of SOD1+CAT. Aged rats with overexpression of CAT spent significant less time than aged control. Examination of the percent of time spent in goal quadrant during the second 30 seconds of probe trial for (C) young and (D) aged rats indicated that aged rats with overexpression of SOD1 continued to perform at chance levels. Examination of the latency for first platform crossing for (E) young and (F) aged rats indicated a treatment effect for aged animals due to an extended latency for the SOD1 group. Age differences were observed for total number of platform crossings between (G) young and (H) aged rats. Asterisk = p < 0.05 group difference. Pound sign indicates a significance (p < 0.05) difference from chance.

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Figure 3-6. Long-term overexpression of SOD1+CAT improved learning in spatial trials

in aged rats. Behavioral measures for young and aged rats during training on the spatial version of the water escape task four months after viral injections. Mean latency (A, B) and mean path length (C, D) to escape during spatial discrimination training. The bar graphs represent the means latency averaged across the last two training blocks. For aged animals better performance was observed for the SOD1+CAT group relative to the SOD1 group. Asterisk indicates a significant (p < 0.05) difference.

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Figure 3-7. Overexpression of SOD1+CAT for four months improves spatial learning in probe test. A single probe trial was given between block 5 and block 6 of spatial training. Mean percent time spent searching the goal quadrant during

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the first 30 seconds of for (A) young and (B) aged rats. Mean percent of time spent in goal quadrant during the second 30 seconds of probe trial for (C) young and (D) aged rats. Examination of the latency for first platform crossing for (E) young and (F) aged rats indicated a treatment effect for aged animals due to an superior performance by the SOD1+CAT group compared to the SOD1 group. Asterisk = p < 0.05 group difference. Pound sign indicates a significance (p < 0.05) difference from chance.

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Figure 3-8. Antioxidant enzymes and oxidative stress markers in hippocampus with 5-

month SOD1 and SOD1+CAT overexpression. Western blot analysis of hippocampal lysates from young and aged rats injected with viral vectors to express (A) SOD1, and (B) SOD1+CAT. For SOD1 (A, C), two bands were observed representing endogenous rat SOD1 (rSOD1) and the human myc

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tagged SOD1 (hSOD1). CAT level did not change in SOD1 rats but increased in SOD1+CAT rats. Lipid peroxidation measured with anti-4-hydroxy-2-nonenal (HNE) antibody was decreased by SOD1 and SOD1+CAT overexpression. Expression of SOD1 did not change the level of GPx1 but expression of SOD1+CAT in aged rats was associated with a decrease in GPx1 (D). GAPDH was used as a loading control. Asterisk indicates a significant (p < 0.05) difference in treatment. ω indicates a significant (p < 0.05) difference in age.

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Figure 3-9. DNA oxidative damage is reduced by overexpression of SOD1. The level of

8-oxodGuo was measured and normalized by young controls (Y-Cont). Bars represent the mean and SEM.

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CHAPTER 4

THE INFLUENCE OF AAV DELIVERED SUPEROXIDE DISMUTASE 1 AND CATALASE ON HIPPOCAMPAL SYNAPTIC PLASTICITY AND NMDA RECEPTOR

FUNCTION

Unlike neurodegenerative disease, cognitive decline in normal aging is not

associated with a significant loss of neurons (Gallagher et al., 1996). Rather, cognitive

decline in normal aging is associated with senescent physiology including changes in

synaptic plasticity (Foster, 2002; Foster and Kumar, 2002; Kumar et al., 2009). Long-

term potentiation (LTP) has been widely considered as a cellular mechanism that

underlies learning and memory because of the characteristics of synaptic modification

(Bliss and Collingridge, 1993; Malenka and Nicoll, 1999). In experimental settings, LTP

is usually initiated by high frequency afferent stimulation (>50 Hz), in order to activate N-

methyl-D-aspartate glutamate receptors (NMDARs). NMDARs play a central role in

LTP-induction and the specificity of synaptic modification. The activation of NMDAR

starts from the neurotransmitters binding to NMDAR and the postsynaptic depolarization

removing magnesium that blocks the channel. Ca2+ thus enters the NMDAR channel at

the synaptic site. Ca2+ will then initiate the signaling cascade that results in LTP-

induction.

During aging, the induction of LTP becomes more difficult, there is an increase in

the level of synaptic activation needed to induce LTP (Foster, 1999, 2012; Kumar, 2011;

Kumar et al., 2007; Milner et al., 2004). The synaptic plasticity shift is due to disruption

of Ca2+ homeostasis (Burke and Barnes, 2010; Foster, 2007; Thibault et al., 2001). The

major sources of intracellular Ca2+ include influx into the cell through NMDA receptors or

voltage-dependent Ca2+ channels (VDCC) and release of Ca2+ from intracellular Ca2+

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stores. Work done in our lab and others indicate that, in the hippocampus, aging is

associated with a decreased involvement of NMDA receptors and an increased role for

VDCC and internal Ca2+ stores in regulating synaptic modifiability (Barnes et al., 1997;

Foster, 2012; Foster and Norris, 1997; Kumar and Foster, 2005; Thibault and Landfield,

1996). The exact mechanism for ROS-mediated regulation of synaptic plasticity is

unknown; however, recent work demonstrates that an age-related shift in the

intracellular oxidation-reduction (redox) state decreases NMDAR function and increases

release of Ca2+ from intracellular Ca2+ stores (Bodhinathan et al., 2010a, b).

The nervous system is highly sensitive to oxidative stress (Halliwell, 1992). The

highly active free radical, superoxide can make irreversible oxidative damage on DNA,

RNA, lipids and proteins. To prevent the irreversible oxidative damage on tissues,

superoxide dismutase catalyzes O2·- into less active H2O2. Hydrogen peroxide per se

does not induce irreversible oxidative damage (Catala, 2010b; Leutner et al., 2001a;

Linden et al., 2008b; Shacter, 2000a). The relatively milder hydrogen peroxide induces

the reversible formation of disulfide bonds between pairs of cysteine residues (thiol

groups: R-S-H) in proteins, shifting protein structure and function, and influencing

multiple signaling cascades including Ca2+ signaling (Foster, 2007). The redox state of

cysteine residues on the extracellular portion of the NMDAR have been implicated in

regulating NMDAR function in cortical neuronal culture and hippocampal slices

(Aizenman et al., 1990; Aizenman et al., 1989; Bernard et al., 1997; Choi and Lipton,

2000; Lipton et al., 2002; Reynolds et al., 1990), suggesting that the age-related decline

in NMDAR function could be regulated by altered redox state acting on extracellular

cysteine residues. Indeed, application of the disulfide reducing agent, dithiothreitol

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(DTT), selectively increased the NMDAR-mediated synaptic responses to a greater

extent and enhanced LTP in hippocampal slides from older rats (Bodhinathan et al.,

2010a). Surprisingly, applying the membrane-impermeable reducing agent, L-

glutathione directly to the hippocampal slices failed to increase the NMDAR response;

instead, an increase in the NMDAR response was observed if L-glutathione was

delivered through the intracellular recording pipette. The results indicate that

intracellular redox state, rather than disulfide bonds of extracellular cysteine residues,

mediates the suppression of NMDAR responses and impaired LTP. The intracellular

redox state mediated NMDAR function decline during aging has recently been

confirmed by other labs (Robillard et al., 2011; Yang et al., 2010).

We recently provided evidence that overexpression of SOD1 in the hippocampi

impaired spatial learning in aged F344BN F1 rats. In addition, overexpression of CAT

together with SOD1 rescued the spatial learning deficit caused by overexpression of

SOD1 alone. The results suggested that the impaired spatial learning in older SOD1

could be due to over production of H2O2 and impaired NMDAR function. The current

study used AAV to deliver SOD1+GFP and SOD1+CAT to the hippocampus of aged

(17-month) F344 rats and examined memory-related behavioral performance one-

month post injection. Following collection of behavioral data, one hippocampus from

each rat was used to examine NMDA receptor function, and the other hippocampus

from each rat was used to determine antioxidant enzyme expression and activity as well

as the level of a major redox buffer, glutathione.

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Results

AAV Delivery of SOD1, CAT and GFP in the Hippocampi of Rats

Animals were killed for examination of vector expression after behavioral testing.

Figure 4-1 illustrates the pattern of AAV-mediated transduction. Immunofluorescence on

brain slides from rats injected with AAV-SOD1 and AAV-GFP revealed that neurons

were co-transduced with AAV-SOD1 (showing in red with anti-myc antibody) and AAV-

GFP (showing in green) (Fig. 4-1a). Injections of AAV-SOD1+ AAV-CAT resulted in co-

transduction of SOD1 and CAT in hippocampal neurons (SOD1 was shown with anti-

myc antibody in red, and CAT was shown with anti-CAT antibody in green) (Fig. 4-1b).

Figure 4-2 shows the increase in enzyme expression and decrease in oxidative

damage associated with viral vector treatment. For quantification, band intensities were

normalized to GAPDH. Two SOD1 bands were observed for SOD1-myc injected rats

(Fig. 4-2A). The band representing endogenous SOD1 was observed at 19 kDa and a

larger band representing the myc tagged human SOD1 was located at 23 kDa.

Densitometry indicated that rats injected with SOD1-myc exhibited a ~2.3 fold increase

in total SOD1 in both SOD1+GFP [F(1,5)=14.36, p <0.05] and SOD1+CAT [F(1,6)=43.0,

p<0.001] rats. Measurement of the 23 kDa band indicated that hSOD1 expression is not

different between SOD1+GFP rats and SOD1+CAT rats [F(1,5)=2.0, p=0.22].

Measurement of the 19 kDa band indicated that SOD1-myc expression did not modify

the level of endogenous SOD1. rSOD1 expression is not different in SOD1+GFP rats

[F(1,6)=0.65, p=0.45] or SOD1+CAT rats [F(1,6)=2.25, p=0.18] compared to rSOD1

expression in GFP rats. C-myc was only detected in rats injected with SOD1+GFP and

SOD1+CAT viruses. GFP expression was observed in rats injected with GFP and

SOD1+GFP virus. In addition, SOD1+GFP and SOD1+CAT overexpression were

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associated with a tendency of reduced HNE-staining. Quantification indicated a

tendency of decrease in HNE-reactive protein for SOD1+GFP [F(1,6)=3.46, p=0.11] and

SOD1+CAT [F(1,6)=2.63, p=0.16]. A tendency for increased CAT expression was

observed in SOD1+CAT rats [F(1,5)=2.78, p=0.16], and no difference was observed for

Cat expression in SOD1+GFP rats compared to GFP rats [F(1,4)=0.025, p=0.88].

SOD overexpression can increase the level of H2O2 and the expression of

downstream antioxidant enzymes (de Haan et al., 1996; Fullerton et al., 1998; Kelner et

al., 1995; Liochev and Fridovich, 2007). A tendency of increase of GPx1 was observed

in SOD1+GFP rats compared to GFP rats [F(1,5)=4.54, p=0.08]; however, no difference

in GPx1 expression was observed in SOD1+CAT rats compared to GFP rats

[F(1,5)=0.001, p=0.98].

SOD1 Overexpression Impairs Spatial Learning while SOD1+CAT Overexpression Improves Spatial Learning in Aged Rats.

Figure 4-3 shows the mean escape latency and escape path length across the five

training blocks during cue discrimination training. A repeated measures analysis of

variance (ANOVA) on latency indicated an effect of training [F(4,136) = 26.25, p <

0.0001] and a treatment effect [F(2,136) = 12.22, p < 0.0005] (Fig 4-3a). Post hoc

analysis indicated that GFP rats required significant longer time to find platform than

SOD1+GFP rats (p<0.0001) and SOD1+CAT rats (p<0.005). ANOVA on mean escape

latency in individual block indicated that the treatment effect was only seen in the early

phase of training including block 2 [F(2, 34)=8.69, p<0.001] and block 3 [F(2, 34)=11.72,

p<0.005]; but not the late phase of training including block 4 [F(2,34)=2.05, p=0.14] and

block 5 [F(2,34)=1.88, p=0.16]. Repeated measures ANOVAS within each group

indicated that all groups exhibited a decrease in escape latency during training (GFP:

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F(4,52)=11.25, p<0.0001; SOD1+GFP: F(4,44)=7.74, p<0.0001; SOD1+CAT:

F(4,40)=10.13, p<0.0001). Similarly, a repeated measure ANOVA on path length

indicated an effect of training [F(4, 136) = 11.43, p < 0.0001] and a treatment effect

[F(2,136)=5.97, p<0.01] (Fig. 4-3b). Post hoc analysis indicated that GFP rats required

significant longer path length to find platform than SOD1+GFP rats (p<0.05). ANOVA on

mean escape path length in individual block indicated that the treatment effect was only

seen in block 2 [F(2, 34)=3.84, p<0.05] and block 3 [F(2,34)=7.54, p<0.005]; but not the

late phase of training like block 4 [F(2,34)=0.47, p=0.63] and block 5 [F(2,34)=1.11,

p=0.34]. Finally, repeated measures ANOVAs within each group indicated that all

groups exhibited a decrease in path length during training (GFP: F(4,52)=11.25,

p<0.0001; SOD1+GFP: F(4,44)=7.74, p<0.0001; SOD1+CAT: F(4,40)=3.64, p<0.05).

Spatial discrimination was initiated 3 days later. A repeated ANOVA on escape

latency indicated significant main effects of training [F(4,124) = 7.96, p < 0.0001] and

treatment [(F4,124)=6.54, p<0.005] (Fig. 4-4a). Post hoc analysis indicated that the

treatment effect was due to decreased escape latency for SOD1+CAT rats, which

exhibited a significant decrease in escape latency relative to GFP (p<0.05) and

SOD1+GFP rats (p<0.05). Repeated measures ANOVAs within each treatment group

indicated that GFP rats (p<0.05) and SOD1+CAT rats (p<0.005) reduced their latency to

find platform during spatial training. In contrast, no effect of training on latency was

observed for SOD1+GFP rats (p=0.51).

Analysis of escape path length indicated effects of training [F(4,124)= 8.87,

p<0.0001], and a tendency of treatment effect [F(2,124)=3.27, p=0.05] (Fig. 4-4b). Post

hoc analysis indicated that the treatment effect was due to decreased escape path

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length for SOD1+CAT rats relative to GFP (p<0.05) rats and a tendency for a decrease

in path length relative to SOD1+GFP rats (p=0.10). Repeated measures ANOVAs within

each treatment group indicated that GFP rats (p<0.005) and SOD1+CAT rats (p<0.005)

exhibited a significant decrease of path length to find platform during spatial training. In

contrast, no effect of training on path length was observed for SOD1+GFP rats (p=0.20).

To determine whether animals acquired a spatial search strategy, a 60 s probe

trial (probe1: acquisition) was delivered immediately following the fifth block of spatial

training. Following probe 1, a refresher training block was delivered, followed one hour

later by a retention probed trial (Probe 2) and a second reminder spatial training block

(block 7). Twenty-four hours later, a training block was delivered followed by a

relearning probed trial (Probe 3). Probe trails were measured by latency to first reach

platform, number of platform crossing and discrimination index score (DI): (% time in

goal quadrant - % time in opposite quadrant) / (% time in goal quadrant + % time in

opposite quadrant). A repeated measure ANOVA across three probe trials on latency to

platform indicated a significant effect on treatment [F(2,64)=3.96, p<0.05] (Fig. 4-5a).

Post hoc analysis indicated that the treatment effect was due to better performance by

SOD1+CAT rats, which reached the platform significant faster than SOD1+GFP rats

(p<0.01). SOD1+CAT rats also had a tendency to reach the platform faster than GFP

rats (p=0.08).

The superior performance of SOD1+CAT rats was confirmed by a repeated

measure ANOVA across three probe trials for platform crossing. A significant effect was

observed for treatment [F(2,68)=4.20, p<0.05] (Fig. 4-5b). Post hoc analysis indicated

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that SOD1+CAT rats made significant more platform crossings than GFP (p<0.05) and

SOD1+GFP (p<0.05) rats.

An ANOVA on DI score from three groups of animals indicated no treatment

effects over the course of testing. To determine if the animals acquired a spatial search

strategy, the DI scores were compared with that expected by chance (i.e., when the

animal spent equal amount of time in goal and opposite quadrants, then DI=0). One

group student t-tests indicated that only GFP and SOD1+CAT rats exhibited DI score

above chance (p<0.05) in probe 1 (acquisition) (Fig. 4-5c). The results indicate that

following the initial training, only GFP and SOD1+CAT rats acquired a spatial search

strategy, focused on the goal quadrant. In probe 2 (one-hour retention), only

SOD1+CAT rats had a DI score above chance (p<0.05) indication that SOD1+CAT rats

retained the spatial search strategy over the one-hour retention interval. In probe 3

(relearning), all groups exhibited DI score above chance (p<0.05), indicating that all

animals, including SOD1+GFP rats can acquire a spatial search strategy with repeated

training. In sum, the results indicate that SOD1 overexpression for 1 month was

associated with impaired spatial learning in aged rats. The fact that the SOD1 +CAT

exhibited superior performance relative to SOD1 group indicates that the impairment

maybe rescued to co-overexpressing CAT. In addition, SOD1+CAT rats exhibited some

benefits relative to GFP animals, exhibiting a decrease in the escape latency during

training, more platform crossings during the probe trial and better memory during the

one hour retention interval.

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NMDAR-Mediated Synaptic Potentials are Reduced in Rats with SOD1 Overexpression

Starting two weeks after finishing water maze study, animals were killed (one per

day) to examine hippocampal synaptic function. SOD1+GFP (n = 8 rats, 12 slices),

SOD1+CAT (n =10 rats, 15 slices), and GFP (n = 12 rats, 20 slices) were used to

examine the input-output function (stimulation intensity-synaptic response) for the total

synaptic response and the N-methyl-D-aspartic acid receptor (NMDAR) mediated

component of the synaptic response, and the level of LTP. Examination of the input-

output curves for the synaptic responses indicated no group difference in baseline

synaptic strength [F(2,444)=0.038, p=0.96] (Fig. 4-6a) suggesting no difference in the -

amino-3-hydroxy-5-methy-4-isoxazolepropionic acid (AMPA) receptor component of

synaptic transmission. Repeated ANOVA on the input-output curves of the NMDAR

synaptic response indicated a significant interaction between treatment and stimulation

intensity [F(24,624)=2.20, p<0.001]. The treatment effect was apparent for the higher

stimulation intensities. In fact, ANOVA on fEPSP at the highest stimulation intensity

indicates a tendency of treatment effect [F(2,52)=2.35, p=0.10] and a post hoc

comparison indicated that SOD1+GFP rats had a significantly reduced NMDAR synaptic

response relative to GFP rats (p<0.05) (Fig. 4-6b). The results suggest that the

influence of redox state regulated by SOD1 and CAT is specific to NMDAR but not

AMPAR.

For the study on LTP, slices were bathed in normal ACSF to record the AMPA and

NMDAR component of the synaptic response. LTP was induced by four episodes of

high-frequency stimulation (HFS; 100 Hz, 1 s with 1 s interval). One group t-tests

indicated that the fEPSP was significantly greater in HFS-induced LTP from GFP

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(p<0.0001), SOD1+GFP (p<0.005) and SOD1+CAT (p<0.005) compared to the baseline

(=100) (Fig. 4-6c). However, there was no difference in the levels of HFS-induced LTP

between treatment groups [F(2,44)=0.242, p=0.78] (Fig. 4-6c).

Effect of Overexpression of SOD1 and CAT on Glutathione Redox State

The level of GSH, GSSG were measured in hippocampus of rats with

overexpression of SOD1+GFP (n = 11), SOD1+CAT (n = 10), or GFP (n = 13) for the

purpose of determining the effects of overexpression of SOD1 or CAT on glutathione

redox state. ANOVA revealed a tendency for a treatment effect for GSH levels

[F(2,31)=1.87, p=0.17]. Post hoc comparison indicated a tendency (p = 0.08) for

decreased GSH in hippocampi of SOD1+GFP compared to GFP rats (Fig. 4-7a). Total

GSH was measured when all the GSSG in the samples were reduced to GSH by adding

extra glutathione reductase to the samples. An ANOVA revealed a treatment effect in

total GSH [F(2,31)=7.55, p<0.005] and post hoc comparisons indicated that

overexpression of SOD1+GFP resulted in a significant decrease (22% compared to

GFP and 17% compared to SOD1+CAT) in total GSH compared to GFP and

SOD1+CAT rats (p<0.001 and p<0.05, respectively). In addition, an ANOVA revealed a

treatment effect in GSSG [F(2,30)=7.449, p<0.005]. Post hoc comparisons indicated

that overexpression of SOD1+GFP resulted in a significant decrease of GSSG

compared to GFP and SOD1+CAT rats (p<0.005 and p<0.005 respectively) (Fig. 4-7c).

Furthermore, an ANOVA revealed a treatment effect in the GSH: GSSG ratio

[F(2,30)=5.19, p<0.05]. Post hoc comparisons indicated that overexpression of

SOD1+GFP in hippocampi resulted in a significant increase in GSH: GSSG ratio

compared to GFP (71% increase; p<0.05) and SOD1+CAT (99% increase; p<0.05) rats

(Fig. 4-5d).

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Effect of Overexpression of SOD1 and CAT on Activities of GSH Peroxidase and GSH Reductase

The activity of glutathione peroxidase (GPx) and glutathione reductase (GR), were

measured in hippocampus of rats with overexpression of SOD1+GFP (n = 12),

SOD1+CAT (n = 10), or GFP (n = 14) for the purpose of determining other enzymes

involved in glutathione redox state affected by overexpression of SOD1 or CAT. GPx

catalyzes the reduction of hydrogen peroxide by reducing glutathione. An ANOVA

revealed a significant treatment effect on GPx activity [F(2,33)=5.96, p=0.006]. Post hoc

comparisons indicated that SOD1+GFP rats had a significant decrease in GPx activity

compared to SOD1+CAT (28 % decrease; p < 0.005) and GFP rats (20 % decrease; p <

0.05). The GPx activity was not different between GFP rats and SOD1+CAT rats (p =

0.23) (Fig. 4-8a). GR catalyzes the reduction of oxidized glutathione (GSSG) to reduced

glutathione (GSH), using β-nicotinamide dinucleotide phosphate (NADPH) as the

hydrogen donor. An ANOVA revealed a significant treatment effect on GR activity

[F(2,33)=4.14, p < 0.05]. Post hoc comparisons indicated that SOD1+GFP rats had a

significant decrease in GR activity relative to GFP rats (10 % decrease; p<0.01);

SOD1+CAT rats exhibited a tendency to have decreased GR activity relative to

SOD1+CAT rats (8 % decrease; p = 0.05). The GR activity was not different between

GFP rats and SOD1+CAT rats (p=0.52) (Fig. 4-8b).

Discussion

In this study we have examined the hypothesis that overexpression of antioxidant

enzymes affects NMDA receptor function via regulating redox state. We used AAV to

deliver SOD1+GFP and SOD1+CAT to the hippocampus of aged (17-month) F344 rats

and examined memory-related behavioral performance one-month post injection. The

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results indicated that SOD1 overexpression was associated with 1) improved cue

discrimination, 2) impaired spatial learning in water maze, 3) a decrease in input-output

curve with NMDAR-mediated fEPSP, 4) reductions in free GSH, total GSH, and GSSG

contents and 5) reductions in GPx and GR activities. On the other hand, overexpression

of SOD1+CAT was associated with improved cue discrimination, spatial learning and 1

hour retention in water maze. In addition, overexpression of SOD1+CAT did not affect

input-output curve of NMDAR-mediated fEPSP. Finally, free GSH and total GSH

contents were decreased in SOD1+CAT rats, but GSSG content did not change. The

activities of GPx and GR were also not altered in SOD1+CAT rats.

Our previous study with viral vector delivery of SOD1, SOD2 and CAT in the

hippocampi of Fischer 344 x Brown Norway F1 (F344BN F1) rats indicated that

overexpression of SOD1 impaired spatial learning and overexpression of CAT together

with SOD1 rescued the learning deficits caused by SOD1 overexpression alone (Lee et

al., 2012). To test the hypothesis that the benefit of SOD1+CAT was not due to

decreased SOD1 expression, as a result of using two viruses, we used a mix of two

viruses containing SOD1 and GFP, respectively, to transduce hippocampal cells. The

results demonstrate spatial learning deficits in SOD1+GFP rats, indicating that the

rescue by CAT was not due to using two viruses. Furthermore, Western blot results

indicate that the expression of hSOD1 was not different between SOD1+GFP rats and

SOD1+CAT rats (Fig. 4-2). In the cue discrimination task, SOD1+GFP and SOD1+CAT

animals exhibited enhanced performance in block 2 and block 3 compared to GFP rats.

The superior performance of the SOD1+GFP rats might due to impaired hippocampal

function, and a reliance or compensation by other neural systems (e.g. basal ganglia).

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One study that examined the effect of brain lesions on the acquisition of the cue

discrimination task found that damage to the basal ganglia, but not the hippocampus,

disrupted acquisition on this task (Packard and McGaugh, 1992). Importantly, for the

current study, no effect of viral treatment was found for behavioral measures after block

4, indicating that all the animals ultimately employed the cue response strategy, the

animals did not exhibit a sensory motor deficit that would prevent them from learning on

the spatial task, and all animals learned the cue task to about the same extent.

Consistent with our previous study, overexpression of SOD1+GFP and

SOD1+CAT reduced the marker of lipid peroxidation, and the decrease in lipid

peroxidation was not associated with improved cognitive function (Lee et al., 2012). The

results indicate that accumulation of oxidative damage is not the only mechanism of

cognitive decline. In this case, other processes influenced by ROS may be more

relevant to age-related cognitive decline, such as intracellular redox state-regulated

synaptic plasticity. Our laboratory previously demonstrated that an age-related shift in

the intracellular redox state was associated with decreased NMDAR function and

increased release of Ca2+ from intracellular Ca2+ stores (Bodhinathan et al., 2010a, b).

Age-related impairment in NMDAR function, including impaired induction of LTP was

reversed by treating the slices with reducing agents. These results have recently been

replicated by several other groups, suggesting that NMDAR function is sensitive to age-

related changes in redox state (Robillard et al., 2011; Yang et al., 2010). To test the

hypothesis that SOD1 overexpression results in redox changes, similar to that observed

during aging, we measured the input-output curve for total fEPSP, NMDAR-mediated

fEPSP, and the level of LTP. Consistent with the idea that SOD1 overexpression

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resulted in a more oxidized redox environment; input-output curve for NMDAR-mediated

fEPSP was reduced in SOD1+GFP rats. In contrast, the input-output curve for total

fEPSP was not affected by overexpression of SOD1+GFP. Since total fEPSP is mainly

due to AMPARs, the result is consistent with previous reports that the reducing agent

DTT had no effect on the AMPAR function of aged neurons (Abele et al., 1998;

Bodhinathan et al., 2010a; Gozlan et al., 1995). Together, the data suggests that the

effects of SOD1 overexpression, like altered redox state, are specific for NMDARs.

The overexpression of SOD1 is thought to influence the redox state through an

increase in H2O2 (Lee et al., 2012). As noted above, viral delivery of SOD1 reduces

oxidative damage in all age groups, but cognitive impairments are age-dependent. Our

previous work involving repeated cognitive testing suggested that deficits began to

emerge starting about 8-9 mo. of age, a time when markers of oxidative stress increase

(Gilmer et al., 2010; Martins et al., 2012). In addition, an increased expression of GPx1

is only observed in older SOD1 animals (Fig. 4-2). Previous work in other tissues

indicates that elevated H2O2 induces an increase in GPx activity (Noack et al., 1998;

Rohrdanz et al., 2001; Wijeratne et al., 2005). Finally, the cognitive impairments

associated with SOD1 overexpression in older animals were ameliorated by co-

expression of SOD1+CAT. Since CAT is one mechanism for the elimination of H2O2, the

results suggest improved cognition resulted from better homeostatic regulation of redox

state due to CAT processing of excess H2O2.

The redox state of tissues shifts towards a more oxidized state during aging

(Rebrin et al., 2004). Many human diseases conditions such as Parkinson’s, HIV/AIDs,

and cystic fibrosis, the more oxidized redox environment is associated with low GSH

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levels (Townsend et al., 2003). Glutathione is considered to be the major thiol-disulfide

redox buffer of the cell (Gilbert, 1990). Measurements of total GSH and/or GSSG levels

have been used to estimate the redox environment of cells. In the current study, we

observed a decrease in glutathione levels in hippocampal tissue that overexpressed

SOD1. The reduction of free GSH, total GSH in SOD1+GFP and SOD1+CAT rats

suggested that the intracellular environments were in a more oxidized state. Normally, a

reduction of GSSG indicates a more reduced environment. However, the combination of

decreased GSSG in addition to loss of free GSH and total GSH in SOD1+GFP rats

suggests a serious loss of GSH, possibly due to deficits in GSH redox cycling.

Glutathione levels are maintained by the GSH redox cycle consisting of glutathione

peroxidase and glutathione reductase. The redox cycle has a high capacity to provide a

constant supply of reduced glutathione through the conversion of GSSG by glutathione

reductase. However, in SOD1+GFP rats, the activities of glutathione peroxidase and

glutathione reductase were both decreased (Fig. 4-8). The results suggest that

glutathione peroxidase and glutathione reductase, which are both redox sensitive

enzymes, might be disrupted/ modified due to the oxidized environment caused by high

level of H2O2 in SOD1+GFP rats. In this regard, overexpression of CAT may provide an

important alternate pathway for elimination of H2O2, preserving glutathione peroxidase

and glutathione reductase activity.

Another possibility for the decrease in both GSH and GSSG in SOD1+GFP rats is

that GSSG may have been actively exported out of the cells. As a result of this transport

process, severe oxidative stress can deplete both cellular GSH and GSSG (Lu, 1999).

Finally, overexpression of SOD1+GFP may have influenced GSH synthesis to decrease

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GSH and GSSG levels. It has been reported that a decrease in GSH in several tissues

during aging is associated with a corresponding fall in the gene expression of glutamate

cysteine ligase and GSH synthase, two enzymes that are important in GSH synthesis

(Liu et al., 2004; Wang et al., 2003a). Future, studies may want to examine GSH

synthesis under conditions of SOD1 overexpression.

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Figure 4-1. Co-overexpression of SOD1+GFP and SOD1+CAT. (A) A rat with co-transduction of AAV-SOD1 and AAV-GFP. A merged image of myc staining (red) and GFP (green). (B) A rat with co-transduction of AAV-SOD1 and AAV-CAT. A merged image of myc staining (red) and CAT staining (green). Calibration bars represent 100 µm

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Figure 4-2. Antioxidant enzymes and oxidative stress markers in hippocampus with 1-month SOD1+GFP and SOD1+CAT overexpression. (A) Western blots of hippocampal lysates from 17-month old rats injected with viral vectors to express SOD1+GFP and SOD1+CAT. For SOD1, two bands were observed representing endogenous rat SOD1 (rSOD1) and the human myc tagged SOD1 (hSOD1 and c-myc). GFP was detected in GFP and SOD1+GFP rats but not in SOD1+CAT rats. CAT level did not change in SOD1+GFP rats but increased in SOD1+CAT rats. Lipid peroxidation measured with anti-4-hydroxy-2-nonenal (HNE) antibody was decreased by SOD1+GFP and SOD1+CAT overexpression. The level of GPx1 was increased in SOD1+GFP rats but was not changed in SOD1+CAT rats. GAPDH was used as a loading control. (B) Quantification of western blot data from SOD1+GFP and GFP rats. (C) Quantification of western blot data from SOD1+CAT and GFP rats. All densitometry was normalized by GAPDH. Asterisk indicates a significant (p < 0.05) difference in treatment. All densitometry was normalized by GAPDH. Error bars represent S.E.M.

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Figure 4-3. Overexpression of SOD1+GFP or SOD1+CAT enhanced cue discrimination in the water maze compared to GFP control. All groups (GFP rats: black filled circles, n=14; SOD1+GFP rats: red square, n=11; SOD1+CAT rats: blue triangle, n=12) learned to reach the visible platform, as indicated by a significant overall decrease in latency (A) and distance (B) across blocks. Asterisk indicates significant difference (p<0.05) from GFP control in a block of cue training by ANOVA. Error bars represent S.E.M.

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Figure 4-4. Overexpression of SOD1+CAT improved spatial learning while overexpression of SOD1+GFP impaired spatial learning in aged rats. Behavioral measures for aged rats during training on the spatial version of the water escape task. Mean latency (A) and mean path length (B) to escape during spatial discrimination training. Only GFP (black filled circles) (n=14) and SOD1+CAT rats (blue triangle) (n=12) had significant training effect indicating they acquired spatial search strategies. No effect of training on latency and path length was observed for SOD1+GFP rats (red square) (n=11) indicating that SOD1+GFP rats did not acquire spatial search strategy. A significant treatment effect due to SOD1+CAT rats find platform quicker compared to GFP and SOD1+GFP rats. Asterisk indicates a significant (p < 0.05) difference from GFP rats by ANOVA. Error bars represent S.E.M.

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Figure 4-5. Overexpression of SOD1+CAT improves spatial learning in probe tests. Three single probe trial was given between block 5 and block 6 of spatial training, 1 hour after block 6 followed by a refresher training block (block 7), and 24 hours later following block 8, respectively. (A) Examination of the latency for first platform crossing across three probe trials, indicated a treatment effect due to an superior performance by the SOD1+CAT group

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(blue triangle) (n=12) compared to the SOD1+GFP (red square) (n=11) (p<0.01) and GFP (black filled circles) (n=14) (p=0.08) group. (B) Examination of the number of platform crossings across three probe trials indicated a significant treatment effect due to SOD1+CAT rats made more crossings than GFP (p<0.05) and SOD1+GFP (p<0.05) rats. (C) No treatment effect was found when examining discrimination index (DI). To determine if the animals acquired a spatial search strategy, the DI scores were compared with that expected by chance. Asterisk sign indicates a significance (p < 0.05) difference from chance (one-group t-test). Error bars represent S.E.M.

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Figure 4-6. NMDAR-mediated synaptic potentials are reduced in rats with SOD1 overexpression. (A) Total field potential (total-fEPSP) in GFP rats (black filled circles) (n=11 rats, 15 slices) was not different from total-fEPSP in SOD1+GFP rats (red square) (n=9 rats, 14 slices) or SOD1+CAT rats (blue

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triangle) (n=8 rats, 11 slices). (B) An interaction of stimulation intensity and treatment for the NMDAR mediated synaptic response (NMDAR-fEPSP) was due a decrease in the response for rats with overexpression of SOD1+GFP (n=10 rats, 21 slices) compared to GFP rats (n=10 rats, 18 slices) at the highest stimulation intensities. (C) Quantification of the mean percent change in the fEPSP slope recorded from the control (white bars) and HFS (grey bars) pathway of the hippocampal slices from GFP (n=12 rats, 20 slices), SOD1+GFP (n=8 rats, 12 slices) or SOD1+CAT rats (blue triangle) (n=10 rats, 15 slices). Asterisk sign indicates that the fEPSP is significantly greater in HFS-induced LTP from GFP (p<0.0001), SOD1+GFP (p<0.005) and SOD1+CAT (p<0.005) compared to the baseline (=100). However, overexpression of SOD1+GFP or SOD1+CAT does not affect LTP in hippocampal area CA1. Error bars represent S.E.M.

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Figure 4-7.Decreased level of reduced GSH (GSH), total GSH and oxidized GSH (GSSG) are observed in rats with overexpression of SOD1+GFP. (A) Free GSH level was measured in hippocampal samples from GFP, SOD1+GFP and SOD1+CAT rats. SOD1+GFP rats had significantly reduced free GSH level than GFP rats. (B) Total GSH level was significantly reduced in SOD1+GFP compared to GFP and SOD1+CAT rats. (C) GSSG level was significantly reduced in SOD1+GFP rats compared to GFP and SOD1+CAT rats. (D) GSH:GSSG ratio was significantly higher in SOD1+GFP rats than GFP and SOD1+GFP rats. Asterisk indicates a significant (p < 0.05) difference by ANOVA. GFP rats (n=13) (black bars); SOD1+GFP rats (n=11) (red bars); SOD1+CAT rats (n=10) (blue bars). Error bars represent S.E.M.

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Figure 4-8. Decreased glutathione peroxidase (GPx) and glutathione reductase (GR) activities are observed in rats with overexpression of SOD1+GFP (A) GPx activity was measured in hippocampal samples from GFP, SOD1+GFP and SOD1+CAT rats. GPx activity was significantly reduced in SOD1+GFP rats relative to GFP and SOD1+CAT rats. (B) GR activity was significantly reduced in SOD1+GFP rats than GFP rats. In addition, there is a tendency of GR reduction in SPD1+GFP rats than SOD1+CAT rats. Asterisk indicates a significant (p < 0.05) difference by ANOVA. GFP rats (n=14) (black bars); SOD1+GFP rats (n=12) (red bars); SOD1+CAT rats (n=10) (blue bars). Error bars represent S.E.M.

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CHAPTER 5

GENERAL DISCUSSION

Summary

Aging is associated with changes in physical performance and cognitive

impairment including deficits in learning and memory (Foster, 1999, 2012), which are

thought to be due to overproduction of free radicals (Harman, 1992). The free-radical

theory of aging indicates that the overproduction of endogenous ROS, including

superoxide, hydrogen peroxide, and hydroxyl radicals result in a pattern of cumulative

damage due to modification of proteins, nucleic acids, and polyunsaturated fatty acids

of the lipid membrane (Leutner et al., 2001a; Liu et al., 2002; Oliver et al., 1987; Smith

et al., 1991). Antioxidant therapy for cognitive function through supplement or

antioxidant enriched diet have little effect on normal aging possibly due in part to poor

penetration of the CNS through the blood brain barrier and (Bowry et al., 1992;

Grodstein et al., 2003; Maxwell et al., 2005). Alternately, antioxidant enzymes are the

major factors for defending ROS in the brain. Studies employing transgenic mice

indicate overexpression of SOD1 improves synaptic plasticity and memory during aging

(Kamsler et al., 2007; Kamsler and Segal, 2003b). Upregulation of SOD2, on the other

hand, has no obvious effect on synaptic plasticity or memory (Hu et al., 2007a). It is

unclear whether the improvement is due to a lifetime of overexpression, decreasing the

accumulation of oxidized molecules, or if increasing antioxidant enzymes in older

animals could reduce oxidative damage and improve cognitive function. In our

preliminary studies, we used lentivirus to modestly increase SOD1 expression in the

CA1 region of young and aged F344BN F1 rats. We tested the rats in various

behavioral tasks and found that modest overexpression of SOD1 in CA1 improved

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learning in the spatial version of water maze in aged rats but had no effect in young rats

(Lee, 2008). Overexpression of SOD1 also did not affect rats’ performance in cue

discrimination, inhibitory avoidance or novel object recognition. However, lentivirus

mediated expression in the hippocampus was generally limited and variable. We

therefore, increased the transduction area using AAV in order to test the hypothesis that

overexpression of antioxidant enzymes (SOD1, SOD2, CAT and SOD1+CAT) in whole

hippocampi will delay age-related memory decline by reducing oxidative damage. This

study examined memory-related behavioral performance of the young (4-month) (19-

month) rats one month and four months post viral injection. The results from this study

indicated that overexpression of antioxidant enzymes reduced oxidative damage;

however, memory function was not related to the level of oxidative damage.

Overexpression of antioxidant enzymes did not affect learning and memory in young

rats. Further, increased expression of SOD2 and CAT did not affect learning and

memory in aged rats. Surprisingly, overexpression of SOD1, initiated in advanced age,

impaired learning. Moreover, increased expression of SOD1+CAT provided protection

from impairments associated with overexpression of SOD1 alone and appears to guard

against cognitive impairments in advanced age. An examination of protein expression

using western blots, revealed that aged rats with overexpression of SOD1 had

increased expression of GPx1. It was concluded that the learning impairment observed

in aged SOD1 rats might be due to overproduction of H2O2.

Previous studies from our laboratory suggest that a more oxidized intracellular

redox state mediates a decrease in NMDAR function, which could underlie impaired

cognition (Bodhinathan et al., 2010a, b). Therefore, we designed a serious of

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experiments to examine the hypothesis that overexpression of antioxidant enzymes in

the hippocampi of aged rats would affect NMDAR function via regulating redox state.

Again, we used AAV to deliver SOD1+GFP or SOD1+CAT to the hippocampus of aged

(17-month) F344 rats and examined memory-related behavioral performance one-

month post injection. The results confirmed that SOD1 overexpression was associated

with impaired performance on the spatial version of the water maze. Importantly, SOD1

overexpression was associated with a decrease in NMDAR-mediated fEPSP.

Biochemical assays indicated that free glutathione, oxidized glutathione and total

glutathione decreased in aged rats with overexpression of SOD1. In addition, both

glutathione peroxidase and glutathione reductase activities decreased in aged rats with

overexpression of SOD1, which might result from altered redox state of the enzymes.

In conclusion, viral vector gene delivery provides a novel approach to test the

hypothesis that increased expression of antioxidant enzymes, specifically in

hippocampal neurons, will provide protection from age-related cognitive decline. The

results indicate that oxidative stress is a likely component of aging; however, it is

unclear whether increased production of ROS or the accumulation of oxidative damage

is the primary cause of functional decline. The results provide support for the idea that

altered redox sensitive signaling rather than the accumulation of damage may be of

greater significance in the emergence of age-related learning and memory.

Discussion

In this series of studies, we asked an important question in the cognitive aging and

memory field. Does ROS/ oxidative damage cause memory impairment during normal

aging? If so, can we rescue the memory deficits by overexpressing antioxidant enzymes

in the hippocampi using viral vectors? Surprisingly, enhanced overexpression of SOD1

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in hippocampi using AAV did not help with memory in aged rats; instead, it impaired

memory. This result led us to examine the importance of tightly controlled antioxidant

system across life span.

One possibility is that SOD1 overexpression is toxic. Mutant SOD1 was

discovered to be the primary cause of 15 to 20 % of familial ALS cases (Rosen et al.,

1993). One possible mechanism proposed for ALS emphasizes reduced SOD activity

arising from instable or imperfectly folded enzymes, which could cause toxic oxidative

stress through an imbalance in oxidative defenses (Deng et al., 1993; Orrell et al., 1995).

However, increased expression of wild-type SOD1 in mice with ALS does not slow

disease progression or diminish the pathological changes in motor neurons (Brown,

1998). The failure of increased SOD1 activity to ameliorate the disease (Brown, 1998),

in addition to the fact that some SOD1 mutants retain high SOD activity (Ratovitski et al.,

1999), and targeted deletion of SOD1 in mice does not induce ALS-like symptoms

(Reaume et al., 1996) challenges the idea that mutant toxicity is linked to oxidative

stress arising from O2·-. Rather, mutant SOD1 is proposed to cause disease by the

acquisition of toxic properties through misfolded mutant SOD1 aggregates (Johnston et

al., 2000; Wang et al., 2003b). A recent study using conformation-specific antibody that

detects misfolded SOD1 (C4F6) found that oxidized wild-type SOD1 and mutant SOD1

share a conformation epitope that is not present in normal wild-type SOD1 but

commonly detected in motor neurons in the lumbosacral spinal cord in a subset of

human sporadic ALS (SALS) cases. The authors suggest that wild-type SOD1 can be

pathogenic in SALS most likely by dysregulating kinase signaling pathways (Bosco et

al., 2010).

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In our current study, the memory impairment observed in aged rats is most likely

not due to SOD1 toxicity for several reasons. First, the memory deficit was limited to

aged SOD1 animals, indicating that it was not simply due to SOD1 overexpression.

Second, one would expect that toxicity should increase over time. On the contrary, we

observed that the memory deficits decreased following four months of SOD1

overexpression in our oldest rats. However, it would be interesting for future studies to

examine the effects of overexpression of wild-type SOD1 under severe oxidative stress

conditions such as in animal models of neurodegenerative diseases.

H2O2 Produced by Overexpression of SOD1 Affects Synaptic Plasticity and Memory in an Age-Dependent Manner

We emphasize the importance of H2O2 as a redox state regulator and suggest that

overexpression of SOD1, which converts O2·- into H2O2 in the hippocampus was

detrimental to learning and memory in aged rats. Several pieces of evidence point to an

increase in H2O2 in aged rats that overexpress SOD1. First, the memory impairment

was only observed in aged rats with overexpression of SOD1, and the impairment could

be rescued by co-overexpression of CAT in the same animal. CAT converts H2O2 to

water, suggesting that the rescue was due to a decrease in an elevated level of H2O2.

Similarly, GPx1 expression, examined by Western blot analysis, was increased in aged,

but not young, rats with overexpression of SOD1. Previous work in other tissues

indicates that elevated H2O2 induces an increase in GPx level (Hussain et al., 2004;

Noack et al., 1998; Rohrdanz et al., 2001; Wijeratne et al., 2005). Finally, the increased

GPx1 expression was not observed in aged rats with overexpression of SOD1+CAT.

The results support the idea that SOD1 overexpression was associated with an

elevated level of H2O2 in aged, but not young rats.

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Young (5-month old) rats with one-month overexpression of SOD1 did not exhibit

memory deficits; however, as the rats aged (9-month old), following four months of

overexpression of SOD1, a mild memory deficit started to emerge. In a study using

superoxide sensitive probes to detect O2·- across life span of mammals and birds, an

age-related increase in O2·--dependent chemiluminescence was observed in the brains

of C56/BL6 mice, Wistar rats, and pigeons, with no change of SOD1 and SOD2 activity

(Sasaki et al., 2008). SOD1 converts O2·- to H2O2, suggesting that overexpression of

SOD1 may have increased H2O2 in the hippocampus as O2·- increased with advancing

age.

GSH is the major redox buffer for cellular H2O2. We observed that GSH, GSSG

and total GSH levels were decreased in SOD1+GFP rats, consistent with the idea that

the intracellular environment was in a more oxidized state, possibly due to an increased

level of H2O2. Although normally a reduction of GSSG indicates a more reduced

environment, the combination of decreased GSSG, in addition to loss of free GSH and

total GSH, in SOD1+GFP rats suggests a serious loss of GSH. A question remains

concerning the mechanism for the decrease in GSH. In this case, an increase in H2O2

may have induced oxidative inactivation of GSH redox cycling proteins such as GPx

and GR. We observed decreased activity of GPx and GR in aged rats with

overexpression of SOD1+GFP. Decreased GSH redox cycling function may have

contributed to the decreased level of GSH, GSSG and eventually insufficient antioxidant

buffering system.

Finally, NMDAR activity is required to control many physiological conditions

including neuronal development, synaptic plasticity and memory. NMDAR function is

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known to be redox sensitive. H2O2 induces the formation of disulfide bonds between

pairs of cysteine residues in proteins, and thus change the structure and function of

these proteins, including proteins involved in multiple signaling cascades including Ca2+

signaling (Foster, 2007). Indeed, application of the cysteine specific reducing agent,

dithiothreitol, increased NMDAR but not AMPAR synaptic response and the magnitude

of LTP in hippocampal slices from older rats (Bodhinathan et al., 2010a). The

membrane-impermeable reducing agent, L-GSH, failed to increase the NMDAR

response when applied on the hippocampal slice; however, an increase of NMDAR

response was observed by intracellular delivery of L-GSH through the intracellular

recoding pipette. The results indicate that the intracellular redox state, rather than the

disulfide bonds on the extracellular cysteine residues, mediates the suppression of

NMDAR function and impaired LTP in aged rats. In our present study using rats with

overexpression of SOD1, the production of H2O2 is from the intracellular space;

however, because H2O2 is membrane permeable, we cannot rule out the possibility that

the newly synthesized H2O2 also affect extracellular cysteine residues. Regardless, the

fact that NMDAR, but not AMPAR synaptic responses were reduced in aged rats with

overexpression of SOD1+GFP provides further support for the idea that H2O2 produced

by SOD1 overexpression resulted in an oxidized redox environment in older animals.

Taken together, the weight of evidence indicates that SOD1 overexpression in aged

animals resulted in an increase in the level of H2O2.

Interestingly, aged (20-month old) rats with one-month overexpression of SOD1

showed severe memory deficits; however, the memory deficit became moderate with

advancing age (24-month old), following four-months overexpression of SOD1. In the

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Sasaki study, the increase in O2·- level from birth until 20-month of age began to decline

with more advanced age in the brains of C56/BL6 mice (Sasaki et al., 2008). Similarly,

in tg-SOD1 mice, overexpression of SOD1 had an age-dependent effect on memory.

SOD1 overexpression impaired or had no effect on learning in young mice and

enhanced spatial memory in older mice (Kamsler et al., 2007; Kamsler and Segal,

2003b). The authors suggest that aged mice adapt to elevated H2O2. The results of the

present study, indicating no effect of memory in young SOD1 rats, severe memory

impairment in old SOD1 rats and moderate memory impairment in more advanced aged

SOD1 rats providing new evidences for the hypothesis that ROS generation increased

with age, and elevated ROS might be beneficial or less detrimental to animals at a more

advanced age.

In fact, one of the main driving forces for the current study was previous work

indicating that overexpression of SOD1 in aged mice was beneficial. Although

overexpression of SOD1 in young transgenic mice was found to be associated with

impaired LTP and impaired memory (Kamsler and Segal, 2003b), overexpression of

SOD1 in aged transgenic mice was associated with enhanced LTP and memory

compared to their age matched wild-type littermates (Kamsler et al., 2007; Kamsler and

Segal, 2003b). The authors proposed that H2O2 regulates synaptic plasticity in a

complex manner that depends on an interaction between the pre-existing redox state

and a transient, tetanic stimulation-induced rise in H2O2. According to their hypothesis,

young SOD1 transgenic mice that overproduce H2O2, contain an intracellular redox

environment with higher level of H2O2. The intracellular proteins therefore are

desensitized to the small rise of H2O2 that follows titanic stimulation, which cannot pass

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the threshold necessary for producing LTP. The aged SOD1 transgenic mice have a

genetic desensitization of H2O2 caused by the overexpression of SOD1, accompanied

by age-dependent mitochondrial leakage (Beckman and Ames, 1998) that produces

more H2O2, resulting a state that is again sensitive to acute change of H2O2, promoting

LTP (Kamsler et al., 2007; Kamsler and Segal, 2003b). This hypothesis may be limited

to explain the age-dependent effects of overexpression of SOD1 on LTP induction in

hippocampal slices. However, if we consider that training and testing in the water maze

involves neural activity and activation of LTP mechanisms, our results could fit into this

hypothesis with a few exceptions. First of all, 4-month SOD1 rats had normal memory,

which is different from young SOD1 transgenic mice that were impaired in LTP and

memory. The different results might be explained by differences in the duration, extent,

or location of SOD1 overexpression. For our studies, SOD1 expression was increased

4-fold for one month in neurons of the hippocampus. In the case of transgenic mice,

SOD1 expression was increased 6-fold during development until 2-month of age in most

of the cells, if not all, including cells in the brain (Epstein et al., 1987). Again it should

be noted that a moderate memory impairment started to show up in our 9-month old

SOD1 rats with 4-month overexpression of SOD1, suggesting increased H2O2 at this

stage. In addition, our 20-month old SOD1 rats were memory impaired, which was

different from old SOD1 transgenic mice that exhibited enhanced LTP and memory.

Thus, age is an important variable. Consider the medium lifespan of SOD1 transgenic

mice is shorter than F344BN F1 (30 months versus 34 month)(Perez et al., 2009b;

Turturro et al., 1999), the 2-year old transgenic mouse was in a more advanced age

compared to a 20-month old F344BN F1 rats. Finally, at 24 months, the SOD1 rats with

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4-month overexpression of SOD1 were not significantly different from the GFP rats at

the same age. It appears that the SOD1 rats may have adapted to the increase in

SOD1, exhibiting similar memory function compared to the GFP rats at this older age.

Future studies may want to examine the correlation of ROS generation and

overexpression of SOD1 in middle age rats, when ROS generation starts to exceed the

antioxidant capacity and in very old rats when ROS generation starts to slow down and

the endogenous level of ROS at this stage may not be sufficient for normal physiological

function.

Co-Overexpression of SOD1 and CAT in the Hippocampi is More Beneficial Than Overexpression of SOD1 or CAT Alone

Overexpression of SOD1 has also been reported to have several deleterious

effects, such as neuromuscular abnormalities and premature thymic involution,

suggesting that upregulation of SOD1 can lead to a toxic buildup H2O2, which can be

eliminated by the additional increase in CAT activity (Peled-Kamar et al., 1995; Peled-

Kamar et al., 1997). In the present study, we demonstrated that relative to animals that

overexpress SOD1, co-overexpression of SOD1 and CAT in hippocampi rescued

memory deficits observed in 20-month old F344BN F1 rats with overexpression for one

month, improved memory in 24-month old F344BN F1 rats with 4-months

overexpression, and improved memory in 18-month old F344 rats with one-month

overexpression.

If a high level of H2O2 during aging impairs memory, can removing H2O2 by

overexpression of CAT alone be beneficial? In our present study, we demonstrated that

overexpression of CAT alone in hippocampi has little effect on memory. The result may

be due to the fact that CAT activity can be inhibited by O2·- (Kono and Fridovich, 1982;

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Shimizu et al., 1984). Therefore, co-overexpression of SOD1 and CAT could interact to

be more beneficial than overexpression of SOD1 or CAT alone by keeping CAT in an

active form through the increased elimination of O2·- and permitting CAT to remove

excess H2O2. Future studies may want to determine the protective effects of

overexpression of SOD1, CAT and co-overexpression of SOD1 and CAT against ROS-

mediated tissue injury including ischemia and reperfusion injury, hypoxic lung injury,

brain trauma, and neurotoxic drugs.

Mitochondrial ROS Has Minimal Effect on Synaptic Plasticity and Memory

Mitochondrial generation of O2·- during normal aerobic metabolism has been proposed

to be an important link between oxidants and aging (Balaban et al., 2005). However, in

a study of overexpression the mitochondrial SOD (SOD2) in transgenic mice, there was

no effect on LTP induction or spatial learning in the water maze (Hu et al., 2007a). The

results from our current study using AAV to upregulate SOD2 expression in hippocampi

were consistent with the SOD2 transgenic mice study. Overexpression of SOD2 in

hippocampi of young or aged rats had no effect on memory. The lack of influence of

SOD2 overexpression on synaptic plasticity and memory may be due to the

phospholipid bilayer structure of the mitochondrial membranes that does not allow free

diffusion of O2·- generated within mitochondria (Zeevalk et al., 2005). One study points

to a mitochondria membrane protein, voltage-dependent anion channels (VDAC), in

controlling the release of O2·- from mitochondria to the cytosol (Han et al., 2003).

Together with our results and the results from SOD2 transgenic mice, mitochondrial

ROS does not appear to contribute in a major way to synaptic plasticity and memory

under normal physiological conditions. In contrast, mitochondrial ROS has been

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reported to play major role in memory dysfunction in neuropathological conditions, such

as Alzheimer’s disease (Dumont et al., 2009; Ma et al., 2011; Massaad et al., 2009).

Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) as Signal Molecules in Synaptic Plasticity

One idea related to SOD1 and H2O2 influences on memory is that H2O2 may act

as a second messenger that is critical for memory. Second messenger molecules are

generated at the time of receptor activation, are short-lived, and act specifically on

effectors to transiently alter their activity (Forman et al., 2004). Indeed, ROS can be

generated at the time of receptor activation and are short-lived, but the specificity of

their action has been difficult to assess. Clearly, a reactive species such as OH cannot

have any specificity, as it reacts at nearly the rate of diffusion with almost any molecule.

H2O2 and O2·-, on the other hand, are less reactive. H2O2, in particular, has gained

credence as a second message as it is a highly diffusible molecule that can be

produced endogenously via receptor-mediated mechanisms in many cell types

(Lambeth, 2002) and is enzymatically metabolized. The targets of H2O2 include thiol

proteins, but these proteins have yet to be fully defined.

Targets for ROS modification include protein kinases, protein phosphatases, and

transcription factors that are involved in long-lasting LTP induction and long-term

memory formation. For example, LTP involves activation of extracellular signal-

regulated kinase (ERK), protein kinase A (PKA), protein kinase C (PKC), and

neurogranin (NG) (a PKC substrate that binds to and sequesters calmodulin (CaM)).

Neurogranin promotes the release of CaM, and thereby promotes Ca2+/CaM signaling

via the activation of CaMKII (Huang et al., 2000; Kishida et al., 2005; Klann et al., 1993;

Li et al., 1999). In contrast, ROS can inhibit protein phosphatases such as calcineurin

134

(protein-phosphatase 2B; PP2B), which has opposite effects to CaMKII and PKC

(Winder and Sweatt, 2001). ROS can also directly activate transcription factors such as

the redox-sensitive transcription factor NF-KB, or indirectly influence transcription factors

via the activity of ERK, PKA and CaMKII (Brigelius-Flohe et al., 2004). Similar to ROS,

NO and RNS can act as signaling molecules and are known to target thiol proteins and

metalloproteins (proteins contain a mental ion cofactor). RNS are capable of chemically

modifying critical thiols on numerous proteins and forming S-nitrosothiols, leading to

disruption and/or regulation of the target protein. Many proteins have been reported to

be at least partially regulated by S-nitrosylation, including NMDARs (Lipton et al., 1993).

Taken together, ROS/RNS signaling is critical for a number of cellular processes,

including synaptic plasticity and memory formation. Thus, indiscriminate suppression of

ROS/RNS may not always be an appropriate therapeutic method for treating

oxidative/nitrosative stress. The current study provides evidence for the complexity of

the ROS system and suggests the futility of modifying a single enzyme. Future studies

may want to examine possible differences in ROS/RNS signaling cascades in memory

impaired and memory unimpaired animals, and determine the proper ratio of ROS/RNS

and antioxidant enzymes as a potential therapeutic avenue.

LTP During Aging

LTP is thought to mediate learning and memory. Furthermore, LTP declines with

advancing age and depends on NMDAR function, which in turn is sensitive to the redox

environment. Therefore, in the current study we examined LTP induction in aged rats

with overexpression of SOD1+GFP, SOD1+CAT and GFP. The results indicated that

the LTP level was not changed in any treatment group and appear to contradict the

135

results in the study using SOD1 transgenic mice, in which overexpression of SOD1 in

aged transgenic mice results in larger LTP than that found in wild-type mice (Kamsler

and Segal, 2003b). The different results might be due to the different protocols used to

induce LTP in these two studies. In Kamsler’s study, LTP was induced by theta burst

stimulation (TBS) (10 trains of 4 pulses at 100 Hz separated by 200 msec intertrain

intervals, at the same intensity as the test stimulation). TBS is patterned after innate

rhythms in the hippocampus and is a relatively mild stimulation that is less likely to

saturate the LTP generating mechanism (Morgan and Teyler, 2001). In contrast, in our

study, LTP was induced by four episodes of high-frequency stimulation (HFS; 100 Hz, 1

s with 1 s interval). Compared to the 40 pulses using the TBS pattern, the 400 pulses

employed in the current study is relatively strong. It has been suggested that aging is

not associated with a difference in the magnitude of LTP, but an increase in the

threshold to induce LTP (Foster, 1999, 2012). Thus, age-related differences are not

observed when relatively strong induction stimulation is employed. In a previous study

examining redox influences on LTP in aged animals, a single burst of 100 Hz for 1 s

was able to distinguish between slices bathed in the reducing agent DTT and control

slices (Bodhinathan et al., 2010a). Therefore, the reason we were not able to see the

difference of LTP between treatment groups might be due to the failure to use a LTP

induction protocol that is near the threshold for LTP induction. Future studies may want

to work on finding the right LTP induction protocols that are suitable to distinguish the

fine synaptic plasticity difference in middle age animals.

Global Versus Local Redox State

The GSH: GSSG ratio is used to describe the global state of oxidized and reduced

species in the cell and is thought to play a major role in maintaining the cellular redox

136

state (Schafer and Buettner, 2001; Schafer et al., 2003). However, it has been argued

that since signal transduction reactions are not global but localized processes, there is

not a direct connection between the global GSH:GSSG ratio and any specific redox

signaling reaction (Forman et al., 2004). The importance of localized events is well

demonstrated by the sudden increase of cAMP which occurs after stimulation and

subsequent activation of the protein kinase A (PKA), even though enough

phosphodiesterase is present in the cell to decrease cAMP to its pre-stimulation level.

Thus cAMP activates PKA only within a short distance allowed by the

phosphodiesterase. Similarly, in our study, it seems that the global reduction of the GSH:

GSSG ratio should not be used to define the local redox state. In other word, the local

concentration of H2O2 may be more important in affecting its target molecules.

In this study, we observed abnormal level of GSH and GSSG and decreased

activities of glutathione peroxidase (GPx) and glutathione reductase (GR) in rats with

overexpression of SOD1+GFP. The combination of decreased GSSG in addition to loss

of free GSH and total GSH in SOD1+GFP rats suggests a serious loss of GSH, possibly

due to deficits in GSH redox cycling. Glutathione levels are maintained by the GSH

redox cycle consisting of glutathione peroxidase and glutathione reductase.

One possibility is that GPx and GR function was modified by the altered redox

environment. GPx’s are a family of peroxidases that contain a rare amino acid,

selenocysteine, which is essential for peroxidase activity and require GSH as a co-

substrate (Rocher et al., 1992; Tappel, 1984). GPx catalyzes the cellular reduction of

inorganic H2O2, lipid peroxide or organic hydroperoxide to water or corresponding

alcohols (Prabhakar et al., 2005). Selenium (Sec) from the active site of GPx is in the

137

same column of elements in the periodic table as sulfur, and thus shares many

chemical properties with it. Therefore, this selenocysteine residue resembles a cysteine

residue in terms of chemical properties but with a higher reactivity (Huber and Criddle,

1967; Stadtman, 1980). The selenocysteine active site of GPx is highly sensitive to

oxidation modification. NO-mediated oxidation of the GPx selenocyeteine (oxidization of

Sec45 to form a selenenyl sulfide (Se-S) with a free thiol) has been shown to inactivate

the enzyme (Asahi et al., 1997). Similar to GPx, GR is another GSH related enzyme.

GR reduces disulfide glutathione (GSSG) to the sulfhydryl form of glutathione (GSH). Its

enzyme activity has been reported to be reversibly inactivated under oxidative stress

induced by myocardial ischemia-reperfusion injury (Yim and Ko, 1999), cardiac arrest

(Sharma et al., 2007), catecholamine (Remiao et al., 2000), and by S-nitrosoglutathione,

an endogenous NO donor (Becker et al., 1995). In conclusion, due to the fact that GPx

and GR are inactivated by a variety of oxidative modifications, it is reasonable to

hypothesize that the reduced activities of GPs and GR observed in rats with

overexpression of SOD1+GFP were at least partially due to chronic exposure to high

level of H2O2. See a model for this hypothesis (Fig. 5-1)

138

Figure 5-1. A model for altered redox state in aged rats with overexpression of SOD1.

Overexpression of SOD1 in aged rats results in an elevated level of H2O2 that cause oxidative modification on the structure and function of GSH-related enzymes, such as GPx and GR. Reduced activities of GPx and GR further influence normal GSH cycling, leading to decreased level of GSH and GSSG. The gray stars indicate oxidative modification

139

APPENDIX

ADDITIONAL FIGURES

Figure A-1. Control images for hippocampal immunofluorescence. (A) Hippocampi from a 25-month old rat injected with AAV-SOD1-myc showed intense myc (green) staining after incubating with myc-tag mouse monoclonal primary antibody and goat-anti mouse Alexa 488 secondary antibody. (B) No primary antibody negative control. Little to no staining was observed in hippocampi incubated with secondary antibody only. (C) Mouse IgG2a was used as an isotype

140

negative control. Little to no staining was shown in hippocampi. There was some non-specific background signal staining which was mainly observed in fiber tracts (e.g. corpus callosum, fimbria) appearing outside the hippocampi. All the images were merged with nuclear DAPI staining (Blue). All the images were taken with exposure time of 250 ms (green channel) and 700 ms (blue channel). Calibration bars represent 500 µm.

141

Figure A-2. SOD1 and myc expression are co-localized in the hippocampus of a rat with

overexpression of SOD1. Immunofluorescence shows a SOD1 staining in red (left), a myc staining in green (center), and a merged image of SOD1 and myc in yellow (right). White boxes in lower left indicate enhanced images of a small part of CA3 area labeled with dotted white box.

142

Figure A-3. Expression of corresponding antioxidant enzymes are higher in the hippocampus of rats with injection of AAV-SOD1, AAV-SOD2, or AAV-CAT. Immunofluorescence of endogenous expression of (A) SOD1 (red) and (E) CAT (red) in AAV-GFP rats. (C) Endogenous expression of SOD2 (green) in AAV-SOD1+CAT rats. (B, D, F) Immunofluorescence of overexpression of SOD1 (red), SOD2 (green) and CAT (red) in AAV-SOD1 rats (B), AAV-SOD2 rats (D) and AAV-CAT rats (F).

143

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BIOGRAPHICAL SKETCH

Wei-Hua Lee was born in 1980 at Taipei, Taiwan. She graduated from National

Taiwan Normal University in 2003, where she received a Bachelor of Science degree in

biology. She came to the United States in 2004 to begin her journey of being a

biological researcher. Wei-Hua Lee enrolled the University of Iowa in 2004 and joined

the lab of Dr. Chun-Fang Wu for her Master of Science degree. After that she joined Dr.

Thomas Foster lab at University of Florida, where she was focusing on studying

memory deficits during normal aging, and eventually dedicated herself into

understanding the relationship between aging, reactive oxygen species, synaptic

plasticity and memory. Wei-Hua Lee was active in her academic career and had

participated in several national conferences. Her scientific discoveries had been

published in different journals. She received her Ph.D. from the University of Florida in

the summer of 2012. In the future she will devote herself to help the community and

human society as a scientist by her research.