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NO mediated neurotoxicity: redox changes and energy failure in a neuroinflammatory model

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NO mediated neurotoxicity: redox changes and energy failure in a neuroinflammatory model

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.
NO MEDIATED NEUROTOXICITY: REDOX CHANGES AND ENERGY FAILURE
IN A NERUOINFLAMMATORY MODEL

by

Li-Peng Yap

_____________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

May 2008

Copyright 2008 Li-Peng Yap

ii
DEDICATION

This thesis is dedicated to my parents Hon and Lily, for their love and dedication to
being the best parents to us girls, to my sisters for their love and the sisterhoods that
we have, and finally, to all the people who have touched and inspired me along the
way.

“The fear of the Lord is the beginning of wisdom, and knowledge of the Holy One is
understanding.” (Proverbs 9:10)

iii
ACKNOWLEDGEMENTS

I would like to express my heartfelt gratitude to my advisor Enrique Cadenas, M.D,
Ph.D for his generosity, guidance, patience and support all through my years in his
laboratory. To Jerome Garcia Ph.D and Derick Han Ph.D, who have always been
willing to teach, help and just be there for me. My appreciation for my current and
former laboratory members, Philip Lam Ph.D, Juliana Hwang PharmD, Allen Chang
Ph.D, Qiong Zhang Ph.D, Ryan Hamilton Ph.D, Lulu Tang, Fei Yin and Li Chan, who
made coming to the laboratory an enjoyment and my graduate years colorful. Ha-Van
Nguyen Ph.D for her invaluable advice and opening my eyes. Joanne Lee, for being
the surrogate mother in the laboratory, constantly taking care of our needs. And last
but not least, Dave, for keeping me sane.

Special acknowledgements also to my dissertation committee, Roberta Diaz-Brinton
Ph.D and Neil Kaplowitz M.D, for their advice and kindness towards me.

iv
TABLE OF CONTENTS

Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures vii
Abstract viii
General Introduction 1
Hypothesis and Specific Aims 1
Background 6
- Nitric Oxide- Role in Brain Function and Pathology 8
- Neuroinflammation - The role of microglia and astrocytes 10
- The Aging Brain – A risk factor for the development of
Neurodegeneration 12
- The cellular redox environment – A determinant of cellular health 18
- Protein thiol modifications – Redox sensitive switches 22

Significance 31

Chapter I:
NO modulation of cellular redox status and protein S- glutathionylation-
Implications for Neurodegeneration 33
Introduction 33
Materials and methods 37
Results 40
Discussion 62

v
Chapter II:
Mitochondrial Energy Axis- A Determinant of Cell Viability 76
Introduction 76
Materials and methods 79
Results 81
Discussion 91

Chapter III: Mitochondria as a cytosolic redox modulator: Topology of
Peroxynitrite generation 99
Introduction 99
Materials and methods 102
Results 103
Discussion 118

Conclusions 122

Future Perspectives 125

Bibliography 128

vi
LIST OF TABLES

Table I: Tabulated values of GSH, GSNO and GSSG formation in primary
neurons and astrocytes 44

Table II: Calculated Nernst Potential for primary cortical neurons and
astrocytes exposed to
.
NO 45

vii
LIST OF FIGURES

Fig. 1: Outline of Specific Aims 8
Fig. 2: Overview of the cytosolic synthesis of Glutathione from precursor
amino acids. 21

Fig. 3: Impact of Oxidative Stress and nitrosative stress on protein function 24
Fig. 4: S-glutathionylation and S-nitrosylation of proteins by GSNO 30

Fig. 5: Intracellular GSNO and GSSG formation after exposure of
primary cortical neurons to
.
NO. 41

Fig. 6: Intracellular GSNO and GSSG formation after exposure of
primary cortical neurons to
.
NO. 42

Fig. 7: Metabolism of GSNO by primary cortical neurons 48

Fig. 8: Metabolism of GSNO by primary cortical neurons 49

Fig. 9: GSNO and GSSG reductase activity 51

Fig. 10: Effects of
.
NO on viability 52

Fig. 11:
.
NO induces glutathionylation in primary cortical
neurons and astrocytes 55

Fig. 12: Glyceraldehyde phosphate dehydrogenase (GAPDH) activity in
primary cortical neurons after exposure to
.
NO 57

Fig. 13: GAPDH is glutathionylated in neurons during
.
NO exposure 59

Fig. 14: GAPDH glutathionylation following treatment with GSSG 61

Fig. 15: Glutathionylation of GAPDH and decrease in activity is age
dependent in the triple transgenic Alzheimer’s disease model 63

viii

Fig. 16: Glutathionylation of GAPDH results in modulation of glucose
metabolism 75

Fig. 17: Effects of
.
NO on viability 82

Fig. 18:
.
NO leads to a depletion of cellular ATP levels 83

Fig. 19: Effects of mitochondrial energy substrates on viability and ATP levels 86

Fig. 20: NAD(P)H formation upon complex I substrate supplementation 88

Fig. 21: Formation of mitochondrial GSH depends on substrate availability
not electron flow through ETC. 90

Fig. 22: Schematic diagram demonstrating the hypothesized effect of
mitochondrial substrates on mitochondrial function 98

Fig. 23: Mitochondrial consumption of nitric oxide 105

Fig. 24: Cu,Zn SOD sensitive consumption of nitric oxide in mitoplasts 106

Fig. 25: Rate of
.
NO consumed by mitoplasts 108

Fig. 26: Effect of
.
NO on DCF fluorescence in brain mitochondria 110

Fig. 27: Formation of peroxynitrite by mitoplasts 112

Fig. 28: Topology of ONOO
-
formation in the intermembrane space 121

Fig. 29: Conceptualization of Risk Assessment Profile of the therapeutic values
mitochondrial energy substrates in
.
NO mediated neurotoxicity 128

ix
ABSTRACT

The nefarious role of
.
NO in neurodegenerative diseases such as Alzheimer’s
disease stems from the excessive production and labile nature of
.
NO. The
mechanisms of
.
NO redox signaling through protein post translational modification is
ill-defined in the neurodegenerative model. S-nitrosylation, which can be an
intermediary step leading S-glutathionylation of proteins has emerged as a well
characterized mechanism through which
.
NO reversibly regulates cell function.
However, there is little evidence concerning the regulation of S-glutathionylation of
proteins by
.
NO, which proteins are glutathionylated in neurons during nitrosative
stress, and the consequence of glutathionylation of proteins on overall cellular and
mitochondrial function. Therefore, the hypothesis to be tested is that nitric oxide
generated during neuroinflammation leads to changes in cellular redox status and
protein post translational modification which play a role in energy impairment
leading to neuronal injury.
Our results indicate that acute exposure of neurons to
.
NO, mirroring
neuroinflammation, led to increase formation of ONOO
-
, oxidation of the cellular
redox environment and the formation of S-glutathionylated proteins. Exposure to
exogenous
.
NO also resulted in an alteration of cellular redox status through the
formation of intracellular GSNO and GSSG. Autoxidation of mitochondrial electron
chain components elicited by
.
NO resulted in the formation of a substantial portion of

x
ONOO
-
in the inter membrane space and outside of mitochondria due to the release of
O
2
.-
from mitochondria even in the presence of
.
NO. Hence, this increase in ROS and
RNS formation by mitochondrial could modulate mitochondrial redox status by
oxidizing GSH. Increasing concentrations of GSNO and GSSG formation as a
consequence of
.
NO exposure correlated with S-glutathionylation of proteins.
Glutathonylation of GAPDH, a key glycolytic enzyme, led to significant inhibition of
its activity. Mitochondrial energy substrates appear important for maintaining
mitochondrial GSH and cellular viability as
.
NO mediated neurotoxicity was
attenuated by the addition of a variety of mitochondrial energy substrates such as
pyruvate, malate and β-hydroxybutyrate. These observations delineate a potential
mechanism through which increase
.
NO production, a key event in neurodegeneration,
could potentially lead to protein S-glutathionylation and neuronal dysfunction seen in
neurodegenerative diseases such as Friedreich's ataxia and Alzheimer’s disease.

1
GENERAL INTRODUCTION

Hypothesis and Specific Aims
The generation of reactive oxygen and nitrogen species by microglia during
neuroinflammation has been implicated in the progression of neurodegenerative
diseases such as Parkinson’s disease and Alzheimer’s disease. It is well established
that when activated, microglia produce superoxide (O
2
.-
) and nitric oxide (
.
NO)
through the activation of NADPH oxidase and inducible isoform of nitric oxide
synthase, respectively.
Microglia-generated O
2
.-
and
.
NO, and their subsequent chemical products
such as ONOO
-
, have the ability to diffuse, and act at sites distal from the site of
production. Reactive oxygen and nitrogen species produced by microglia are involved
in the regulation of the redox status of neurons through the modulation of glutathione
(GSH), S-nitrosoglutathione (GSNO) and glutathione disulfide (GSSG) pools thereby
affecting neuronal function through (i) redox buffering capacity of the GSH/GSSG
couple, (ii) redox modulation of protein sulfhydryl groups and (iii) mitochondrial
function. Impairment or perturbations to the communication networks between these
components underlies the mechanism inherent in cell death and loss of cell function
relevant for neurodegeneration.

2
Hypothesis – The hypothesis to be tested is that the nitric oxide generated during
neuroinflammation leads to changes in cellular redox status and protein post
translational modification which play a role in energy impairment leading to neuronal
injury. This hypothesis is based on results obtained in this and other laboratories and
is supported by the following evidences:
a) Increased generation of Nitric Oxide during neurodegeneration and aging.
Excessive production of
.
NO in the brain has been implicated in a number of
neurodegenerative diseases including Alzheimer’s disease (AD) and
Parkinson’s disease (PD) (Duncan and Heales 2005; Brown 2007). Glial cells,
such as microglia and astrocytes, express the inducible isoform of nitric oxide
synthase and are significant sources of
.
NO in the brain (Bal-Price, Matthias et
al. 2002; Brown 2007). Microgliosis is related to the pathology of numerous
neurodegenerative diseases. As such, over activation of microglia precedes
symptomatic development and neutrophil destruction in AD and is localized
with degenerating dopaminergic neurons in PD. Additionally, a simultaneous
increase in activated microglia (Block, Zecca et al. 2007) and nNOS levels
occurs during aging (Lam and Cadenas 2007). This is important considering
that most neurodegenerative diseases are associated with aging. Taken
together, it may be surmised that during age associated neurodegenerative
diseases, there is an increase in intracellular and extracellular generation of
.
NO.

3
b) NO as a modulator of GSH homeostasis and cellular redox status. The cellular
redox status, as defined by the GSH/GSSG ratio, is an important indicator of
cellular health (Schafer and Buettner 2001; Maher 2006). Significant loss of
GSH is the earliest detectable biochemical event in PD and precedes, as well as
facilitate, mitochondrial impairment and dopaminergic cell death (Sian, Dexter
et al. 1994; Chinta, Kumar et al. 2007). A decrease in GSH content from red
blood cells isolated from male AD patients was also observed and was
attributed to decreased de novo synthesis of GSH (Liu, Harrell et al. 2005).
Exposure of neurons to
.
NO resulted in a significant loss of GSH levels (Gegg,
Beltran et al. 2003) and increased GSNO and GSSG formation (see Chapter
II). Furthermore, neurons lack the ability to upregulate glutamate-cysteine
ligase (GCL), the rate limiting enzyme in glutathione synthesis, during
nitrosative stress, compounding NO induced GSH loss (Gegg, Beltran et al.
2003). It may be surmised that changes in cellular GSH levels paralleled by
increase GSSG and GSNO formation (see section II), elicited by
.
NO exposure
may be a precipitating event in the pathological development of
neurodegenerative diseases.
c)
.
NO modulation of protein function through protein post translational
modifications. Alteration of redox status and increased ROS and RNS can lead
to chemical post-translational modification of redox sensitive cytosolic and
mitochondrial proteins such as aconitase (Han, Canali et al. 2005) and

4
mitochondrial complex I (Murray, Taylor et al. 2003; Taylor, Hurrell et al.
2003). Glutathionylation of ATP synthase and aconitase resulted in significant
loss of enzymatic activity (Han, Canali et al. 2005; Garcia, Han et al. 2007)
which may adversely affect overall mitochondrial bioenergetic capacity.
Additionally, an increase in S-glutathionylated proteins were observed in the
inferior parietal lobe of individuals afflicted with AD (Newman, Sultana et al.
2007), implying that alterations of protein function through glutathionylation
might play a role in altered cellular function in Alzheimer’s disease.
d) Mitochondria are direct targets of
.
NO. While almost all components of the
mitochondrial respiratory chain as well as ATP synthase may be inhibited by
.
NO in pathological conditions, cytochrome c oxidase remains the most
sensitive to NO induced inhibition (Cadenas 2004; Moncada and Bolanos
2006). Inhibition of cytochrome c oxidase by
.
NO results in an increase in
superoxide generation and inhibition of mitochondrial respiration (Boveris and
Cadenas 2000; Moncada and Erusalimsky 2002). Astrocytic derived
.
NO and
.
NO donors resulted in inhibition of mitochondrial respiratory chain and
irreversible damage in neurons (Stewart, Sharpe et al. 2000; Gegg, Beltran et
al. 2003).

5
Specific Aims – The validity of the hypothesis rationalized above will be tested
through three specific aims, which incorporate the following:
• Specific Aim 1: Characterize
.
NO modulation of cellular GSH redox status
in neurons and astrocytes. Determine the changes in the cellular GSH redox
status through (i) identify and quantify the GSH, GSNO and GSSG formed
intracellularly, (ii) measure the activity of GSNO and GSSG reductase activity
in neurons and astrocytes, and (iii) ascertain the changes in cellular Nernst
Potential driven by
.
NO.
• Specific Aim 2: Investigate the role of Protein Post Translational
Modification in the regulation of glucose metabolism. Determine the effects
of altered redox status on (i) the chemical modifications of sulfhydryl groups
of proteins relevant to glycolysis (ii) the consequences of these protein post
translational modifications on protein function, and (iii) overall cellular
function. The experimental designs within this specific aim will employ a
proteomic approach to identify redox sensitive proteins in neurons with
specific emphasis on proteins likely to modulate energy utilization in the cell
or mitochondrial function.
• Specific Aim 3: Modulation of mitochondrial function and neuronal viability
by
.
NO. Determine the consequence of reactive oxygen and nitrogen species
generated by activated microglia on neuronal mitochondrial function by
assessing changes in (i) the bioenergetic functions of mitochondria (ii) the

6
generation of reactive oxygen and nitrogen species within neurons and (iii) the
release of apoptotic factors. Results from specific aim 2 will be further
expanded herein to establish the significance of mitochondrial-cytosol
interactions in neurons within the context of metabolic, redox, and apoptotic
signaling models.
• Specific Aim 4: Characterize mitochondrial generation of reactive oxygen
and nitrogen species in the presence of
.
NO. Determine the (i) temporal and
(ii) quantitative generation of reactive oxygen and nitrogen species that is/are
critical for inducing changes in cellular redox status during nitrosative stress.

Background
Neurodegenerative diseases such as Alzheimer’s disease are estimated to affect 4.5
million Americans and the numbers are expected to grow to anywhere between 11.3
and 16 million by 2050 (Association; Foundation). There are also an estimated 1.5
million Americans suffering from Parkinson’s disease currently, with an additional
60,000 new cases each year (Foundation). These debilitating diseases are expected to
increase due to the aging of the baby boom population from the 1950s, considering
that aging is the most important risk factor for the development of neurodegenerative
diseases (Swerdlow and Khan 2004; Yankner, Lu et al. 2007). The idea that reactive
oxygen and nitrogen species generated during inflammation by microglia contributes

7
to the etiology of neurodegenerative diseases (Koutsilieri, Scheller et al. 2002; Liu
and Hong 2003) introduces the concept of an imbalance between pro-oxidants and
antioxidants and subsequent neuronal dysfunction and death. This concept is
exceptionally important in light of the reduced capacity for cellular regeneration of
post mitotic cells in the brain (Koutsilieri, Scheller et al. 2002; Andersen 2004),
making the organ highly susceptible to oxidative and nitrosative stress. The
identification of a fundamental role for oxidative and nitrosative stress in
neurodegeneration requires the recognition that the fate of a cell is inherently linked to
its redox status. Alterations in the redox status of the cell can lead to activation of
redox sensitive signaling cascades by the chemical modifications of key proteins, and
subsequently either directly or indirectly modulates the function of the mitochondria.
Impairment of a cell’s ability to buffer oxidative or nitrosative stress underlies the
mechanism inherent in death pathways and the loss of cell function relevant to the
process of neurodegeneration. The information below supports the formulation of the
hypothesis and specific aims. Emphasis will be placed on the role of iNOS expressed
in glial cells as well as nNOS in neurons as a source of
.
NO.

Nitric Oxide - Role in brain function and pathology
Activation of microglia and astrocytes leads to increased expression of inducible
isoform of nitric oxide synthase and the generation of
.
NO (Bal-Price and Brown
2001; Brown 2007). Nitric Oxide Synthases are flavohaem enzymes that

8

Figure 1. Outline of Specific Aims

9
catalyze the NADPH-and O
2
-dependent oxidation of L-arginine to Nitric Oxide (NO)
and citrulline, with N
ω
-hydroxyl-L-arginine formed as an intermediate (Vallance and
Leiper 2002). There are three isoforms of NOS, inducible or inflammatory NOS
(iNOS), neuronal NOS (nNOS) and endothelial NOS (eNOS) and these enzymes are
responsible for the biological production of nitric oxide. These enzymes have
approximately 50% sequence homology and are encoded by different genes. The
amount of NO generated by each isozyme varies with the inducible form (iNOS)
generating NO in the micromolar range while nNOS generates NO in the nanomolar
range (Martinez-Ruiz and Lamas 2004). The differential expression, activation state
and cellular and sub-cellular distribution of the NOS isoenzymes determine the
concentration and duration of NO production (Boyd and Cadenas 2002). It is also
noteworthy that NOS activity in the brain is higher than any other tissue(Duncan and
Heales 2005) and up to 1-4 μM of
.
NO was detected in vivo during ischemia
reperfusion in rat brain (Malinski, Bailey et al. 1993).
Nitric Oxide is a fairly unreactive radical that exists as a colorless gas and is
moderately soluble in water (~ 2-3 mM) (Fukuto 1995). Due to its non-polar nature, it
is able to freely diffuse through membranes and readily between and within cells. NO
has been estimated to be able to diffuse a distance of 10 μm in less then one second
(Halliwell and Gutteridge 1999). The ability for NO to function as a secondary
signaling molecule stems from its ability to bind to or react with transition metals or
metal-containing proteins and the subsequent activation of signaling cascades

10
(Boyd and Cadenas 2002). This is classically exemplified by the binding of NO to
the iron heme group of guanylate cyclase and subsequently activating the enzyme,
initiating the signaling cascade (Davis, Martin et al. 2001; Boyd and Cadenas 2002).
As important as its ability to act as diffusible signaling molecule, is its ability to
undergo numerous chemical reactions in aerobic aqueous solution. In aerobic aqueous
solution, NO undergoes autoxidation to form higher oxides of nitrogen, dinitrogen
trioxide (N
2
O
3
), which can partly dissociate into a caged radical species [NO
+
..NO
2
]
which is touted to be the quintessential nitrosating species that can chemically modify
protein sulfhydryls through the formation of an S-NO bond (Martinez-Ruiz and Lamas
2004). Aside from the formation of nitrosating species, another biologically significant
reaction of nitric oxide is with the ubiquitous radical, superoxide. In fact, the two
radicals react at a diffusion controlled rate to form peroxynitrite (ONOO
-
) a highly
reactive oxidant capable of modifying proteins through an interaction with tyrosine
and cysteine residues (Fukuto 1995). The biological effects of NO are determined by
its concentration. At low concentrations of NO, the direct effects of NO prevails but
when the concentration of NO increases, the indirect effects of NO (i.e. through the
formation of reactive nitrogen species and higher oxides of nitrogen) prevail (Davis,
Martin et al. 2001). Whether acting directly through the interaction of NO with metal
centers of proteins or indirectly through the formation of reactive nitrogen species that
can chemically modify proteins, the role of NO within the cell is numerous.

11
Neuroinflammation- The role of Microglia and Astrocytes
Although the etiologies of neurodegenerative diseases are complex and varied,
neuroinflammation is a common underlying component in their pathologies (Block,
Zecca et al. 2007; Brown 2007). Injection of amyloid β (A β) protein into the cerebral
cortex of rhesus monkeys resulted in profound neuronal loss and microglial
proliferation (Geula, Wu et al. 1998). Additionally, up regulation of genes involved in
immune function as well as increase microglial activation occur as a function of age
(Yankner, Lu et al. 2007). First defined and christened by the neuroanatomist Rio
Hortega (Streit 1995; Kreutzberg 1996) in 1932, Microglia represent about 5 to 12%
(Kreutzberg 1996; Gonzalez-Scarano and Baltuch 1999) of the glial cell population in
the entire central nervous system and are estimated to equal the number of neurons
(Streit 1995; Block, Zecca et al. 2007). Dubbed as the resident innate immune cells of
the brain, microglia in the adult nervous system can be clearly identified in three states
(i) resting ramified in non-pathological central nervous system (ii) activated or
reactive microglia in the pathological state and (iii) phagocytic microglia that
represent the full-fledged brain macrophage (Streit 1995). The nature of microglia are
as vigilant sensors of the microenvironment of the central nervous system, being
rapidly activated not only by changes in the brain’s structural integrity but also by
very subtle alterations in the microenvironment such as imbalances in ion
homeostasis. The activation of microglia is rapid and precedes the activation of other
cell types in the brain (Kreutzberg 1996). Of the plethora of inflammatory triggers,

12
notably A β and α synuclein can lead to the recruitment and activation of microglia and
astrocytes (Geula, Wu et al. 1998; Brahmachari, Fung et al. 2006; Block, Zecca et al.
2007). Recent data from our laboratory demonstrate that dopamine, a physiological
neurotransmitter, can lead to the up-regulation of iNOS in astrocytes as well. When
activated, microglia and astrocytes produce a plethora of neurotoxic factors such as
cytokines, tumor necrosis factor alpha (TNF α) and reactive oxygen and nitrogen
species (Gonzalez-Scarano and Baltuch 1999; Wood 2003). Microglia over activation
is an early pathogenic event that precedes neutrophil destruction and cluster with A β
aggregates before the development of symptoms (Block, Zecca et al. 2007),
suggesting that the release of neurotoxic factors by microglia play a precipitating role
in the development of at least Alzheimer’s disease. Additionally, recent evidence
demonstrates that astrocytes, which traditionally play a neuro-supportive role, can act
as immunocompetent cells as well through vigorous astrogliosis (Bal-Price and Brown
2001; Brahmachari, Fung et al. 2006). Activation of astrocytes with bacterial
lipopolysacharide and IFN- γ resulted in a 96-fold increase in iNOS expression(Chock
and Giffard 2006). Neuroinflammation seems to be responsible for a vicious cycle
where
.
NO released during glial activation initiates increase
.
NO production in neurons
through glutamate neurotoxicitiy (Bal-Price and Brown 2001; Golde, Chandran et al.
2002; Duncan and Heales 2005).

13
The aging brain – A risk factor for the development of Neurodegeneration
The fundamental aging process is the most significant variable that influences the
development of age-related neurodegenerative diseases (Hayflick 2007; Yankner, Lu
et al. 2007). Therefore, understanding the changes that occur during
neurodegenerative diseases, such as Alzheimer’s disease, must take into the account
changes that occur in biological pathways that are altered as a function of age. This
point is encapsulated in non human primate studies demonstrating that aging renders
the brain susceptible to amyloid β neurotoxicity (Geula, Wu et al. 1998). As such, lies
the golden question: what changes have to occur in which biological pathways that
transforms normal brain aging to pathological aging, giving rise to neurodegenerative
disease. Although the mechanisms that drive this transformation is unclear, we do
know that the distinguishing feature between normal brain aging and
neurodegenerative diseases is the extensive loss of neurons observed in the latter
(Yankner, Lu et al. 2007). Mitochondrial dysfunction with age may be accelerated in
neurodegenerative diseases or the threshold of cell impairment or energy thresholds in
brain mitochondria might be lowered in neurodegenerative diseases (Atamna and Frey
2007; Calabrese, Mancuso et al. 2007). As such, given the central role of mitochondria
and cellular redox status in neuronal function and apoptosis, it is safe to say that
impairment in mitochondrial functions is a key event in neurodegenerative diseases.
a) Mitochondrial energy axis - Dubbed as the cellular powerhouse of the cell,
mitochondria generate most of the energy needed for cellular function through

14
the conversion of fuel molecules into chemical energy through oxidative
phosphorylation. The conversion of fuel molecules into ATP is achieved
through three steps (1) incorporation of carbon into acetyl-CoA (2) oxidation
of carbon to produce reducing equivalents (NADH, FADH
2
), small amount of
ATP and CO
2
through the TCA cycle and (3) donation of electrons from
NADH and FADH
2
for the reoxidation of electron carriers in the electron
transport chain for the synthesis of additional ATP (Mathews, van Holde et al.
2000). The pyruvate to acetyl-CoA conversion, the entry of the latter to the
TCA cycle, and the reducing equivalents (NADH, FADH
2
) thereby generated
and channeled into the electron-transfer chain to generate energy (ATP) in a
proton-motive force-dependent manner constitutes the energy axis of the
mitochondria. Energy hypometabolism, specifically impaired glucose
utilization, is one of the most consistent and earliest abnormalities seen in mild
cognitive impairment and AD(Atamna and Frey 2007). Impaired glucose
utilization becomes significant considering that the brain almost strictly utilizes
glucose as its sole energy source. A 35% decrease in complex I activity in
brain and liver of old rats was observed and this decrease is close to the limit
of tolerable functional damage in terms of basal energy production. This
decrease is further compounded by a lower mitochondrial mass in neurons,
where aged neurons are unable to respond to increase in ATP demands
(Navarro and Boveris 2004). Changes in components of the electron transport

15
chain and the TCA cycle has been documented both in aging (Atamna and
Frey 2007; Lam and Cadenas 2007; Navarro and Boveris 2007), as well as
AD(Atamna and Frey 2007), highlighting once again a common mechanism of
impaired of mitochondrial energy utilization and generation.
b) Mitochondria are the major cellular source of oxygen radicals: – The
mitochondrial respiratory chain is the most significant redox system in aerobic
organelles and account for 98-99% of all O
2
consumption(Jones 2006). The
standard reduction potential for the conversion of O
2
to O
2
.-
is -0.16V. Given
the highly reducing environment of the mitochondria, many components of the
respiratory chain are thermodynamically capable of univalent reduction of
oxygen (Yap, Han et al. 2007). Following initial reports by Chance and
Boveris on the production of H
2
O
2
by intact mitochondria (Boveris, Oshino et
al. 1972), subsequent work in our laboratories established that O
2
.–
was the
stoichiometric precursor of mitochondrial H
2
O
2
and that it was primarily
generated during ubisemiquinone autoxidation (Boveris and Cadenas 1975;
Boveris, Cadenas et al. 1976; Cadenas, Boveris et al. 1977) and secondarily,
NADH-dehydrogenase activity (Turrens and Boveris 1980). The physiological
generation of O
2
.-
occurs due to the oxidation of the components of the electron
transport chain (complex I and III) (Nichollas and Ferguson 2002). O
2
._
formed
during electron transfer at the inner membrane, can also be vectorially released
into the intermembrane space (Han, Williams et al. 2001) where it is converted

16
to H
2
O
2
by the Cu,Zn-superoxide dismutase present in this compartment. The
mitochondrial production of H
2
O
2
is regulated by the mitochondrial metabolic
state and intramitochondrial steady-state concentration of nitric oxide. The
effects of aging on respiration are more prominent in tissues whose
parenchyma contains mostly post mitotic cells such as brain, heart and skeletal
muscle rather than tissues that contain slowly dividing cells such as liver.
Complex I, III and IV activities were demonstrated to decrease with age in
mitochondria isolated from numerous organs (Navarro and Boveris 2007). A
decline in state 3 respiration, respiratory control ratios and uncoupled
respiration rates using either NAD-linked or FAD-linked substrates was
observed during aging in Drosophila Melanogaster (Ferguson, Mockett et al.
2005). The age associated loss of function of complex I, III and IV may
promote higher oxidant production. This is further exacerbated by low
mitochondrial turnover in brain, leading to increased accumulation of
dysfunctional mitochondria with respect to aging (Navarro and Boveris 2007).
c) Regulation of mitochondrial functions by nitric oxide – The role of nitric oxide
within the mitochondria bears significant importance in regulation of
mitochondrial function as (i) complex III and IV can be inhibited by nitric
oxide, (ii) nitric oxide can bind to haem group of proteins altering their
function, and (iii) NO can react with O
2
.-
at diffusion-controlled rates to

17
generate ONOO
-
, a oxidant capable of inhibiting important enzymes (Turrens
2003; Cadenas 2004). Cytochrome c oxidase (COX) is the terminal component
of the electron transport chain and catalyses the oxidation of cytochrome c and
the reduction of O
2
to water in a process that is linked to proton pumping out
of the matrix.
.
NO binds to the binuclear center(Antunes, Boveris et al. 2004)
of COX and inhibits its activity in a reversible and O
2
competitive
manner(Brown G.C 1994). The IC
50
of
.
NO for COX is predicted to be ~ 20
nM, indicating that the physiological concentrations of
.
NO in tissues (10-450
nM) are sufficient for O
2
competition intracellularly. A new concept of the
regulation of cellular respiration is advanced; it retains the classical concept
that energy demands drive respiration but kinetic control of both respiration
and energy supply is dependent upon the availability of ADP to F
1
-ATPase and
O
2
and
.
NO to COX (Boveris, Costa et al. 1999). Hence, in addition to
regulating COX activity, NO brings about a metabolic hypoxia where due to
increases in NO, such as during inflammation or degenerative disease,
available O
2
cannot be adequately used(Moncada and Erusalimsky 2002). At
physiological concentration of
.
NO, inhibition of mitochondrial respiration
occurred with a concomitant increase in O
2
.–
and ONOO
–
formation (Poderoso,
Carreras et al. 1996). Inhibition of mitochondrial respiration by
.
NO should be
viewed as paradoxical, where the tonic reversible inhibition of mitochondrial
respiration during hypoxic conditions lead to a conservation of cellular O
2
and

18
transient adaptation to hypoxic conditions. Additionally, the release of a small
amount of H
2
O
2
during transient inhibition leads to the activation of redox
signaling pathways that can lead to upregulation of genes. Persistent
irreversible inhibition of mitochondrial respiration that occurs as a result of
prolonged exposure to higher concentrations of
.
NO, such as during
inflammation, results in increased H
2
O
2
production and ONOO
-
production
leading to a state of oxidative and nitrosative stress(Yap, Han et al. 2007).
d) Chemical modification of redox-sensitive mitochondrial proteins – Many
mitochondrial proteins such as Mn SOD (Comhair, Xu et al. 2005), complex
I(Brown and Borutaite 2004) and aconitase(Han, Canali et al. 2005) have been
shown to be chemically modified during oxidative and/or nitrosative stress. In
vitro studies using isolated mitochondria showed that complex I can undergo
post-translation modifications such as nitration by ONOO
-
and
glutathionylation, resulting in loss of complex I activity (Murray, Taylor et al.
2003; Taylor, Hurrell et al. 2003) Our lab has also shown that mitochondrial
aconitase was nitrated at tyrosine residues 151 and 472 and the cysteines 126
and 385 were oxidized to sulfonic acid. These modifications lead to a loss of
enzyme activity. Aside from the above modifications, aconitase was also able
to undergo glutathionylation, indicating that this modification may be an
important means in modulating aconitase activity under oxidative and
nitrosative stress (Han, Canali et al. 2005). Additionally recent work in our

19
laboratory demonstrated an age dependent decrease in Succinyl-CoA:3-
oxoacid Co-A transferase activity, a key mitochondrial matrix enzyme for
ketolysis that provides the only alternative energy for brain during glucose
starvation (Lam and Cadenas 2007). Impairment of mitochondrial protein
function has specific consequences which include increase ROS and RNS
production, energy deficit, collapse of mitochondrial membrane potential that
leads to loss of ATP production and mitochondrial permeability transition, an
initiation of the commitment phase of mitochondrion-driven apoptosis.

The cellular redox environment: A determinant of cellular health
The glutathione system- Glutathione (GSH) is a small hydrophilic molecule that is
synthesized in the cytosol from glycine, glutamate and cysteine in a two step process
by the enzymes γ-glutamylcysteine synthetase and GSH synthase(Griffith 1999)
(Fig.2) As the most abundant non-protein thiol (Han, Canali et al. 2003), GSH plays a
central and important role as an antioxidant. The oxidation product of GSH, GSSG is
reduced back to GSH by glutathione reductase, a NADPH-dependent enzyme
ubiquitously distributed throughout the cell and in tissues. Under non oxidative or
nitrosative stress conditions, the concentration of GSSG is negligible at 1/100
th
of the
total GSH pool (Schafer and Buettner 2001). Measurement of GSH and or GSSG
levels has been used to define the redox environment of the cell. The redox state of a
redox couple can be defined by the half-cell reduction potential and its capacity to

20
reduce. The redox potential can be calculated using the Nernst equation (E
hc
= E
o
–
RT/nF ln Q, where R is the gas constant, T is the temperature in Kelvins, and F is the
Faraday constant, n is the number of electrons exchanged and Q is the mass action
exchanged). Thus, for GSH/GSSG, at 25
o
C, pH7.0, the redox potential for GSH is
defined as E=-240 –(59.1/2) log([GSSG]/[GSH]
2
)(Schafer and Buettner 2001). This is
extremely useful, as this allows comparison of the reducing force available from the
GSSG/2GSH couple with respect to other redox couples. As the concentration of
GSH far exceeds any other redox couple (~100-10,000 greater) (Han, Hanawa et al.
2006), the redox status of the cell can be assessed by taking the redox potential for
GSH/GSSG. The redox status of the cell is associated with a cell’s progression
through its life cycle. It has been shown that as the redox environment becomes
increasingly oxidized, the cell progresses from proliferation to differentiation to
apoptosis and necrosis (Schafer and Buettner 2001). Work done in our laboratory
showed that at low concentrations of H
2
O
2
, where the redox status is less oxidized,
cells undergo apoptosis; however, at higher concentrations of H
2
O
2
, the cellular redox
status becomes more oxidized, shifting the mode of cell death from apoptosis to
necrosis (Antunes and Cadenas 2001). In the situation of oxidative or nitrosative
stress, this balance is altered due to the excessive production of ROS or RNS and/or an
impairment of the antioxidant capacity of the cell (Klatt and Lamas 2000).
Traditionally, the formation of GSSG from GSH through the glutathione peroxidase
catalyzed reduction of peroxides was thought to be the predominant pathway whereby

21

Figure 2. Overview of the cytosolic synthesis of Glutathione from
precursor amino acids.

22
the GSH/GSSG ratio is altered. However, recent progress in this area suggests that the
formation of S-nitrosoglutathione can also alter the redox environment of the cell by
affecting the GSH/GSSG ratio. As described in the above section, the generation of
NO results in formation of NO
+
(Martinez-Ruiz and Lamas 2004) and S-
nitrosoglutathione (GSNO) through nitrosylation of GSH. As the concentration of
GSH is important in determining the reduction potential for the GSH/GSSG couple
(Schafer and Buettner 2001), formation of GSNO, detracts from the GSH
concentration of the cell thereby altering the reduction potential of the GSH/GSSG
couple. Formation of ONOO
-
can also alter the GSH/GSSG ratio mainly through the
formation of GSSG and a minimal amount of GSNO (Radi, Cassina et al. 2002).

Protein thiol modifications: redox-sensitive switches
The biological consequences of changes in the cell redox and energy status during
situations such as oxidative or nitrosative stress are the chemical modifications of
target proteins which may be associated with either reversible (e.g. S-nitrosylation or
glutathionylation) or irreversible (e.g. nitration, carbonylation) loss of protein function
(Fig. 3). Recent recognition that protein sulfhydryls can undergo reversible oxidation
or nitrosation has revealed a sensitive mechanism that modulates redox pathways in
cell signaling whereby oxidative or nitrosative stress is translated into a mechanism to
which the cell can recognize (Klatt and Lamas 2000). Hence, redox sensitive cysteines
entrench themselves in redox signaling as environmental rheostats, monitoring

23
changes in GSH/GSSG levels as well as increase in reactive oxygen and nitrogen
species generation. The glutathione system keeps the cellular environment reduced,
thus keeping most proteins in the reduced state. The sulfhydryl group in the vast
majority of protein cysteine residues (cys-SH) have a pK
a
of > 8.0 (Cumming, Andon
et al. 2004). However, redox sensitive proteins have cysteines that are ionisable such
that thiolate anion can be stabilized by acid base interactions with neighboring amino
acid residues, resulting in the lowering of their pK
a
values at neutral pH (Cumming,
Andon et al. 2004; Martinez-Ruiz and Lamas 2004). These cysteine residues are
redox sensitive because they are more vulnerable to chemical modifications or
oxidation during redox changes in the cellular environment (Cumming, Andon et al.
2004).

S-nitrosylation- S-nitrosothiols are thiol-esters of nitric oxide with the generic
structure of R-S-N=O and are direct analogs of nitrite esters of alcohols (Hogg 2002)
and not adducts of
.
NO. In principle, the direct reaction of
.
NO with a thiol does not
result in the formation of nitrosothiols (Hogg 2002; Martinez-Ruiz and Lamas 2004)
but generates a thiol disulfide (Hogg 2002).
2
.
NO + O
2
→ N
2
O
3
[5]
Pr-SH + NO
+
→ Pr-S-NO [6a]
N
2
O
3
+ Pr-SH → Pr-S-NO + 2NO
2
-
+ 2H
+
[6b]

24
Figure 3. Impact of oxidative stress and nitrosative stress on protein
function.

25
Instead, prior reaction of
.
NO with O
2
to form higher nitrogen oxides is necessary for
the S-nitrosylation of thiols. In the presence of oxygen,
.
NO is oxidized to dinitrogen
trioxide (N
2
O
3
) (Eq.5) which is thought to be the quintessential nitrosating agent
(Hogg 2002; Martinez-Ruiz and Lamas 2004). N
2
O
3
partially dissociates into a caged
species [NO
+
..NO
2
] and the reaction of the nitrosonium (NO
+
) moiety with the
nucleophilic sulfur atom (Eq.6a and 6b)(Martinez-Ruiz and Lamas 2004) yields a
covalent bond between the sulfur and nitrogen yielding a relatively stable bond not
partial to homolysis (i.e. bond breakage to form radicals)(Hogg 2002). Hence, S-
nitrosylation is actually the transfer of NO
+
instead of
.
NO. This reaction also occurs
during trans-nitrosation whereby the S-nitrosylation is transferred between a
nitrosothiol and another thiol (Martinez-Ruiz and Lamas 2004). Stamler and
colleagues have suggested (Stamler, Lamas et al. 2001) that there is a consensus motif
that affords specificity to nitrosylation. However, it has become quite clear recently
that instead, the local electrostatic and hydrogen-binding interactions may play a more
important role in determining the kinetics of transnitrosation by affecting the pKa of
the thiol (Hogg 2002). In many instances where S-nitrosylation has been described,
the protein cysteines are also oxidized resulting in the subsequent formation of
disulfide bonds (S-thiolation)(Martinez-Ruiz and Lamas 2004). In vivo, the most
abundant protein thiol is glutathione which exist in millimolar concentration
(Halliwell and Gutteridge 1999) in the cytoplasm. Hence, S-nitrosylation is likely to
promote S-glutathionylation (incorporation of glutathione in proteins via mixed

26
disulfide bonds) of proteins which is also viewed as an important post translational
modification especially during oxidative stress (Ghezzi and Bonetto 2003; Martinez-
Ruiz and Lamas 2004) (Fig.4). The astoundingly high degree of specificity of S-
nitrosylation and its reversibility has led some to speculate of a role in signaling for
this post translational modification. This concept lends itself to the idea that S-
nitrosylation works just like phosphorylation, that when the modification is made,
protein functions are altered, thus modulating signaling cascades (Stamler, Lamas et
al. 2001). However, unlike phosphorylation, denitrosylation enzymes/ proteins that
remove this modification have yet to be identified. Additionally, the equilibrium
constant of the reaction is close to unity, where in an in vivo situation where the
amount of glutathione grossly outnumbers the thiols of any one protein, a protein with
an equilibrium constant of one could never be stably or extensively modified. This
might suggest that the biological manifestation of this chemistry is perhaps a stress
response to high and prolonged exposure to nitric oxide (Hogg 2002). S-nitrosylation
should not be confused with tyrosine nitration which is the addition of a nitro group (-
NO
2
) into position three of the phenolic ring of tyrosine residues. This results in
irreversible modification of the protein which often leads to protein damage. It is
generally accepted that nitration of tyrosine residues is a biological marker of
peroxynitrite formation (Martinez-Ruiz and Lamas 2004).

27
S-glutathionylation -. The GSH/GSSG redox couple can dynamically regulates protein
function through the reversible formation of mixed disulfides between protein cysteine
sulfhydryls and GSH in a process termed glutathionylation. Mixed disulfide
formation can occur mainly through three mechanisms. (i) thiol disulfide exchange
between protein sulfhydryl groups and GSSG (equation 7a, 7b) (ii) GSH reduction of
protein sulfenic acids (equation 8a, 8b)and (iii) the nucleophilic attack of thiolate
anion on the S-NO bond of GSNO (equation 9)(Klatt and Lamas 2000; Schafer and
Buettner 2001).
GSH + H
2
O
2
→ GSSG + H
2
O [7a]
P-SH + GSSG → P-SSG [7b]
P-SH + H
2
O
2
→ Pr-SOH [8a]
Pr-SOH + GS
.
→ Pr-SSG +
.
OH [8b]
Pr-SH + GSNO → Pr-SSG + NO
-
[9]
In some regard, the formation of mixed disulfides should in some way reflect
the redox status of the cell (Schafer and Buettner 2001). Unlike S-nitrosylation,
protein-mixed disulfides are specifically reduced by glutaredoxins which catalyzes the
reversible transfer of the glutathionyl moiety via an enzyme glutathione mixed
disulfide intermediate. The reversible formation of mixed disulfides has been
suggested as a mechanism that protects critical sulfhydryls from irreversible damage
and also has significance with regard to redox regulation of signal transduction (Klatt
and Lamas 2000). It should be further pointed out that the generation of reactive

28
nitrogen species can also promote glutathionylation through the formation of GSNO
and likewise, GSH itself may also undergo reactions with S-nitrosylated thiols.
Whether a protein remains nitrosylated or become glutathionylated depends on the
structural environment of the protein (Klatt and Lamas 2000). The possible inter
conversion of both chemical modifications might initially seem to be redundant.
However, upon further analysis, S-glutathionylation may serve to integrate oxidative
and nitrosative stress into a common functional response by independent pathways
(Klatt and Lamas 2000). Unlike S-nitrosylation, protein-mixed disulfides are
specifically reduced by glutaredoxins, which catalyze the reversible transfer of the
glutathionyl moiety via an enzyme glutathione mixed disulfide intermediate. The
reversible formation of mixed disulfides has been suggested as a mechanism that
protects critical sulfhydryls from irreversible damage and also has significance with
regard to redox regulation of signal transduction (Klatt and Lamas 2000; Dalle-Donne,
Milzani et al. 2007). In intact rat liver mitochondria, the proportions of exposed and
reactive thiols are 5-fold higher than that of GSH suggesting that the relationship
between exposed protein thiols and GSH plays a key role in mitochondrial thiol
metabolism (Hurd, Costa et al. 2005). A number of proteins in mitochondria have
been identified to be glutathionylated when the GSH was oxidized: complex I of the
electron transport chain (Taylor, Hurrell et al. 2003), aconitase (Han, Canali et al.
2005), ATP synthase (Garcia, Han et al. 2007), cytochrome oxidase subunit Va and
Vb, α-ketoglutarate dehydrogenase, and the E
2
subunit of pyruvate dehydrogenase

29
(Hurd, Costa et al. 2005). Additionally, glutathionylation of proteins were observed in
the inferior parietal lobe of individuals afflicted with AD, suggesting that significant
redox changes occur such that glutathionylation occurs during AD and that
glutathionylation of proteins might affect cellular function during AD (Newman,
Sultana et al. 2007). The role of glutathionylation in the etiology of AD remains to be
investigated.

30

Figure 4. S-glutathionylation and S-nitrosylation of
proteins by GSNO.
Figure 4. S-glutathionylation and S-nitrosylation of proteins by GSNO.

31
SIGNIFICANCE

A central tenant of my hypothesis is that generation of nitric oxide during
neuroinflammation leads to an alteration in the redox status in neurons which
subsequently leads to neuronal death associated with neurodegeneration. The research
proposed here has several novel features and it will contribute to the elucidation of the
processes involved in the redox sensitive pathways that controls cell survival and
mitochondria function and dysfunction in neurodegeneration associated with
inflammation.
a) Although the deleterious role of
.
NO in neurodegenerative diseases is
irrefutable, the mechanisms which NO regulates the cellular redox homeostasis
and the effect of the redox environment on regulating energy utilization
through glycolysis and oxidative phosphorylation is still ill defined.
Understanding how the redox environment regulates energy utilization during
nitrosative stress is of paramount importance considering that NAD
+
is
converted either to NADH, to generate energy during glycolysis and oxidative
phosphorylation, or NADPH, the only electron donor for the glutathione and
thioredoxin system in the cytosol and mitochondria. Understanding the
energy-redox relationship would provide novel therapeutic targets for
alleviating symptoms associated with cognitive impairment during aging and
neurodegenerative diseases as demonstrated recently by the development of

32
Ketasyn™ by Accera, Inc a ketone body drink that has shown promising
results with respect to reducing cognitive decline in AD(Biospace 2007).
b) The chemical modifications of proteins in neurons during neuroinflammation
are still largely unknown. Characterization of such modifications will
inarguably be valuable in understanding the redox regulation of protein and
mitochondrial functions in the process of neurodegeneration. Hence, a purpose
of this study is to provide a biochemical basis – upon the mechanistic analysis
of signaling cascades- for the causation of neurodegeneration associated with
inflammation.
The regulatory mechanisms that regulate such signaling cascades are no doubt
fundamental in the development of neurodegenerative diseases. Perturbations in these
signaling cascades play a critical and functional role in the progression in
neurodegeneration associated with inflammation. Characterization and elucidation of
these perturbations will undoubtedly shed light on the underlying mechanisms of
neurodegeneration associated with inflammation. Information obtained in these
studies will provide novel perspectives into the development of neurodegeneration
associated with inflammation and the potential development of intervention therapies
that might delay the progression of neurodegenerative diseases such as Parkinson’s
and Alzheimer’s disease.

33
CHAPTER I:
.
NO – Meditated Glutathionylation: Implications for
neurodegeneration

Introduction
There is now considerable body of evidence regarding the role on excessive
generation of nitric oxide in neurodegenerative diseases such as Alzheimer’s disease
(Duncan and Heales 2005; Block, Zecca et al. 2007). Although the etiologies of
neurodegenerative diseases are complex and varied, neuroinflammation is a common
underlying component in their pathologies (Block, Zecca et al. 2007; Brown 2007).
Injection of amyloid β (A β) protein into the cerebral cortex of rhesus monkeys
resulted in profound neuronal loss and microglial proliferation (Geula, Wu et al.
1998). Microglia over activation is an early pathogenic event that precedes neutrophil
destruction and cluster with A β aggregates before the development of symptoms
(Block, Zecca et al. 2007), suggesting that the release of neurotoxic factors by
microglia play a precipitating role in the development of at least Alzheimer’s disease.
When activated, microglia and astrocytes produce a plethora of neurotoxic factors
such as tumor necrosis factor alpha (TNF α) and reactive oxygen and nitrogen species,
in particular nitric oxide (
.
NO) (Gonzalez-Scarano and Baltuch 1999; Wood 2003).
Co-culture of primary cortical neurons and glial cells resulted in neuronal death
mediated through
.
NO generated by activated glia (Bal-Price and Brown 2001). As

34
such, the generation of
.
NO and it’s toxic effect on neuronal viability has been a
central tenant of the neuoroinflammatory model in neurodegeneration (Liu, Gao et al.
2002; Block, Zecca et al. 2007; Brown 2007; Whitton 2007).
The reactivity of
.
NO in biological systems is governed by complex reactions,
in particular the formation of NO derived reactive nitrogen species (RNS) such as
dinitrogen trioxide (N
2
O
3
) and S-nitrosothiols (Miranda, Espey et al. 200). The
diverse biological effects of RNS can be attributed in part through their ability to
modify and regulate the activity of target proteins through the reaction with redox
sensitive thiols (Klatt, Pineda Molina et al. 2000). The cellular redox status , as
determined by the GSH/GSSG ratio, is an important determinant of cellular health and
regulates many cellular functions (Schafer and Buettner 2001). Altered redox status
has been described in AD, where lymphocytes from AD patients exhibited a reduced
GSH levels and increased GSSG levels (Calabrese, Sultana et al. 2006). Treatment of
primary cortical neurons with
.
NO resulted in a loss of cellular glutathione levels that
correlated with increased cell death and impaired mitochondrial function (Gegg,
Beltran et al. 2003). However, the authors did not investigate the effect of
.
NO
mediated GSH loss on protein post translational modification.
A biological consequence of changes in the cell redox status during oxidative
and nitrosative stress is the formation of protein mixed disulfides, S-glutathionylation,
between proteins and GSH the most abundant non-protein thiol present in the cell
(Klatt and Lamas 2000; Maher 2006; West, Hill et al. 2006). Traditionally viewed as

35
a marker of oxidative stress (Sullivan, Wehr et al. 2000; Ghezzi, Romines et al. 2002),
it is becoming apparent that glutathionylation of proteins is a mechanism through
which changes in the cellular redox state and the increased generation of reactive
oxygen and nitrogen species is translated into a functional modality through which the
cell can sense or adapt to such changes (Klatt, Pineda Molina et al. 2000). Numerous
proteins such a mitochondrial aconitase (Han, Canali et al. 2005), ATP synthase,
succinyl CoA: 3-oxoacid CoA-transferase (SCOT) (Garcia, Han et al. 2007)were
identified by our laboratory as being glutathionylated in isolated mitochondria.
Furthermore, S-glutathionylation of numerous other proteins such as glyceraldehyde-
3-phosphate dehydrogenase (Mohr, Hallak et al. 1999), actin (Pastore, Tozzi et al.
2003), adenine nucleotide transporter (West, Hill et al. 2006) have been demonstrated.
The presence of protein S-glutathionylation under normal physiological (Ghezzi 2005;
Dalle-Donne, Milzani et al. 2007) and pathological circumstances, such as
Alzheimer’s disease, has incited further clarification of the role of protein S-
glutathionylation in cellular function (Pastore, Tozzi et al. 2003; Dalle-Donne, Milzani
et al. 2007; Newman, Sultana et al. 2007). Reversible S-glutathionylation of cysteine
residues have been suggested as a protective mechanism against irreversible oxidation
of cysteine residues, however, S-glutathionylation of proteins often lead to altered
function and thus, mediate redox signaling pathways. The formation of GSNO,
through the reaction of
.
NO with GSNO, has been demonstrated as a S-
glutathionylating agent (Mohr, Hallak et al. 1999; Klatt, Pineda Molina et al. 2000) in

36
in vitro models. Recently, NO-induced protein glutathionylation in intact COS-7and
aortic smooth muscle cells was demonstrated. Overexpression of inducible NOS
resulted in increase glutathionylation of proteins in the hearts of iNOS transgenic mice
(West, Hill et al. 2006).
Substantial evidence supports the role of S-glutathionylation as a general
signaling modality. However, there is little evidence concerning the ability of increase
generation of exogenous NO, such as during inflammation, to modulate the function
of redox sensitive proteins through glutathionylation in neurons. Hence the impetus
for this study was (i) to develop a sensitive method for detecting intracellular
formation of GSNO and GSSG (ii) determine if S-glutathionylation of redox sensitive
proteins is a consequence of increase GSNO and GSSG formation and (iii) the role of
.
NO modulated S-glutathionylation of proteins in neuronal cell death.
Our results indicate that acute exposure of neurons to
.
NO, mirroring
neuroinflammation, can lead to S-glutathionylation of specific proteins. Increasing
concentrations of GSNO and GSSG formation as a consequence of
.
NO exposure can
lead to the formation of protein mixed disulfides. Loss of protein function was a
consequence of protein S-glutathionylation. These observations delineate a potential
mechanism through which increase
.
NO production, a key event in neurodegeneration,
could potentially lead to protein S-glutathionylation and neuronal dysfunction seen in
neurodegenerative diseases such as Friedreich's ataxia (Pastore, Tozzi et al. 2003) and
Alzheimer’s disease (Newman, Sultana et al. 2007).

37
Materials and Methods
Chemicals – MTT, BCNU, protease inhibitor cocktail, PMSF, NP-40, L-
Glutamine, L-Glutamate, O-metaphosphoric acid, N-ethylmalemide, Ammonium
Sulfamate, acetonitrile, sodium monobasic phosphate, 1-octanesulphanic acid, o-
phosphoric acid, NADH, NADPH, GSNO, GSSG, glyceraldehyde-3-phosphate, were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). ATP assay Kit,
Neurobasal media, B-27 Supplements and penicillin/streptomycin were purchased
from Invitrogen (USA). Purified rabbit GAPDH was purchased from Roche
Chemicals. (Indianapolis, IN, USA).
Primary Cortical Neurons and Astrocytes- Primary cortical neurons were
isolated according to the methods described previously (Zhou, Lam et al. 2008).
Briefly, cortical neurons were isolated from timed pregnant Fisher 344 rats and plated
at a density of ~ 1 x 10
6
cells per well in 6 well dishes and maintained in Neurobasal
media supplemented with B-27 supplements, penicillin, streptomycin, L-Glutamate
and L-Glutamine for the first 3 days. Thereafter, they were maintained in Neurobasal
media supplemented with B-27 supplements, penicillin, streptomycin and L-
Glutamine until they were ready to be used. All neurons experiments were carried out
on neurons 10-14 days old. Primary cortical astrocytes were isolated from pups 2-4
days old and maintained in DMEM/F12 until the cultures reached an age of 3 weeks
old. At which, they were trypsinized, recounted and seeded according to the
requirements of the experiments.

38
Measurement of GSH/GSSG - GSH, GSNO and GSSG will be detected using
HPLC with electrochemical detection as described previously (Harvey, Ilson et al.
1989) with slight modifications. Briefly, cells were treated with either 2 mM NEM 10
min before lysis or 5% ο-metaphosphoric acid with 25 mM Ammonium Sulfamate to
prevent artifactual formation of GSNO. Mobile phase consisted of 3% acetonitrile, 25
mM Sodium monobasic phosphoric acid, and 0.5 mM 1-octanesulphanic acid, p.H to
2.7 with O-phosphoric acid.
MTT assay –MTT measurements were carried out as descried previously (Zhou,
Lam et al. 2008). Briefly, 0.5 mg/ml of MTT was dissolved in HEPES toxicity Buffer
and added to the wells at the end of the experiment. Cells were incubated for 1½ hour
at 37
0
C and lysed in DMSO. MTT reduction was measuring absorption at 490 nm.
Values were expressed as percent reduction as compared to controls. All
measurements were done in duplicates for each treatment.
Western blotting and Immunoprecipitation – Cells were scraped in RIPA buffer
with (non)reducing SDS sample buffer, separated by Laemmli SDS/PAGE, and
transferred onto PVDF membranes. Using appropriate antibodies, the immunoreactive
bands will be visualized with an enhanced chemiluminescence reagent. Gels will be
stained with Sypro Ruby protein gel or blotted onto PVDF membrane and probed
against appropriate antibodies. Gel images will be acquired using VersaDoc1000
imaging system (Bio-Rad). For immunoprecipitation, cell lysates were prepared as
mentioned above and ran through Nanosep columns (VWR). Lysates were incubated

39
with agarose conjugated anti-glutathione antibody at 1:10 for 48 hrs at 4
o
C. Samples
were ran on a 10% Laemmli SDS/PAGE and transferred onto PVDF membrane and
probed with appropriate antibodies as specified in the figure legends.
GSNO/ GSSG Reductase Activity - Reductase activities were measured according
to the method described previously with slight modifications (Liu, Hausladen et al.
2001). Cells were lysed in reductase buffer (20 mM Tris.HCl, 0.5 mM EDTA, 0.1%
NP-40 and 1 mM PMSF, pH to 8.0) and sonicated 3 times to disrupt and solubilize all
proteins. Protein concentration was determined using the Bradford assay. GSNO
reductase activity was measured by a decrease in NADH fluorescence at 340 nm
spectrophotometrically in the presence of GSNO. GSSG reductase activity was
measured as a decrease in NADPH fluorescence spectrophotometrically at 340 nm in
the presence of GSSG. 20 mM BCNU was added as an inhibitor of GSSG reductase
activity.
GAPDH Activity- GAPDH activity in neurons was measured according to the
methods briefly described with some slight modifications (Liu, Hausladen et al. 2001).
Briefly, cells were lysed in GAPDH reaction buffer (100 mM Tris.HCl, 5 mM Sodium
Arsenate, pH 8.6) with the addition of protease inhibitors and 2 mM N-ethylmaleimide
and undergo 3 cycles of freeze-thaw. Purified GAPDH activity was measured in lysis
buffer. Increase in NADH was monitored spectrophotometrically at 340 nm
wavelength in the presence of NAD
+
and glyceraldehyde-3-phosphate solution.

40
Results
Exogenous Nitric Oxide leads to intracellular formation of GSNO in Primary Cortical
Neurons and Astrocytes
The intracellular redox status of a cell is governed by the GSH/GSSG ratio and is an
important indicator of cellular health as well as a modulator of protein-thiol redox
status (Schafer and Buettner 2001). Astrocytes have demonstrated greater resiliency
to
.
NO challenges than neurons and this may be in part due to differential changes in
their GSH/GSSG ratio. It has been demonstrated that NO reacts with GSH to produce
GSNO. To examine
.
NO-mediated changes in GSH, GSSG and GSNO levels upon
exposure to
.
NO, primary cortical neurons and astrocytes were exposed to 3.4 -13.8
mM DETA-NO, a relatively pure
.
NO donor that decomposes into only
.
NO and the
free amine diethylenetriamine (Borutaite and Brown 2003) (corresponding to a steady
state release of NO at 0.061-0.25 μM/s) as determined using a ISO clark type
.
NO
electrode), for 1 hour. The cells were either treated with N-ethylmalemide to chelate
free thiols or ammonium sulfamate to chelate nitrite before they were lysed in 5% o-
metaphosphoric acid. The samples were then subjected to reverse phase HPLC and
GSH, GSSG and GSNO were detected using electrochemical detection method. The
respective concentrations of GSH, GSNO and GSSG were calculated based on
injected standards. This method is highly sensitive for detecting cellular
concentrations of GSNO and GSSG, allowing detection to as low as pico molar
concentrations.

41

A
B
Figure 5. Intracellular GSNO & GSSG formation after exposure of
primary cortical neurons to
.
NO. Primary cortical neurons were treated
with increasing concentrations of DETA-NO for 1 h in neurobasal media.
Cells were rinsed twice with ice cold PBS and lysed (A) in 5 %
Metaphosphoric acid with 25 mM Ammonium Sulfamate. GSNO ( ■) and
GSH ( ▲) levels were determined by HPLC. (B) Cells were pre-treated with 1
mM NEM for 10 min and then lysed with 5% Metaphosphoric acid. GSNO
( ■) and GSSG( ●) levels were determined by HPLC.

0.00
0.05
0.10
0.15
0.20
0.25
0.19 0.25 0.12 0.061
GSNO
GSSG
dNO/dt ( μM/s)
GSNO Concentration (nmol/10
6
cells)
0
0.00
0.05
0.10
0.15
0.20
0.25
GSSG Concentration (nmol/10
6
cells)
0
2
4
6
8
10
12
14
GSH
GSNO
dNO/dt ( μmol/s)
GSH Concentration (nmol/10
6
cells)
0
0.061 0.12 0.19 0.25
0.0
0.2
0.4
0.6
0.8
1.0
GSNO Concentration (nmol/10
6
cells)

42

A
B
Figure 6. Intracellular GSNO & GSSG formation after exposure of
primary cortical astrocytes to
.
NO. Primary cortical astrocytes were treated
with increasing concentrations of DETA-NO for 1 h in neurobasal media.
Cells were rinsed twice with ice cold PBS and lysed (A) in 5 %
Metaphosphoric acid with 25 mM Ammonium Sulfamate. GSNO ( ■) and
GSH ( ▲) levels were determined by HPLC. (B) Cells were pre-treated with 1
mM NEM for 10 min and then lysed with 5% Metaphosphoric acid. GSNO
( ■) and GSSG ( ●) levels were determined by HPLC.

0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
GSNO
GSSG
dNO/dt (μmol/s)
GSNO Concentration (nmol/10
6
cells)
0 0.061 0.12 0.19 0.25
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
GSSG Concentration (nmol/10
6
cells)
0
2
4
6
8
10
12
14
16
18
20
22
24
26
GSH
GSNO
dNO/dt ( μM/s)
GSH Concentration (nmol/10
6
cells)
0
0.061
0.12 0.19 0.25
0.0
0.2
0.4
0.6
0.8
1.0
GSNO Concentration (nmol/10
6
cells)

43
Total GSH concentrations for control neurons were 14.32 nmol/10
6
cells and 26.61
nmol/10
6
cells. Increasing concentrations of
.
NO lead to a dose dependent increase in
GSNO and a parallel decrease in GSH concentrations in both primary cortical neurons
(Fig. 5A) as well as primary astrocytes (Fig. 6A). Upon exposure to 0.25 μM/s rate of
release of
.
NO, GSH concentrations in neurons fell by 55% while GSH concentrations
in astrocytes fell by only 14%. This dramatic decrease in GSH levels in neurons is
similar to the levels observed in neurons exposed to 1 μM
.
NO for 24 hr (Gegg,
Beltran et al. 2003). GSNO concentrations increased by 14% from non detectable
levels to 0.14 nmol/10
6
and 46% from non detectable levels to 4.60 nmol/10
6
cells in
neurons and astrocytes, respectively while GSSG concentrations increased by 7%
from 0.02 nmol/10
6
cells to 0.09 nmol/10
6
in neurons and 3% from 0.10 nmol/10
6
to
0.13 nmol/10
6
cells in astrocytes (Fig. 5B & 6B). Even exposure to lower levels of
.
NO at 0.061 μM/s led to a 35% decrease in GSH concentrations in neurons while only
a 7% dropped occur in astrocytes (Table 1).

Exposure of Nitric Oxide leads to changes in Nernst Potential
The redox state of a cell can be quantified by the Nernst equation, E
hc
. Changes in the
half cell potential of the GSSG/2GSH couple appear to correlate with the biological
status of the cell. As the Nernst potential becomes increasingly oxidized, cells shift
towards an apoptotic state and upon further oxidation, undergo necrosis (Schafer and
Buettner 2001). Although the equation does not take into the absolute concentration of

44
NEURONS
GSNO
(nmol/10
6
cells)
GSSG
(nmol/10
6
cells)
GSH
(nmol/10
6
cells)
Rate of
.
NO
( μM/s)
0 26.62 0.00 0.10
0.061 24.75 2.45 0.12
0.12 25.70 2.01 0.14
0.25 22.78 4.60 0.13
GSNO
(nmol/10
6
cells)
GSSG
(nmol/10
6
cells)
GSH
(nmol/10
6
cells)
Rate of
.
NO
( μM/s)
0 14.32 0.00 0.02
0.061 8.54 0.04 0.03*
0.12 9.33 0.09 0.04
0.25 5.98 0.14 0.09

Table 1. Tabulated values of GSH, GSNO and GSSG formation in primary
neurons and astrocytes. Near pure primary cortical neurons and astrocytes were
exposed to different concentrations of DETA-NO for 1 hr at 37
o
C. The cells were
then harvested utilizing two different methods 1) 5% Ο-Metaphosphoric acid + 25
mM Ammonium Sulfamate or 2) Pre-treated with 5 mM N-ethylmaleimide for 10
min. before the addition of 5% Ο-Metaphosphoric acid. Supernatants were then
passed through reverse phase HPCL and GSH, GSNO and GSSG were detected
using electrochemical detection. GSH values are obtained from samples treated
with ammonium sulfamate method and GSNO and GSSG values were obtained
from samples treated with N-ethylmaleimide.
ASTROCYTES

45

Rate of
.
NO ( μM/s) Nernst potential (mV)
Neurons
0 - 263.68
0.061 - 245.21
0.12 - 243.79
0.25 - 221.96
Astrocytes
0 - 273.51
0.061 - 266.45
0.12 - 263.72
0.25 - 250.22
Table 2: Calculated Nernst Potential for Primary Cortical Neurons and
Astrocytes exposed to
.
NO. Primary cortical neurons and astrocytes were treated
with increasing concentrations of DETA-NO for 1 h in neurobasal media and
DMEM/12 respectively. Cells were rinsed twice with ice cold PBS and lysed in 5
% Metaphosphoric acid with 25 mM Ammonium Sulfamate or pre-treated with 1
mM NEM for 10 min and then lysed with 5% Metaphosphoric acid. GSNO, GSH
and GSSG levels were determined utilizing HPLC electrochemical detection.
Nernst Potential was calculated utilizing the equation, E
hc
= -240-
(59.1/2)log([GSH]
2
/[GSSG]). Concentrations were calculated on cell volume at the
value of 10 μl per mg of protein (Jones 2002).

46
GSNO or GSNO/GSH ratio, but the formation of GSNO from GSH is reflected in the
overall absolute concentration of GSH.
Exposure of primary cortical neurons to
.
NO led to an increase in the Nernst
Potential, i.e. the redox status of neurons became increasing oxidized with respect to
increasing concentrations of
.
NO. The redox states in neurons were -263.68 mV in
control samples and were oxidized to -221.96 mV upon exposure to a rate of
.
NO
released at 0.25 μM/s. However, astrocytes exposed to the same concentrations of
.
NO
did not show significant change in their Nernst potential (Table 1) increasing from ~ -
273.51mV in control to ~ -250.22 mV in astrocytes treated with 0.25 μM/s rate of
release of
.
NO. This suggests that astrocytes are able to maintain their glutathione
redox buffering pool more effectively in the face of nitric oxide challenge as compared
to neurons.

GSNO reductase activity in Primary Cortical Neurons and Astrocytes
It has been demonstrated previously that Alcohol dehydrogenase Class III enzyme,
also known as GSH-dependent formaldehyde dehydrogenase, exhibits a NADH
dependent GSNO reductase activity and constitutes a metabolic pathway that is
important in protecting against nitrosative stress (Liu, Hausladen et al. 2001). In the
presence of NADH, GSNO reductase converts GSNO into glutathione disulfide
(GSSG) and ammonia. To determine if primary cortical neurons and astrocytes are
able to metabolize GSNO, GSNO was incubated with cellular lysates from primary

47
cortical neurons and astrocytes in the presence or absence of NADH and GSNO
concentrations were analyzed at different time points. Neurons and astrocytes
demonstrated the ability to metabolize GSNO in a NADH dependent reaction (Fig. 7A
and 8A). However, most of the GSNO was metabolized completely by astrocytes by
15 min (Fig. 48A) while it took neurons twice the time to metabolize most of the
GSNO (Fig 7A). To determine if this metabolism is likely through ADH III, GSSG
formation was monitored. Incubation of GSNO with cellular lysates from neurons
and astrocytes lead to an NADH dependent increase of GSSG and the increase in
GSSG formation was also faster in astrocytes than in neurons, doubling within 15 min
for astrocytes while taking almost 30 min for neurons to reach the same amount. The
concomitant NADH dependent increase in GSSG during GSNO metabolism indicates
that this GSNO metabolism is most likely through the ADH III as previously reported
by other groups.

Higher GSNO and GSSG reductase activity confers cellular tolerance to NO
Glutathione reductase (GR) is a critical enzyme for the maintenance for a reduced
glutathione pool. Utilizing NADPH as an electron donor for the reduction of GSSG to
GSH (Schulz, Schirmer et al. 1978), the activity of this enzyme governs the rate of
conversion of GSSG to GSH and thus, the GSH/GSSG ratio within the cell (Halliwell
and Gutteridge 1999). GSNO reductase, on the other hand, regulates the

48
0 5 10 15 20 25 30
50
100
150
200
250
300
350
Percent increase in GSSG
Time (min.)
+ NADH
-NADH
B
A
0 5 10 15 20 25 30
0
20
40
60
80
100
Percent Decrease in GSNO
Time (min)
+ NADH
- NADH
Figure 7. Metabolism of GSNO by primary cortical neurons. 1.0 mg/ml of
primary cortical astocytes lysate and 100 μM of GSNO was incubated ± 200 μM
NADH .Aliquots were taken at indicated time points and mixed with ice cold 5%
Metaphosphoric acid. GSNO and GSSG concentrations were determine using
HPLC method. (A) GSNO concentration without NADH ( ●) and with 200 μM
NADH ( ■). (B) GSSG levels with 200 μM NADH ( ■). Data were expressed as
percent of GSNO at time zero (immediately after addition of NADH). The data
represent mean ± SEM (n=2).

49
0 2 4 6 8 101214 16
0
50
100
150
200
250
300
Percent Increase in GSSG
Tim e (m in.)
- NADH
+ NADH
0 2 4 6 8 101214 16
0
20
40
60
80
100
120
140
Percent Decrease in GSNO
Time (min.)
- NADH
+ NADH
Figure 8. Metabolism of GSNO by primary cortical astrocytic lysate. 1.0
mg/ml of primary cortical astocytes lysate and 100 μM of GSNO was incubated ±
200 μM NADH .Aliquots were taken at indicated time points and mixed with ice
cold 5% Metaphosphoric acid. GSNO and GSSG concentrations were determine
using HPLC method. (A) GSNO concentration without NADH ( ■) and with 200
μM NADH ( ●). (B) GSSG levels with 200 μM NADH ( ●). Data were expressed as
percent of GSNO at time zero (immediately after addition of NADH). The data
represent mean ± SEM (n=2).
A
B

50
amount of GSNO present through the conversion of GSNO to GSSG (Liu, Hausladen
et al. 2001). In concert, these two enzymes may regulate GSH, GSNO and GSSG
levels formed during nitrosative stress. To determine if the differential ability to buffer
significant changes in the redox potential in face of a nitric oxide challenge between
astrocytes and neurons might be due to the ability to efficiently recycle GSSG and
GSNO back to GSH, the enzymatic activity of GR and GSNO reductase were
measured both in astrocytes and in neurons by monitoring the decrease in NADPH
absorbance in the presence of GSSG or the decrease in NADH absorbance in the
presence of GSNO (Liu, Hausladen et al. 2001).
GSNO reductase activity in neurons were approximately 4-fold lower than in
astrocytes, consuming 1.88 ± 0.20 nmol/min/mg of NADH in the presence of GSNO
as compared to 8.21 nmol/min/mg of NADH in astrocytes (Fig 5A). GSSG reductase
activity was measured by monitoring the consumption of NADPH in the presence of
GSSG. GSSG reductase activity was 11-fold higher in astrocytes than in neurons,
where the activity of GSSG reductase in astrocytes was 7.41±0.5 nmol/min/mg of
protein as opposed to 0.63± 0.14 nmol/min/mg of protein in neurons. NADPH
consumption in the presence of GSSG was inhibited upon pretreatment of cells with
BCNU, a GSSG reductase inhibitor (Smith, Alberts et al. 1987) (data not shown). If
the ability to regulate cellular redox status upon exposure to
.
NO is an important
mechanism through which cells maintain cellular health, astrocytes having
demonstrated a higher GSNO and GSSG reductase activities should be less susceptible

51
B
A
Neurons Astrocytes
0
20
40
60
80
100
120
140
160
NADH Consumption (pmol/mg*min)
GSNO Reductase Activity
Neurons Astrocytes
0
20
40
60
80
100
120
140
NADPH Consumption (pmol/mg*min)
GSSG Reductase Activity
Figure 9. GSNO and GSSG Reductase activity. Primary cortical neurons and
astrocytes were lysed in reductase buffer and sonicated. (A) The rate of NADH
(200 μM) consumption was measured as a decrease in absorption at 340 nm with
the addition of GSNO. The initial velocity was calculated as nmol NADH/min/mg
of protein. A best fit line was fitted to the data points. (B) The rate of NAD(P)H
(200 μM) consumption was measured as a decrease in absorption at 340 nm with
the addition of GSSG. The initial velocity was calculated as pmol NADH/s/mg of
protein. A best fit line was fitted to the data points. The data represent mean ±
SEM (n= 3 independent experiments)

52
Figure 10. Effects of
.
NO on viability. Primary cortical neurons and astrocytes
were exposed to increasing concentrations of DETA-NO for 6 h. Viability was
determined using a MTT assay. Values were expressed as percent of the viability
in control treated cells. Data represent mean ± SEM (n=3).
0
20
40
60
80
100
Percent Survival
dNO/dt ( μM/s)
Astrocytes
Neurons
Control 0.061 0.12
0.25

53

to
.
NO induced cell death. MTT assay was used to determine cellular viability in
primary neurons and astrocytes exposed to varying steady state concentrations of NO
for 6 h. There was a dose dependent loss in cellular viability in neurons not seen in
astrocytes. The absence of dose dependent loss of cellular viability in astrocytes could
be due to a threshold effect. As expected, astrocytes were more tolerant to
.
NO
exposure as 49.7% ± 16.16 of astrocytes were viable at the highest rate of release of
.
NO, 0.25 μM/s, as opposed to 9.7%± 6.64 in neurons.

NO exposure leads to glutathionylation of proteins
.
NO can lead to protein glutathionylation through three possible mechanism; (i)
disulfide-thiol exchange reaction with GSSG, (ii) reaction of GSH with nitrosylated
proteins, Pr-SNO, and (iii) transnitrosation reaction between GSNO and cysteine
thiol(Martinez-Ruiz and Lamas 2007). Incubation of primary cortical neurons and
astrocytes led an increased formation of GSNO and GSSG (Table 1), to examine if
NO mediated redox changes could lead in protein glutathionylation, primary neurons
and astrocytes were incubated with DETA-NO (corresponding to rates of release of
.
NO at 0.12-0.5 μM/s) for 1 hr. The cells were lysed and the total protein-glutathione
formation was assessed by immunoblotting using an anti-glutathione antibody.
Incubation of primary cortical neurons led to a dose dependent increase of
glutathionylated proteins (Fig 11A). Coomassie blue staining of gels showed equal

54
loading of samples. Treatment of samples treated with 0.25 μM/s of
.
NO with DTT,
which reduces disulfide bonds, resulted in the loss of immuno-reactive bands,
indicating that the antibody recognized glutathionylated proteins. Addition of
diamide, a widely used thiol-specific oxidant (Kosower and Kosower 1995) initially as
a positive control, led to a slew of glutathionylated proteins while
.
NO treatment led to
only a small increase in glutathionylated proteins (only three detectable bands
corresponding to approximately 37 kDa, 40 kDa and 50 kDa) suggesting that
.
NO
induced glutathionylation led to glutathionylation of specific proteins and a
mechanism likely different from thiol oxidation. Treatment of primary astrocytes with
NO for 1 hr also led to glutathionylation of proteins (Fig 11B). However, only one
band (~ 90 kDa) increased in intensity in a dose dependent manner. Immuno reactive
bands at 40 kDa and 37 kDa did not disappear upon treatment with DTT and showed
no change in intensity upon NO treatment suggesting, that these proteins were unlikely
glutathionylated and rather non specific binding of the antibody. Treatment with
diamide resulted in a laddering pattern, indicating that
.
NO induced glutathionylation
occurs in a highly specific manner, affecting a discrete number of proteins in
astrocytes as well. To prevent GSH adduction to proteins during cell lysis, cells were
treated with 1 mM NEM, an alkylating agent, prior to cellular lysis.

55
A
dNO/dt ( μM/s) 0 0.25 0.12 0.25 0.5 0
Diamide mM - - - - - 200
DTT mM - 100 - - - -
6
31
40
82
210
M.W. (kDa)
Figure 11:
.
NO induces glutathionylation in Primary Cortical Neurons and
Astrocytes. Western blot analysis of glutathionylated proteins of cellular lysate
from (A) neurons and (B) astrocytes treated with increasing concentration of
DETA-NO for 1 h. Glutathionylated proteins were detected with anti-glutathione
antibodies (1:500). A representative blot of three individual westerns are shown
here.
B
dNO/dt ( μM/s) 0 0.25 0.12 0.25 0.5 0
Diamide mM - - - - - 200
DTT mM - 100 - - - -
6
31
40
82
210
M.W. (kDa)

56
Modulation of GAPDH activity by
.
NO
Glyceraldehyde-3-phosphate dehydrogenase, GAPDH, is a classical glycolytic
enzyme responsible for generating the first high energy intermediate and a pair of
reducing equivalents (Mathews, van Holde et al. 2000) in glycolysis. Macrophage
cells exposed to
.
NO resulted in an inhibition of glycolysis and loss of cellular ATP
levels (Borutaite and Brown 2003). To determine if
.
NO could lead to impairment of
glycolysis in primary cortical neurons, GAPDH activity was measured. GAPDH
activity was inhibited by
.
NO in a dose dependent manner (Fig. 12) within an hour of
exposure to
.
NO donor. Exposure to 0.12 μM/s rate of release of
.
NO for an hour
resulted in a 17 % decrease in GAPDH activity, while treatment at a higher release
rate of 0.25 μM/s resulted in a 53% decrease of GAPDH activity. Glutathionylation of
GAPDH has been demonstrated under oxidative stress (Sullivan, Wehr et al. 2000)
and is susceptible to GSNO induced glutathionylation (Mohr, Hallak et al. 1999).
Recently, COS-7 or rat aortic smooth muscle cells exposed to
.
NO donors resulted in
an increase in protein glutathionylation (West, Hill et al. 2006). To determine if
.
NO
treatment resulted in glutathionylation of GAPDH, anti-glutathione
immunoprecipitates of lysates prepared from
.
NO treated primary cortical neurons
were separated on non reducing SDS-PAGE. Although the concentration of
.
NO used
is higher than what cells would normally see in a physiological setting, based on
previous results (Fig. 11A), these proteins were glutathionylated at lower
concentrations of
.
NO. Thus to circumvent any sensitivity issues during

57

Figure 12. Glyceraldehyde phosphate dehydrogenase (GAPDH) activity in
primary cortical neurons after exposure to
.
NO. Cells were treated with
increasing concentrations of DETA and GAPDH activity was measured in cell
extracts after just one hour of exposure by monitoring the formation of NADH in
the presence of glyceraldyhyde-3-phosphate. Data represents mean ± SEM (n=3)
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.12
NADH Formation (nmol/s/mg of protein)
Control
0.061
dNO /dt (μM/s )

58
immunoprecipitation, the highest concentration of
.
NO was used to ensure a good
identification of glutathionylated proteins. Western blotting using anti-GAPDH
antibody confirmed that
.
NO treatment resulted in an increase in glutathionylation of
GAPDH in primary cortical neurons (Fig. 13). A detection of GAPDH in control
conditions indicated that either GAPDH is endogenously glutathionylated or due to
non-specific binding of the anti-glutathione antibody. Further experiments with
purified rabbit muscle GAPDH demonstrated binding of the anti-glutathione antibody
with the protein itself in western blotting, suggesting some degree of non-specific
binding. But nonetheless, the increase in GAPDH precipitated by anti-glutathione
antibody in neurons exposed to
.
NO demonstrates that glutathionylation of GAPDH is
increased during nitrosative stress.

Glutathionylation of GAPDH resulted in a loss of activity
GAPDH consists of four identical 37 kDa subunits, each containing four cysteines;
two of which (cys-149 and cys-153) are located in the catalytic site. Cys149 forms a
highly reactive thiolate group due to its interaction with histidine and is necessary for
GAPDH activity (Mohr, Hallak et al. 1999). An important mechanism for
glutathionylation of protein thiols by GSSG is through disulfide-thiol exchange (eq.6).
Protein-SH + GSSG → protein-SSG + GSH [6]

59
Figure 13. GAPDH is glutathionylated in neurons during
.
NO exposure.
Primary cells were incubated with 40 μM steady state of
.
NO for one hour and
immunoprecipitated with an anti-glutathione antibody. Immuno precipitates were
ran on a non-reducing SDS-PAGE and transferred onto PVDF membranes.
Western blotting with an anti-GAPDH antibody confirmed that GAPDH was
increasing glutathionylated during NO exposure. Densitometric analysis of bands
were carried out using Quantity one Software (Bio Rad, CA)
Co n t r o l N O
0
20000
40000
60000
80000
100000
120000
140000
INT/mm
2
Anti-GAPDH

60
Incubation of purified rabbit GAPDH with increasing concentrations of GSSG
led to a dose dependent increase in glutathionylation of GAPDH as ascertained by
western blot analysis. Addition of 1 mM DTT resulted in a loss of signal (Fig. 14 A)
Glutathionylation of GAPDH correlated with a decrease in GAPDH activity with
maximal inhibition (60%) observed with ~ 2 mM GSSG. Addition of 1 mM of DTT
rescued GSSG elicited inhibition of GAPDH activity (Fig. 14B). Kinetic analysis of
GSSG modulated inhibition of GAPDH showed a sigmoid curve, with a linear range
between 1-2 mM suggesting that GAPDH inhibition might be an all or nothing
phenomena (Fig. 14C).

Glutathionylation of GAPDH occurs in the Alzheimer’s disease model
To determine if GAPDH glutathionylation occurs in a neurodegenerative disease
model, we utilized a triple transgenic Alzheimer’s disease mice model developed by
Frank LaFerla’s group (Oddo, Caccamo et al. 2003). In this specific model, the mice
develop no overt pathology or cognitive impairment until age 4 months. At 6 months
of age, the mice develop intra and extra cellular accumulation of beta-amyloid (A β)
and plaque formation that is associated with retention and contextual fear deficits.
Western blot analysis using an anti-glutathione antibody revealed that
glutathionylation of GAPDH occurred in an age specific manner at 6 months in the
AD transgenic mice and was absent at 3 months (Fig 15A).

61
B
00.1 0.5 0.75 1
0
5000
10000
15000
20000
25000
30000
INT/mm
2
GSSG concentration (mM)
A
Figure 14. GAPDH glutathionylation following treatment with GSSG. (A)
Purified rabbit GAPDH (10 μg) was incubated with varying amounts of GSSG in
GAPDH assay buffer (100 mM Tris.HCl and 5 mM Sodium Arsenate, p.H 8.6) for
10 min before activity measurements were made. GAPDH activity was measured
in the presence of 250 μM NAD
+
and 100 mg/ml of Glyceraldehyde-3-phospahte
(GAP). GAPDH activity was calculated using linear rates 30 s after the addition of
NAD
+
and rates were calculated using the extinction coefficient of NADH (B)
Addition of 1 mM DTT lead to a rescue of (2mM) GSSG induced inhibition of
GAPDH (C) 10 μg of purified rabbit GAPDH was incubated with increasing
concentrations of GSSG in GAPDH assay buffer. The GAPDH preparation was
run on a 10% SDS-PAGE non reducing gel. (Upper panel) Western blot analysis
of glutathionylated proteins using an anti-glutathione antibody. (Lower panel)
Semiquantative analysis (densitometry) of Western blots.

0
25
50
75
+ D T T 2 m M G SSG
GAPDH Activity (% Inhibition)
0.0 0 .5 1.0 1 .5 2.0 2 .5 3.0 3 .5 4.0 4 .5 5.0
0
20
40
60
80
GAPDH Activity (% Inhibition)
G SSG (m M )
C
Anti-GSH

62
Measurement of GAPDH activity from the brain cytosolic fraction showed that there
was no difference in GAPDH activity between control and transgenic mice at 3
months. However, a 24% decrease in GAPDH activity at 6 months, as compared to
age matched controls, was observed (Fig.15B). Western blot of using anti-GAPDH
antibody showed that loss of GAPDH activity was not due to degradation of GAPDH
proteins (Fig.15C). Based on previous data presented and the correlation of age
dependent increase in GAPDH glutathionylation with loss of GAPDH activity, it may
be surmised that in the AD model, glutathionylation of GAPDH occurs in the
relatively earlier stages of disease progression and results in a loss of GAPDH activity.

Discussion
The impetus for this study was (i) to develop a sensitive method for detecting
intracellular formation of GSNO and GSSG, (ii) determine if S-glutathionylation of
redox sensitive proteins is a consequence of increase GSNO and GSSG formation, and
(iii) the role of
.
NO modulated S-glutathionylation of proteins in neuronal cell death.
It has been previously demonstrated that neurons and astrocytes demonstrate
differential susceptibility to
.
NO toxicity (Gegg, Beltran et al. 2003). Hence, astrocytes
were used here as a comparative model to illustrate the effects of impaired GSH
homeostasis regulation with respect to cell survival. The results in this study
demonstrate that excessive exposure to exogenous
.
NO can lead to perturbations of the
cellular redox status of neurons as well as astrocytes through the increased formation

63
A
Control 3Tg
3Tg
Control
3Tg
3 months
6 Months
Anti-GSH
Anti-GAPDH
B
c ontro l T r a n s gen ic
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
NADH Formation (nmol/s/mg)
C ontr o l T r ans genic
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
NADH Formation (nmol/s/mg)
C
Figure 15. Glutathionylation of GAPDH and decreased activity is age
dependent in the triple transgenic Alzheimer’s disease model. (A) Brain of age
matched control and transgenic mice were homogenized in mitochondrial isolation
buffer and the cytosol was separated from the mitochondrial using discontinuous
percoll gradients. Cytosolic fractions were mixed with one part RIPA with non-
reducing sample buffer and loaded onto a non-reducing 12% SDS-PAGE. Gels
were transferred onto PVDF membrane. Western blot analysis of glutathionylated
proteins was achieved using anti-glutathione antibody. Membrane was stripped
and re-probed with anti-GAPDH antibody to identify GAPDH. GAPDH activity
was measured as mentioned above using 200 μg of cytosolic extract of (B) 3 month
old control and transgenic mice and (C) 6 month old control and transgenic mice.

64
of GSNO and GSSG. We also demonstrate that
.
NO can regulate protein-glutathione
mixed disulfide (glutathionylation) formation in neurons and astrocytes. Preferential
glutathionylation of a small subset of proteins in neurons and astrocytes suggests a
highly specific process rather than an indiscriminate consequence of redox changes
during nitrosative stress. Increased formation of glutathionylated proteins correlated
with increasing neuronal cell death. GAPDH, a glycolytic and pro-apoptotic protein,
(Hara, Cascio et al. 2006) was glutathionylated in neurons during nitrosative stress
most likely as a consequence of increase GSNO and GSSG formation.
Glutathionylation of GAPDH led to a significant inhibition of its activity.
The overall redox environment is determined mainly by the GSH/GSSG
couple and estimation of the redox potential of GSH requires measurement of GSH
and GSSG levels (Schafer and Buettner 2001; Han, Hanawa et al. 2006).
Measurement of GSSG levels can be difficult due to the lack of sensitive methods
capable of measuring very low levels of GSSG (Maher 2006) as well as GSNO. We
have utilized a highly sensitive HPLC method that can detect very low concentrations
of GSSG and GSNO at pico molar levels. Dose dependent increase in GSNO with
respect to increasing concentrations of
.
NO demonstrates sensitivity of the method for
detecting GSNO. The data presented in this study is in accordance with previous
studies (Gegg, Beltran et al. 2003) that neurons are more susceptible to
.
NO toxicity
(Fig 6), as judged by MTT reduction, as compared to astrocytes. We demonstrate that
.
NO neurotoxicity is mediated in part through alteration of neuronal redox status and

65
subsequent glutathionylation of proteins such as GAPDH as pretreatment of neurons
with N-acetylcysteine, a thiol antioxidant and GSH precursor (Ghezzi, Romines et al.
2002), attenuated NO induced cell death (data not shown).
Following exposure to increasing concentrations of
.
NO, GSH concentrations
in neurons fell by 41% while GSH concentrations in astrocytes remained relatively the
same. Decreases in GSH concentrations correlated with a linear increase of GSNO
and GSSG concentrations with respect to
.
NO concentrations in both astrocytes and
neurons (Fig. 5 & 6), suggesting that
.
NO exposure does indeed lead to the cellular
formation of GSNO possibly through nitrosylation of GSH. (eq. 1 & 2). Additionally,
a concomitant increase in GSSG was observed and this could be due in part from (a)
the conversion of GSNO to GSSG through the GSNO reductase (Liu, Hausladen et al.
2001) or (b) oxidation of GSH by ONOO
-
(Radi, Denicola et al. 2000) formed through
rapid reaction between
.
NO and O
2
.-
generation due to
.
NO inhibition of cytochrome c
oxidase of the electron transport chain (Cadenas 2004) (Garcia, Yap 2007). This
dramatic loss of GSH levels (Table 1) seen only in neurons exposed to
.
NO becomes
increasingly problematic when considering that during
.
NO exposure, glutamate-
cysteine ligase (GCL), the rate limiting enzyme in GSH synthesis, activity is not
upregulated (Gegg, Beltran et al. 2003) therefore limiting additional synthesis of GSH
to maintain cellular redox status. Taking into account the total amount of GSH and
GSH derivatives (GSNO and GSSG), there is little or significant change to total
amounts of GSH levels between astrocytes exposed to the highest concentration of

66
.
NO for one hour as compared to control. This suggests that the steady state levels of
the total glutathione pool, as a function of GSSG export and GSH synthesis, is
maintained more efficiently in astrocytes. A change in total GSH pool in neurons
indicates a change in steady state levels of GSH, which could be due to either increase
efflux of GSSG or a slower rate of GSH synthesis. Nonetheless, loss of GSH from
neurons might further impede the removal of
.
NO through the GSH-GSNO reductase
pathway (He, Wang et al. 2007) or affect the activity of GSSG reductase (Barker,
Heales et al. 1996) and glutaredoxin (Maher 2006).
An important mechanism in maintaining GSH homeostasis is through the
recycling of the oxidized (GSSG) and nitrosylated form (GSNO) of GSH, by GSSG
reductase and GSNO reductase respectively. This recycling mechanism is critical for
maintaining cellular redox status through the GSH/GSSG ratio. Under physiological
conditions, the ratio of GSH/GSSG is 100:1 (Schafer and Buettner 2001). The activity
of both the GSSG reductase and GSNO reductase were much higher in astrocytes as
compared to neurons (Fig 5), suggesting that during nitrosative stress, the subsequent
formation of GSNO and GSSG can be recycled back to GSH more rapidly in
astrocytes than in neurons thereby maintaining cellular GSH levels. Aside from
metabolizing GSNO, GSNO reductase regulates the level of S-nitrosylated proteins
during nitrosative stress (Que, Liu et al. 2005; He, Wang et al. 2007). Recent work
done by He et.al demonstrated that down regulation of GSNO reductase in cerebellar
cells led to increased susceptibility to NO- mediated neurotoxicity. Lower GSNO

67
reductase activity observed in neurons could therefore affect the removal of
.
NO
(through GSNO formation) and result in higher sensitivity towards nitrosative stress.
Additionally, inhibition of GSSG reductase by BCNU blocked the recovery of
glutathione levels and dethiolation of proteins in endothelial cells exposed to
.
NO
(Padgett and Whorton 1998). Lower GSSG reductase activity in neurons could
therefore affect the recovery of cellular GSH and deglutathionylation rate of proteins.
Conventionally, the reduction potential of GSH is dependent on the
GSH/GSSG ratio as well as the absolute concentration of GSH. Although the
equation does not take into consideration the formation of GSNO, the equation can
still be applicable as changes in the absolute concentration of GSH will change the
redox potential even without changing the GSH/GSSG ratio (Schafer and Buettner
2001; Maher 2006). Cells that have higher concentrations of GSH have a greater
reducing capacity than cells with lower GSH levels. HPLC analysis demonstrates that
astrocytes have a higher concentration of GSH (~ 27 nmol/10
6
cells) than neurons
(~14 nmol/10
6
cells), indicating a higher reducing capacity. The importance of
reducing capacity is reflected in the changes in their respective Nernst Potentials
during nitrosative stress. Calculation of Nernst potentials showed an approximately ~
41 mV change in the Nernst Potential in neurons exposed to a rate of release of
.
NO at
0.25 μM/s of
.
NO for an hour while the Nernst potential in astrocytes changed by ~ 23
mV (Table 2). This is further substantiated by lower sensitivity of astrocytes to
nitrosative stress as demonstrated by MTT reduction.

68

Aside from its importance in cellular survival and adaptability to oxidative or
nitrosative stress, the GSH/GSSG ratio is important in the regulation of redox
sensitive protein thiols. Thiol groups of cysteines that are ionisable exist as thiolate
anions due to acid-base interactions with neighboring amino acid residues, resulting in
the lowering of their pK
a
values at neutral pH (Cumming, Andon et al. 2004;
Martinez-Ruiz and Lamas 2004). Changes in the redox state of these protein cysteinyl
groups can regulate protein function if the cysteines are of functional importance to
the overall structure and activity of the protein (Dalle-Donne, Milzani et al. 2007;
Kemp, Go et al. 2007). Glutathionylation of proteins have been observed in cells
undergoing oxidative (Sullivan, Wehr et al. 2000) and nitrosative duress (West, Hill et
al. 2006) as well as under physiological conditions (Ghezzi 2005). The conundrum
that glutathionylation poses in terms of overall mechanism as well as significance with
respect to cellular physiology and pathophysiology remains to be fully resolved.
The most straightforward mechanism for glutathionylation of proteins is a
direct thiol disulfide exchange reaction between protein thiols and GSSG (eq.6).
However, this mechanism seems unlikely as the redox potentials of most protein
cysteinyl residues are such that only 50 % would be glutathionylated at very low
GSH/GSSG ratios and this remains unlikely because it requires significant increases in
GSSG concentrations (Dalle-Donne, Milzani et al. 2007; Martinez-Ruiz and Lamas
2007). Rather, “activation” of the thiol through electron loss before the incorporation

69

of GSH, could be an alternative mechanism for glutathionylation. Of interest to us, is
the reaction of redox sensitive cysteinyl thiols with
.
NO derivatives and or GSNO
(eq.7a-7c), where nitrosothiols represent the activated form of the cysteinyl thiol
(Martinez-Ruiz and Lamas 2007):
PS-N=O + GSH → PS-SG + HNO [7a]
PSH + GSNO → PS-SG + HNO [7 b]
PSNO + GSH ↔ PSH + GSNO [7c]
In this present study, we have demonstrated that exposure to extra cellular
.
NO, such
as during neuroinflammation, led to increased formation of intracellular GSNO and
GSSG, both of which could lead to glutathionylation of proteins. As such, exposure
of neurons and astrocytes to increasing concentrations of
.
NO correlated with
increasing levels of glutathionylated proteins indicating that glutathionylation of
proteins is a consequence of nitrosative stress (Fig 11).
.
NO modulated
glutathionylation of proteins is a highly specific process, as demonstrated by the small
number of proteins that could be detected by anti-glutathione antibody as opposed to
the number of glutathionylated proteins induced by a strong thiol oxidizing agent such
as diamide. The small number of proteins that are glutathionylated in response to
nitrosative stress indicates that GSNO and or GSSG might be an important mediator of
glutathionylation during nitrosative stress. In the presence of GSNO, both S-
nitrosylation as well as glutathionylation can occur, as to which modification is the

70
end result is determined by the nucleophilicity of the thiolate residue and the stability
of the modification. As such, depending on the ability of the thiolate anion to break
the S-NO bond (Mohr, Hallak et al. 1999) and the overall structure of the protein
(Dalle-Donne, Milzani et al. 2007), not all redox sensitive cysteinyl thiols are capable
of forming mixed disulfides with GSNO. This could be reflected by the low numbers
of glutathionylated proteins we observe as opposed to a higher number observed in
previous studies where glutathionylation was induced by hydrogen peroxide (Sullivan,
Wehr et al. 2000). In this case, glutathionylation of proteins by GSNO is not likely to
occur. Additionally, the detection of a small subset of proteins that were
glutathionylated during nitrosative stress could be due to sensitivity of the antibody or
the microenvironment. Models using transgenic mouse over expressing nitric oxide
synthase in the heart demonstrated greater degree of glutathionylation of proteins
(West, Hill et al. 2006). This may be in part due to a local rapid increase in
intracellular GSNO or
.
NO. The proteins that were glutathionylated in neurons were
somewhat different than in astrocytes (one additional band at ~90 kDa in astrocytes),
suggesting that some of the proteins modified by glutathionylation may be cell type
specific (West, Hill et al. 2006) or due to expression of cell type specific proteins.
Inhibition of GAPDH by
.
NO has been previously demonstrated in
macrophages (Borutaite and Brown 2003) and by glutathionylation during oxidative
(Ghezzi, Romines et al. 2002) and nitrosative stress in bovine aortic endothelial cells
(Padgett and Whorton 1997). GAPDH has been demonstrated previously by Mohr

71
et.al to be glutathionylated by GSNO and GSSG, albeit preferentially by GSNO in
vitro (Mohr, Hallak et al. 1999; Klatt, Pineda Molina et al. 2000). Treatment of
neurons with increasing concentrations of
.
NO resulted in a dose dependent inhibition
of GAPDH, which correlated with
.
NO induced glutathionylation of GAPDH in
neurons (Fig. 12 & 13). Incubation of purified GAPDH with increasing
concentrations of GSSG showed a dose dependent inhibition of GAPDH, with a
maximal inhibition of 60%. Glutathionylation of GAPDH by GSSG follows a
sigmoid curve, showing linearity within a fairly narrow range of GSSG concentrations
(1-2 mM), plateauing at a maximal inhibition of 60%. Suggesting that there exists a
threshold level with respect to inhibition of GAPDH by glutathionylation during
oxidative or nitrosative stress. Incubation of GAPDH with its substrate,
glyceraldehyde-3-phosphate, did not protect against glutathionylation induced
inhibition suggesting that glutathionylation by GSSG did not necessarily occur on the
active site cysteine 149 or a higher affinity for the active site cysteine for GSSG (pKa
of cys149 of GAPDH is 5.5, suggesting strong nucleophilic properties at neutral pH
(Dalle-Donne, Milzani et al. 2007)) instead of its physiological substrate. Purified
rabbit muscle GAPDH contains four cysteines per monomer, cys 149 (active site),
153, 244 and 281, of which at least 281 has been demonstrated to be sensitive to
oxidation (Nakajima, Amano et al. 2007), although the other cysteines are capable of
being glutathionylated as well (Klatt, Pineda Molina et al. 2000). Glutathionylation of
other cysteines might lead to allosteric modulation of GAPDH, decreasing its affinity

72
for its physiological substrates. This is supported by our observations that higher
concentrations of glyceraldehyde-3-phosphate, was needed to overcome decrease in
NADH formation by glutathionylated GAPDH (data not shown). Inhibition of
GAPDH activity by glutathionylation during nitrosative stress might account for
impaired glucose metabolism observed in Alzheimer’s disease. Complementary to our
findings that glutathionylation of GAPDH might lead to neuronal cell death is the
recent work done by Solomon Snyder’s group. They identified a novel apoptotic
pathway involving
.
NO activation of GAPDH and its association with Siah1, an E3
proteosomal ligase. Nitrosylation of GAPDH augmented its binding with Siah1 and
together this complex translocates into the nucleus, promoting the degradation of
nuclear targets of Siah1, resulting in neuronal death. Hydrogen peroxide treatment
also induced GAPDH binding to Siah1 although the association is not as strong,
suggesting that the oxidation status of the cysteine is important for the association
(Hara, Agrawal et al. 2005). As such, glutathionylation of GAPDH seen here might
mask the nitrosylation status of GAPDH, through transnitrosation reaction between
GSH and nitrosylated GAPDH and it is also possible that glutathionylation and
nitrosylation can occur at the same time on GAPDH as the cysteine in the active site is
more sensitive to direct reaction with
.
NO (Hara, Agrawal et al. 2005; Lopez, Wink et
al. 2007), while glutathionylation can occur on other cysteines (Klatt and Lamas
2000).

73
Glutathionylation of GAPDH and a decrease in its activity has been
demonstrated in the inferior parietal lobule of Alzheimer’s disease patients (Newman,
Sultana et al. 2007). Inhibition of GAPDH leads to impairment in the glycolytic
pathway and the generation of pyruvate, the key energy substrate for mitochondrial
function and generation of ATP. To determine if glutathionylation of GAPDH
observed in pathogenic AD brain plays a significant role in AD development or is
simply a marker of altered oxidative/nitrosative stress, we measured the
glutathionylation status and activity of GAPDH in a triple transgenic model of AD.
The glutathionylation of GAPDH and decrease in GAPDH activity occurred in an age
specific manner relatively early in the disease progression before any overt neuronal
degeneration. At 6 months of age, the mice are neuropathologically characterized by
diffuse amyloid plaques in the neocortex and intra-neuronal build up of beta- amyloid
(A β) in the pyramidal neurons of the cortex, hippocampus and amygdala. However,
this precedes any tangle formation (Billings, Oddo et al. 2005). Loss of GAPDH
activity observed at 6 months also correlated with retention and contextual fear
deficits (Billings, Oddo et al. 2005), suggesting that decreased GAPDH activity might
play a role in impaired glucose utilization in patients with mild cognitive impairment
and in the earlier stages of AD (Atamna and Frey 2007). This suggests that
glutathionylation of GAPDH is not just simply a marker of oxidative/nitrosative stress
but rather, plays a role in the development of the disease. As glutathionylation of
GAPDH occurs after accumulation of A β, the role of A β in modulating cellular

74
glutathionylation poses an intriguing question that needs to be further elucidated.
Recent work in our laboratory demonstrated that mitochondrial energy substrates,
glutamate/malate, modulated mitochondrial redox status through deglutathionylation
of mitochondrial proteins, and conferred greater resistance to hydrogen peroxide
(Garcia, Han et al. 2007). Loss of mitochondrial energy substrates might affect
overall mitochondrial functions and lead to mitochondrial driven apoptosis. As
demonstrated by the data presented herein, loss of GSH from neurons is a
consequence of nitrosative stress and the inability of neurons to replenish GSH during
nitrosative stress (Gegg, Beltran et al. 2003) might affect deglutathionylation rate of
proteins (Ghezzi, Romines et al. 2002), which might account for the presence of
glutathionylation of proteins in neurodegenerative diseases such as Alzheimer’s
disease.
In conclusion, the data presented herein demonstrates that excessive
production of
.
NO, such as during neuroinflammation associated with
neurodegeneration or activation of glutamate excitotoxicity pathway, can lead to
glutathionylation of important proteins such as GAPDH through increase formation of
GSNO and GSSG. These findings further highlight an important mechanism through
which
.
NO can modulate cellular death through altering the thiol status of a key
glycolytic enzyme which can affect neuronal metabolic status and mitochondrial
function through disruption of redox signaling, introducing a new perspective in the
role of glutathionylation in neurodegenerative disease (Fig. 16)

75

. .
N NO O
SSG
SH
Active
GADPH
Glucose
Pyruvate
Inactive
GADPH
T TC CA A
NADH
I
IV
c V
Q
III
Formation of GSNO, GSSG
Figure 16. Glutathionylation of GAPDH results in modulation of glucose
metabolism. Glutathionylation of GAPDH due to alterations in the cellular
GSH redox status leads to inhibition of it’s activity. Decrease GAPDH activity
may result in a decrease in pyruvate levels, therefore affecting mitochondrial
oxidativte phosphorylation capacity.

76
Chapter II:
Mitochondrial Energy Axis - A Determinant of Cell Viability
Introduction
The production and delivery of energy in the form of ATP and NAD
+

oxidizing power within cellular organelles forms the basis of cellular bioenergetics
(Mattews, Van Holde et al. 2000; Parihar and Brewer 2007). Dubbed as the cellular
powerhouse of the cell, mitochondria generate most of the energy needed for cellular
function through the conversion of fuel molecules into chemical energy through
oxidative phosphorylation. The pyruvate acetyl-CoA conversion, the entry of the
latter to the TCA cycle, and the reducing equivalents (NADH, FADH
2
) thereby
generated and channeled into the electron-transfer chain to generate energy (ATP) in a
proton-motive force-dependent manner constitutes the energy axis of the mitochondria
(Mattews, Van Holde et al. 2000). Altered mitochondrial energy metabolism, or
mitoenergetic failure, contributes to the pathophysiology of acute brain injury and
chronic neurodegenerative diseases (Parihar and Brewer 2007; Soane, Kahraman et al.
2007; Small, Bookheimer et al. 2008). Energy hypometabolism is an invariant factor
and one of the earliest abnormalities observed in AD and mild cognitive impairment.
Reduction of cerebral metabolic rate for glucose and O
2
and decreased cerebral blood
flow are the best documented abnormalities in AD and other dementias (Blass 2001).
Decrease in glucose metabolism appears before the onset of memory deficits and

77
sensitizes neurons to energy deficiency (Atamna and Frey 2007). In the previous
chapter, we have demonstrated that glyceraldehyde-3-phosphate dehydrogenase was
sensitive to redox regulation during nitrosative stress. Glutathionylation of GAPDH
resulted in inhibition of its activity and impairment of glycolysis (Chapter II). In non
dividing bacterial cells, GAPDH has the largest control strength of all the glycolytic
enzymes for metabolic regulation (Solem, Koebmann et al. 2003). GAPDH is the first
enzyme that catalyzes the first step in the energy harvesting portion of glycolysis,
generating NADH in the process (Mattews, Van Holde et al. 2000). Alteration in
patterns of glucose utilization in the brain becomes burdensome as the brain has low
levels of stored glycogen and almost strictly depends of the glucose metabolism for
the generation of ATP (Parihar and Brewer 2007).
Cell culture studies have demonstrated the favorable use of mitochondrial
energy substrates as neuroprotective agents against oxidative stress (Desagher,
Glowinski et al. 1997; Wang, Perez et al. 2007), nitrosative stress (Frenzel, Richter et
al. 2005) as well as A β toxicity (Alvarez, Ramos et al. 2003; Wang, Takata et al.
2007). Addition of pyruvate to neurons during H
2
O
2
challenge attenuated
mitochondrial membrane depolarization, decreased mitochondrial O
2
.-
production
(Wang, Perez et al. 2007) and prevented caspase-3 release during A β toxicity
(Alvarez, Ramos et al. 2003). Initially demonstrated as a non enzymatic scavenger of
H
2
O
2
(Desagher, Glowinski et al. 1997), pyruvate has been demonstrated to directly
affect mitochondria function through modulating mitochondrial NAD(P) redox status

78
(Alvarez, Ramos et al. 2003), generation of O
2
.-
(Wang, Perez et al. 2007)

and increase
aconitase function (Wang, Takata et al. 2007). Furthermore, the therapeutic value of
mitochondrial energy substrates as feasible treatments for neurodegenerative diseases
has been recently explored. Ketone bodies such as β-hydroxybutyrate, which are
alternative fuel source for the brain during fasting (Parihar and Brewer 2007), have
been shown beneficial effects in improving cognitive defects in recent phase IIb
clinical trials in AD patients without the APOE 4 genotype (Biospace 2007) as well as
in a rodent Parkinson’s disease model (Kashiwaya, Takeshima et al. 2000; Tieu, Perier
et al. 2003). Additionally, we have shown that the addition of mitochondrial energy
substrates modulated mitochondrial GSH redox status through the release of protein
bound GSH in isolated brain mitochondria and protected mitochondria for H
2
O
2
toxicity (Garcia, Han et al. 2007). Most of the studies regarding the neuroprotective
effects of mitochondrial energy substrates had been evaluated with respect to A β,
MPTP and oxidative stress toxicity. However, the value of mitochondrial energy
substrates as neuroprotectants in a nitrosative stress model and the mechanisms
through which mitochondrial energy substrates during nitrosative stress affect
mitochondrial redox status is ill-defined. Hence, in the present study, we attempt to
address the value of mitochondrial energy substrate supplementation in
.
NO toxicity
and mitochondrial redox status.

79
Materials and Methods
Chemicals – MTT, L-Glutamine, L-Glutamate, Sodium Pyruvate, Sodium
Hydroxy-3-butyrate, Malic acid, Niacinamide, DTT, percoll, metaphosphoric acid,
CHAPS, rotenone, potassium cyanide (KCN), antimycin were from Sigma Chemical
Co. (St. Louis, MO, USA). ATP assay Kit, Neurobasal media, B-27 Supplements and
penicillin/streptomycin were purchased from Invitrogen (USA).
Primary Cortical Neurons- Primary cortical neurons were isolated according to
the methods described previously(Zhou, Lam et al. 2008). Briefly, cortical neurons
were isolated from timed pregnant Fisher 344 rats and plated at a density of ~ 1 x 10
6

cells per well in 6 well dishes and maintained in Neurobasal media supplemented with
B-27 supplements, penicillin, streptomycin, L-Glutamate and L-Glutamine for the first
3 days. Thereafter, they were maintained in Neurobasal media supplemented with B-
27 supplements, penicillin, streptomycin and L-Glutamine until they were ready to be
used. All neurons experiments were carried out on neurons 10-14 days old.
Mitochondrial Isolation – Mitochondria from rat brain were isolated by
previously described procedures for differential centrifugation (Sciamanna and Lee
1993) and discontinuous percoll gradient (Anderson and Sims 2000; Schroeter, Boyd
et al. 2003)
Measurement of NAD(P)H Oxidation/Reduction State – Reduced NAD(P)H
was measured flourimetrically with a PerkinElmer LS 55 luminescence spectrometer
using an excitation wavelength of 346 nm and an emission wavelength of 460 nm.

80
NAD(P)H standard curves were done prior to each experiment and used to determine
the concentration of NAD(P)H reduction in the mitochondria.
Measurement of GSH/GSSG. GSH and GSSG will be detected using HPLC with
electrochemical detection as described previously (Harvey, Ilson et al. 1989).
MTT assay –MTT measurements were carried out as descried previously(Zhou,
Lam et al. 2008). Briefly, 0.5 mg/ml of MTT was dissolved in HEPES toxicity Buffer
and added to the wells at the end of the experiment. Cells were incubated for 1½ hour
at 37
0
C and lysed in DMSO. MTT reduction was measuring absorption at 490 nm.
Values were expressed as percent reduction as compared to controls. All
measurements were done in duplicates for each treatment.
ATP measurements – ATP measurements were done according to the
manufacturer’s specifications as previously described (Zhou, Lam et al. 2008) with
slight modifications. Cells were lysed in RIPA and the pellet was spun down at 6,000
g for 10 min at 4
0
C. The lysate was taken and ATP concentrations were determined
based on a standard curve generated. ATP concentrations were determined either as
percent decrease as compared to controls or by absolute values per 10
6
cells. All
measurements were done in duplicates for each treatment.

81
Results
Exposure of Primary Cortical Neurons to
.
NO leads to cell death
Different steady state concentrations of exogenous
.
NO were added to primary cortical
neurons to establish the neurotoxicity of
.
NO. Cell viability was measured 6 h later
using MTT assay, a colorimetric assay. The reduction of the yellow tetrazole, MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the purple
formazan occurs only in viable cells (Bernhard, Schwaiger et al. 2003). Addition of
increasing rates of release of
.
NO resulted in a dose dependent loss of viability in
neurons. Addition of 0.12µM/s steady state concentrations of
.
NO, which has been
demonstrated to occur during brain ischemia (Malinski, Bailey et al. 1993), resulted in
a 65 % loss in cell viability. While higher concentrations of
.
NO, at 0.25 μM/s,
resulted in almost complete cell death at 6 h (88%) (Fig. 17).

Exposure to NO results in loss of cellular ATP levels
Cytochrome c oxidase (COX) is the terminal component of the electron transport
chain and catalyses the oxidation of cytochrome c and the reduction of O
2
to water in a
process that is linked to proton pumping out of the matrix.
.
NO binds to the binuclear
center (Antunes, Boveris et al. 2004) of COX and inhibits its activity in a reversible
and O
2
competitive manner (Brown G.C 1994). Inhibition of mitochondrial electron
chain leads to a loss of ATP generation due to the dissipation of the proton gradient
(Moncada and Erusalimsky 2002). To determine if addition of

82
Figure 17. Effects of
.
NO on viability. Primary cortical neurons were
exposed to increasing concentrations of DETA-NO for 6 h. Viability was
determined using a MTT assay. Values were expressed as percent of MTT
reduction in control treated cells. (n=3)

0
20
40
60
80
100
Percent MTT Reduction
d.NO/dt( μmol/s)
Control 0.061 0.12 0.25

83

Figure 18. NO leads to a depletion of cellular ATP levels. Primary cortical
neurons were exposed to increasing concentrations of
.
NO for (a) 3 h or (b) 6 h
before lysis with RIPA buffer. ATP measurements were made using a
commercially available kit from Invitrogen, USA, as detailed in Materials and
Methods. (n = 2, each set is a duplicate)
0
20
40
60
80
100
120
Percent decrease in ATP
dNO/dt ( μM/s)
Control 0.01 0.029
0.061 0.12
6 Hour Treatment
A
B
0
20
40
60
80
100
120
0.12
0.061 0.029 0.01
Percent decrease in ATP
dNO/dt ( μM/s)
Control
3 Hour Treatment

84
.
NO resulted in a rapid loss of ATP levels in neurons, ATP levels were determined
using a bioluminescence kit which utilizes the requirement of recombinant luciferase
enzyme for ATP for the conversion of luciferin. Addition of increasing concentrations
of
.
NO lead to a dose dependent decrease in cellular ATP levels. Addition of
.
NO at
rate of 0.01 μM/s resulted in a 20% decrease in ATP levels, at 3 h (Fig 18A) which
decreased further to 40% (Fig 18B) at 6 h. A rate of release of
.
NO at 0.029 μM/s
resulted in a dramatic decrease in ATP levels which corresponded to approximately
80% decrease at 3 h and 6 h. Intriguingly, at 0.029 µM/s of NO, there was no
significant difference in ATP levels between 3 h and 6 h time point. Addition of
increasing concentrations of NO resulted in complete collapse of ATP levels (Fig 18A
and 18B).

Monocarboxylates or mitochondrial energy substrates supplementation ameliorates
.
NO toxicity but does not restore ATP levels
Previous studies have demonstrated the efficacy of pyruvate, β-hydroxybutyrate and
malate in protection against numerous modes of neurotoxicity involving oxidative
stress such as hydrogen peroxide and zinc neurotoxicity (Desagher, Glowinski et al.
1997; Sheline, Behrens et al. 2000; Alvarez, Ramos et al. 2003; Massieu, Haces et al.
2003). Based on previous data (Fig. 17 and 18), the effect of mitochondrial energy
substrates on neuronal viability were determined at a rate of release of
.
NO at 0.029

85
μM/s for 3 h. We anticipate at these conditions, that approximately a 30-40% decrease
in viability is observed and ATP levels are drastically affected but not completely
depleted. Primary cortical neurons were pre-treated for 20 min with 5 mM of
pyruvate, malate and β-hydroxybutyrate and then exposed to
.
NO at a rate of release of
0.029μM/s. Treatment with 5 mM malate and β-hydroxybutyrate resulted in
amelioration of
.
NO induced toxicity as determined by MTT assay, while pyruvate
seemed to have lesser protective effect on protection against
.
NO toxicity. Addition of
.
NO resulted in approximately 50 % cell death, whereas in the presence of malate and
β-hydroxybutyrate there was no detectable difference in cell death as compared with
control (Fig.19A). To determine if addition of mitochondrial energy substrates
rescued ATP levels, we determined ATP concentrations in neurons pre-treated with 5
mM pyruvate, malate or β-hydroxybutyrate and then exposed to 0.029 µM/s of
.
NO.
Addition of 0.029 µM/s of
.
NO resulted in approximately 70% loss in ATP levels,
consistent with previous measurements (Fig.18A). However, the addition of any of
the mitochondrial substrates pyruvate, malate or β-hydroxybutyrate did not rescue
ATP levels (Fig. 19B).

86

0
200
400
600
800
1000
1200
1400
+ BOH + Mal
ATP pmol /10
6
cells
Control 0.029 μM/s NO
+ Pyr
Control NO Pyruvate M alate BOH
0
20
40
60
80
100
120
Percent MTT Reduction
Figure 19. Effects of mitochondrial energy substrates on viability and
ATP levles. Primary cortical neurons were pre-treated with 5 mM with
respective mitochondrial energy substrates for 20 min before being exposed to
0.029 μM/s of DETA-NO for 3 h. Viability was determined using MTT
assay. Values were expressed as percent of the viability in control treated
cells. (n = 2, each set is a duplicate) Primary cortical neurons were pre-treated
with 5 mM with respective mitochondrial energy substrates for 20 min before
being exposed to 0.029 μM/s of DETA-NO for 3 h. ATP measurements were
made using a commercially available kit from Invitrogen, USA, as detailed in
Materials and Methods. (n = 2, each set is a duplicate)

A
B

87
Addition of mitochondrial substrates results in the increase generation of
mitochondrial NAD(P)H
The pyridine nucleotide pool, NAD
+
/NADH and NADP
+
/NADPH, represent
important redox pairs that control mitochondrial generation of energy and the
antioxidant system respectively. Previous work demonstrated that the protective
ability of pyruvate, malate and β-hydroxybutyrate was related directly to their ability
to directly modulate mitochondrial function (Alvarez, Ramos et al. 2003; Tieu, Perier
et al. 2003). To confirm if the mitochondrial substrate supplementation resulted in an
increase generation in mitochondrial reducing equivalents, the formation of NAD(P)H
was measured fluorometrically at absorbance 340 nm. Addition of complex I
substrates in the absence of ADP (state 4) to isolated brain mitochondria,
glutamate/malate, resulted in a generation of 6.43 nmol/mg of NAD(P)H. Substrate
supplementation of mitochondrial with glutamate/malate resulted in the presence of
rotenone, a complex I inhibitor, resulted in a higher generation of 8.01 nmol/mg of
NAD(P)H, representing maximal NAD(P)H production. Addition of ADP (state 3)
resulted in a rapid decrease in NADP(H) fluorescence, leveling off at a basal level of
1-2 mol/mg of NAD(P)H (data not shown), most likely due to consumption of NADH
by complex I (Fig.20).

88

02468
0
5
10
15
20
B
A
6.43 nmol / mg
G/M Mitochondria
8.01 nmol / mg
Flourescence (a.u.)
Time (min)
Figure 20. NAD(P)H formation upon complex I substrate
(glutamate/malate-G/M) supplementation. Reduced NAD(P)H was
measured flourimetrically using an excitation wavelength of 346 nm and an
emission wavelength of 460 nm. Intact mitochondria were incubated at room
temperature in a respiration buffer for a total of 8 minutes and
glutamate/malate was added at the 4 min mark. Standard curves for NAD(P)H
were generated before every experiment to determine concentration. Maximal
NAD(P)H production was ascertained using complex I inhibitor, rotenone

89
Regulation of mitochondrial GSH pool is independent of electron transport chain
activity
Previous work carried out in our laboratory demonstrated that supplementation of
mitochondria with complex I substrates, glutamate/malate, resulted in an increase in
mitochondrial GSH levels mediated through deglutathionylation of mitochondrial
proteins (Garcia, Han et al. 2007). The generation of NAD(P)H is important for the
regulation of the mitochondrial GSH pool, based on the fact that it is the only electron
source for the GSH pool in the mitochondria. In mitochondria, ~ 50% of NADPH is
formed through the conversion of NADH to NADPH by the proton linked
nicotinamide nucleotide transhydrogenase (Rydstrom 2006). In the presence of a
proton gradient, the formation of NAD(P)H is favored on a magnitude of 5-10 fold.
To determine if a functional electron transport chain is required for the increase GSH
observed during substrate supplementation, GSH levels were measured in isolated
brain mitochondria in the presence of various mitochondrial respiratory complex
inhibitors with or without substrate supplementation with glutamate/malate. Addition
of glutamate/malate and ADP resulted in a 10 fold increase in GSH levels. GSH
levels were not as high in mitochondria supplemented with glutamate/malate in the
absence of ADP, increasing only 4 fold. In the presence of antimycin A, a complex III
inhibitor, there was a ~ 6 fold increase in GSH levels. Addition of rotenone, a
complex I inhibitor, or potassium cyanide, a complex IV inhibitor, also resulted in an
approximately 6 fold increase in GSH levels (Fig.21). The data suggests that release

90
0
2
4
6
8
10
12
nmol/mg
Glutamate/Malate - + - + + + + +
ADP - + + - - - - -
Antimycin A - - - - + - + -
Rotenone - - - - - + + -
KCN - - - - - - - +
Fig. 21. Formation of mitochondrial GSH depends on substrate
availability not electron flow through the ETC. Brain mitochondria isolated
by discontinuous percoll gradient and incubated for 10 min. at 37
o
C with the
appropriate factors. All mitochondrial complex chain inhibitors were added
prior to supplementation with glutamate/malate, succinate, or ADP. At 10 min,
mitochondria were pelleted, and resuspended in 5% metaphosphoric acid and
analyzed for GSH and GSSG by electrochemical detection using reverse phase
HPLC. GSH, GSSG concentrations were calculated based on GSH and GSSG
standards run alongside the samples. (n=3)

91
of mitochondrial GSH is independent of electron flow through the electron transport
chain, but rather the generation of mitochondrial reducing equivalents, NADH and the
formation of NADPH through the nicotinamide nucleotide transhydrogenase catalyze.

Discussion
This study demonstrates that mitochondrial energy substrates are important
modulators of cellular viability and mitochondrial redox status during nitrosative
stress. Exposure of primary cortical neurons to
.
NO resulted in increased cell death
which was attenuated by the addition of pyruvate, malate and β-hydroxybutyrate.
Significant loss of cellular ATP levels was a consequence of
.
NO exposure, most
likely due to
.
NO inhibition of cytochrome oxidase. The attenuation of cell death by
mitochondrial energy substrates is independent of the ability to modulate
.
NO induced
loss of cellular ATP levels but rather through the modulation of mitochondrial redox
couples NAD(P)
+
/NAD(P)H and GSH/GSSG.
The protective effects of pyruvate against H
2
O
2
mediated toxicity was
attributed to its ability to act as a scavenger through the non enzymatic reaction
between the α-ketoacid with H
2
O
2
yielding the carboxylic acid and water (see eq.8)
(Desagher, Glowinski et al. 1997).
R-COCOOH + H
2
O
2
→ R-COOH + CO
2
+ H
2
O
2
[8]
Additionally, it has been recently suggested that pyruvate can act as an
intracellular scavenger of increased reactive oxygen species generated during
.
NO

92
exposure in Müller cells (Frenzel, Richter et al. 2005). Utilizing the
.
NO electrode,
we demonstrate that there was no direct chemical reaction between
.
NO and pyruvate
as well as the other mitochondrial energy substrates, malate and β-hydroxybutyrate
(data not shown). Hence, in concordance with other studies, the protective effects of
pyruvate, malate and β-hydroxybutyrate during nitrosative stress are likely due to their
ability to modulate the mitochondrial redox state and bioenergetic capacity
(Kashiwaya, Takeshima et al. 2000; Alvarez, Ramos et al. 2003; Nakamichi, Kambe et
al. 2005). This is supported by other studies demonstrating that addition of pyruvate
to neuroblastoma SK-N-SH cells prevented H
2
O
2
induced mitochondrial membrane
depolarization. Addition of pyruvate to submitochondrial particles also attenuated the
formation of O
2
.-
in the presence of rotenone or antimycin D (Wang, Perez et al.
2007). Additionally, malate and β-hydroxybutyrate are not monocarboxylates but
strictly mitochondrial energy substrates and were able to attenuate
.
NO induced
neurotoxicity. What was interesting was that the protection afforded by malate and β-
hydroxybutyrate (~100% rescue) was higher as compared to pyruvate (~ 20-30%
rescue). This could be due to either (i) the reaction between pyruvate and H
2
O
2

formed from the dismutation of O
2
.-
generated as a consequence of NO induced
autoxidation of ubiquinol (Poderoso, Carreras et al. 1996), reducing its concentrations
reaching mitochondria or (ii) sensitivity of pyruvate dehydrogenase complex (PDHC)
to
.
NO or oxidative damage. Post ischemic hyperoxia in canine models resulted in a
loss of PDHC activity due to nitration (Richards, Rosenthal et al. 2006) and oxidation

93
of PDHC was observed in rodent models with traumatic brain injury (Opii, Nukala et
al. 2007). Previously, we and others have demonstrated that addition of 1µM of
.
NO
resulted in a generation of ONOO
-
into the mitochondrial matrix space (Poderoso,
Carreras et al. 1996) (chapter III). Hence, the lower efficacy of pyruvate maybe
mediated by decrease activity due to nitration of the complex. This effect was not
observed in other models utilizing H
2
O
2
due in part to the absence of
.
NO, limiting the
formation of ONOO
-
. 3-oxoacid CoA transferase (SCOT), which is part of the
metabolic pathway that catalyzes the conversion of β-hydroxybutyrate into acetyl Co-
A was also nitrated as a function of aging (Lam and Cadenas 2007). However, the
efficacy of β-hydroxybutyrate in protection against
.
NO mediated neurotoxicity was
not affected. This poses an intriguing question and may be answered by measuring
their sensitivity to ONOO
-
and the extent of inhibition of the enzymes by ONOO
-
.
This may be reflected by the protective effects of β-hydroxybutyrate in Parkinson’s
and Alzheimer’s disease model (Kashiwaya, Takeshima et al. 2000).
Loss of cellular ATP is a key event in neurodegeneration due to the heavy
dependence of neurons on ATP for cellular function (Parihar and Brewer 2007).
Addition of pyruvate, malate and β-hydroxybutyrate did not lead to an increase in
ATP levels as observed in other studies (Sheline, Behrens et al. 2000; Massieu, Haces
et al. 2003). This is likely due to inhibition of oxidative phosphorylation by
.
NO.
.
NO
can bind to the binuclear center of Cytochrome oxidase (COX), the terminal electron
acceptor of the electron transport chain, and inhibit its activity in a reversible and O
2

94
competitive manner (Brown and Cooper 1994; Cleeter, Cooper et al. 1994).
Additionally
.
NO can elicit an antimycin like effect at complex III, stemming electron
transfer from cytochrome b to ubiquinol further inhibiting electron flow through the
electron transport chain (Poderoso, Carreras et al. 1996). The dissipation of the proton
gradient due to the loss of proton extrusion of the matrix leads results in the loss of
electrochemical energy required for conversion of ADP to ATP by the ATP synthase.
Hence, addition of mitochondrial substrates in the presence of
.
NO should not lead to
an increase generation of ATP due to inhibition of the electron transport chain which
is what we observe. The inability for additional supplementation of glucose, pyruvate
and lactate to increase ATP levels, while improving neuronal survival was also
observed in hippocampal slices exposed to A β
25-35
(Wang, Takata et al. 2007). This
could be attributed to the selective inhibition of COX by A β directly (Canevari, Clark
et al. 1999) or through A β modulated increase in
.
NO generation through NMDA
receptor mediated pathways (Parks, Smith et al. 2001). Rather, addition of
mitochondrial energy substrates, glutamate/malate, resulted in an increase in GSH
levels in isolated brain mitochondria independent of mitochondrial respiration.
The mitochondrial GSH pool can apparently function autonomously from the
cytosolic GSH pool. Isolated hepatocytes treated with BSO, an inhibitor of GSH
synthesis, showed a slower depletion of mitochondrial GSH pools as opposed to the
cytosolic pool. This suggests that the mitochondrial GSH/GSSG ratio can vary
independently of that of the cytosol, in response to local changes in the production of

95
mitochondrial oxidants (Hurd, Costa et al. 2005). In cellular necrotic models utilizing
GSH-depleting agents, loss of cytoplasmic GSH was less consequential in determining
cell death as opposed to loss of mitochondrial GSH (Uhlig and Wendel 1992).
Previous work in our laboratory demonstrated that addition of glutamate/malate to
isolated brain mitochondria resulted in an increase in mitochondrial GSH levels. Due
to the inability of mitochondria to synthesize GSH in situ, the increase GSH levels
were determined to come from protein bound GSH (i.e. deglutathionylation.).
However, this increase in mitochondrial GSH was measured at state 3 respiration in
the presence of glutamate/malate and ADP (Garcia, Han et al. 2007). To determine if
release of protein bound GSH was dependent upon state 3 respiration, we determined
the concentration of GSH and GSSG in the presence of mitochondrial complex
inhibitors utilizing a HPLC method. Addition of individual mitochondrial complex
inhibitors did not affect the release of protein bound GSH, although the extent of GSH
increase was not as substantial as GSH increase observed during state 3 respiration
(Fig. 21). Increase in GSH and GSNO levels were observed in isolated brain
mitochondria in the presence of 1 µM steady state concentration of
.
NO incubated
with glutamate/malate and ADP as opposed to just
.
NO (Chang 2006), confirming that
release of protein bound GSH in the mitochondria is independent of mitochondrial
respiration. This increase in mitochondrial GSH elicited by mitochondrial energy
substrates might account for the intracellular “ROS and RNS scavenging” effects of
pyruvate (Desagher, Glowinski et al. 1997). Hence, although the loss of cellular ATP

96
during inhibition of the mitochondrial electron transport chain, temporary modulation
of the mitochondrial GSH pool by mitochondrial energy substrates-linked formation
of NAP(D)H plays an important role in initial adaptation to
.
NO challenge.
NADPH is the major source of electrons for reductive biosynthesis and is the
only source for reduction of the glutathione and thioredoxin pool in the mitochondria.
The conversion of NADH to NADPH by the nicotinamide nucleotide
transhydrogenase, NNT, is coupled to the proton gradient. Lower mitochondrial
membrane potential due to loss of electron flow through the electron transport chain
might affect the rate of conversion of NADH to NADPH and (Olausson, Fjellstrom et
al. 1995) hence affect the rate of deglutathionylation catalyzed by NADPH dependent
Glutaredoxin 2 (Maher 2006). NADPH supply has been suggested to be limiting factor
for the glutathione (Vogel, Wiesinger et al. 1999) and thioredoxin system (Patenaude,
Ven Murthy et al. 2004; Zhang, Go et al. 2007). A lower increase in GSH levels in the
presence of mitochondrial respiratory chain inhibitors might be due to a slower
generation of NADPH from NADH by the NNT, which is responsible for 50% of
NADPH generation in the mitochondria. The dependence of mitochondrial GSH redox
status on mitochondrial energy substrates was also demonstrated elsewhere. Recovery
of GSH from GSSG during H
2
O
2
exposure in isolated mitochondria was markedly
improved in the presence of mitochondrial energy substrates or intermediates of the
tricarboxylic acids. Differential inhibition of mitochondrial metabolic enzymes and
electron transport chain indicated that regeneration of NADPH by NADP
+
isocitrate

97
dehydrogenase, malic enzyme and NNT all contribute to the regeneration of NADPH
required for the reduction of GSH (Vogel, Wiesinger et al. 1999).
The ability of a wide range of (glucose derived, ketone body derived or TCA
derived) mitochondrial energy substrates to attenuate
.
NO induced toxicity through
modulation of the mitochondrial redox status highlight the importance of
mitochondrial energy failure in neurodegeneration. The effects of mitochondrial
energy substrates are varied and can be classified accordingly; (i) donation of
electrons from NADH and FADH
2
for the reoxidation of electron carriers in the
electron transport chain for the synthesis of additional ATP (Mathews, vn Holde et al.
2000), (ii) catalyzing the release of protein bound GSH, thereby maintaining
mitochondria in a reduced state and (iii) generate tricarboxylic intermediates such as
citrate which can protect aconitase from ONOO
-
mediated damage (Han, Canali et al.
2005). This study highlights the importance of availability of mitochondrial energy
substrates for normal cellular function and stresses the importance of therapeutic
targets aimed at maintaining mitochondrial bioenergetic and redox status (Fig.22).

98

β-OH
Malate
PDH
Glucose
Pyruvate
Glycolysis
Pyruvate
Inactive
Active
P P
Acetyl-CoA
TCA
cycle
PDH-Pase
PDH-K
CO
2
. NADH
HSCoA.
NAD
+
2ATP
I III IV
c
V
Q
PDH
Major energy source
Ketones, alternative energy source
Acetoacetate
+
Succinyl CoA
S SC CO OT T
Acetoacetyl-CoA
NADP
+

.
NO
.
NO
NADPH
Modulate
Thioredoxin/Glutathione
System
NNT
Malic
Enzyme
NAD
+
NADH
MITOCHONDRIA
Figure 22. Schematic diagram demonstrating the hypothesized effect of
mitochondrial substrates on mitochondrial function. Typically the energy
intensive brain depends on glucose derived pyruvate to sustain oxidative
phosphorylation. In the advent of decrease glycolysis of inhibition of PDHC,
alternate utilization of ketone bodies such as β-hydroxybuterate or
acetoacetate, can provide acetyl-CoA and intermediates of the TCA cycle.
Generation of NADH can lead to the formation of NADPH through the NNT.
Consumption of NAPDH by the thioredoxin and glutathione system drives the
regulation of protein disulfide and mixed disulfide formation as well as
regeneration of GSH from GSSG formed during the non-enzymatic reaction
between GSH and reactive oxygen and nitrogen species such as ONOO
-
and
H
2
O
2
(Yap, Han et al. 2007).

99
Chapter III :
Mitochondria as a Cytosolic Redox Modulator:
Topology of Peroxynitrite Generation

Introduction
Over the last few decades, the complex and disparate role that mitochondria play
within the cell has become increasingly well defined. Classically recognized as the
cellular powerhouses that produce the energy required to drive all endergonic process
for cellular life, mitochondria are also important cellular sources of reactive oxygen
species and signaling molecules that regulate cell cycle, proliferation, and apoptosis
(Cadenas 2004). As such, over 40 diseases such aging and neurodegenerative diseases
have been linked to impaired mitochondrial function and to an increase of
mitochondrial free radical generation and subsequent macromolecule damage,
activation of signaling pathways and/or apoptotic or necrotic cell death (Storz 2006).
Mitochondria are recognized as the major cellular sources of O
2
.–
, largely originating
from the autoxidation of ubisemiquinone – a mobile carrier that (a) transfers electrons
from complex I and II to complex III of the mitochondrial respiratory chain and from
(b) rotenone-sensitive complex I (Yap, Han et al. 2007).
Aside from its role as a cellular source of O
2
.-
, mitochondria are important
sinks of nitric oxide (
.
NO) (Radi, Cassina et al. 2002). Initial work demonstrated that
.
NO led to inhibition of mitochondrial respiratory chain complexes I and III, without

100
affecting complex IV activity. This effect was initially thought to occur due to direct
interactions between
.
NO and components of the complexes, however, further work
utilizing sub mitochondrial particles (inverted mitochondria) demonstrated that long
term inhibition of complex I (Riobo, Clementi et al. 2001) and complex III (Radi,
Cassina et al. 2002) was dependent upon the secondary formation of ONOO
-
.
ONOO
-
formation in mitochondria occurs primarily through the diffusion controlled
reaction between
.
NO and O
2
.-
(k ~ 10
10
M
-1
s
-1
) (Radi, Denicola et al. 2000) and
constitutes the major pathway for
.
NO metabolism in the mitochondria (Cadenas
2004). Formation of ONOO
-
can be influenced by the ubiquinol pool (Poderoso,
Carreras et al. 1999), through the formation of ubisemiquinone radical through
reaction of
.
NO with ubiquinol. Autoxidation of the ubisemiquinone radical leads to
the univalent reduction of O
2
, generating O
2
.-
which precedes to rapidly react with
.
NO
to form ONOO
-
(Poderoso, Carreras et al. 1996). Alternate pathways of ONOO
-

formation in mitochondria can occur through the reaction of nitroxyl anion (NO
-
),
formed during one electron reduction of
.
NO by mitochondrial electron donors such as
ubiquinol or reduced cytochrome c, with molecular oxygen (Radi, Cassina et al.
2002).
The formation of ONOO
-
is dependent upon the generation of O
2
.-
and hence,
influenced by the topology of O
2
.- generation. The topology of complex III (bc
I

complex) in the mitochondrial electron transport chain involves an inner (UQ
I
) and
outer pool of ubisemiquinone (UQ
o
), facing the matrix and inner membrane space

101
respectively. It has been established by our laboratory that the majority of O
2
.-

generated upon ubisemiquinone autoxidation

is released vectorially

into the matrix
(70-80%) (Cadenas 2004) and a smaller portion into the inter membrane space (20-
30%) (Han, Antunes et al. 2003; Cadenas 2004). Given the differential generation of
O
2
.-
, into the inter membrane space, we anticipate that the formation of ONOO
-
would
reflect the topological distribution of O
2
.-
. The presence of millimolar levels of
reduced cytochrome c, which reacts at considerably efficient rates (k= 2.5 x 10
5
M
-1
s
-
1
) with O
2
.-
(Han, Williams et al. 2001)

and ONOO
-
(Batthyany, Souza et al. 2005)
might also influence the formation of ONOO
-
in the inter membrane space. It has also
been speculated that although most of the ONOO
-
generated in the mitochondria will
react with targets at the site of production, the diffusion of ONOO
-
in and out of the
mitochondria would be possible. The half life of ONOO
-
is approximately 3-5 ms
which is a diffusion distance of about 3-4 μm, a distance slightly larger than the
average size of mitochondria of 2-3 μm (Radi, Cassina et al. 2002).
At present, the generation of ONOO
-
into the matrix is well characterized
(Poderoso, Carreras et al. 1996; Riobo, Clementi et al. 2001), however the formation
of ONOO
-
in the inter membrane space has not been addressed yet. This study was
aimed at evaluating the topological site(s) of the release of ONOO
-
in the inter
membrane space using mitoplasts (devoid of portions of the outer mitochondrial
membranes) and membrane impermeable Cu,Zn-SOD. Furthermore, we served to
address the question of whether mitochondria could be significant sources of ONOO
-

under physiological concentrations of
.
NO.

102
Materials and Methods
Chemicals – 5,5’-Dimethyl-1-pyrroline-N-oxide (DMPO), percoll, Cu,Zn-
superoxide dismutase, digitonin, Glutamate, malic acid, dimethyl sulfoxide (DMSO).
(St. Louis, MO, USA). DCFH-DA was purchased from Invitrogen (Carlsbad, CA,
USA)
Mitochondrial Isolation – Mitochondria from rat brain were isolated by
previously described procedures for differential centrifugation (Sciamanna and Lee
1993) and discontinuous percoll gradient (Anderson and Sims 2000; Schroeter, Boyd
et al. 2003). Mitoplasts- Mitoplasts were derived from intact isolated brain
mitochondria according the methods previously established in our laboratory (Han,
Williams et al. 2001). Briefly, mitochondria were treated with digitonin to a final
concentration of 0.2% in isolation buffer for 15 on ice. The sample was diluted 6 fold
using mitochondrial isolation buffer and centrifuged at 10,000 g for 10 min.

.
Nitric Oxide levels –Measurement of steady state levels of
.
NO was
performed

using a Clark-type NO electrode (World Precision Instruments)

inserted
through the top of a thermostated, stirred chamber (Brown and Cooper 1994). The
.
NO
electrode was calibrated with aliquots

of NO-saturated water, assumed to contain
2 mM
.
NO. Concentrations of
.
NO were calculated based on calibration curves.
Electron paramagnetic resonance (EPR) measurements – EPR spectra were
obtained with a Bruker ECS 106 spectrometer (operating at X-band) equipped with a
cylindrical room temperature cavity operating in TM
110
mode. Aliquots (150 μl) of the

103
reaction mixtures were transferred to bottom-sealed Pasteur pipettes and measured at
room temperature under the instrument settings described in the figure legends.
Oxidant Formation in Mitochondria – Formation of oxidants in intact
mitochondria was assessed by monitoring DCF fluorescence(LeBel, Ischiropoulos et
al. 1992).

Results
Mitochondrial NO Consumption
NO consumption has been attributed to (i) direct reaction with O
2
in the phospholipid
bilayer, (ii) reaction with cytochrome oxidase (iii) matrix thiol pool and (iv) reduction
by ubiquinol (Radi, Cassina et al. 2002). Recent work carried out in our laboratory by
Han et.al demonstrated that 20-30% of superoxide generated by ubisemiquinone
autoxidation (eq.9) is released into the intermembrane space and then further released
into the cytosol through VDAC (Han, Williams et al. 2001; Han, Antunes et al. 2003).
A major route of NO metabolism or consumption is the rate diffusion controlled
reaction between NO with O
2
.-
to generate ONOO
-
(k ~ 10
10
M
-1
s
-1
) (Koppenol,
Moreno et al. 1992; Radi, Denicola et al. 2000; Cadenas 2004). The steady state levels
of NO reflects the continuous generation of NO from DETA-NO a pure donor that
decays into NO and a triamine, and the consumption of NO by mitochondrial
components. Addition of intact mitochondria resulted in a dip in steady state NO
concentrations as monitored using an NO electrode. Addition of complex I substrates

104
(glutamate/malate), which leads to O
2
.-
generation (Han, Williams et al. 2001; Riobo,
Clementi et al. 2001), resulted in further consumption of NO indicating ONOO
-

generation.
UQ
.-
+ O
2
.-
→ UQ + O
2
.-
[9]

In the presence of 20 µM superoxide dismutase, NO consumption was inhibited and
returned to initial steady state concentrations before addition of intact mitochondria,
suggesting that formation of ONOO
-
is a significant route of NO metabolism. As
Cu,ZnSOD is membrane impermeable, the restoration of NO levels by Cu, ZnSOD
suggests that ONOO
-
formation occurs on the cytosolic side of mitochondria. The
ability of SOD to modulate the levels of ONOO
-
suggests that ONOO
-
is not leaking
out from the mitochondria from sites of ONOO
-
formation such as the ubiquinol pool
(Poderoso, Carreras et al. 1996) but rather ONOO
-
is formed from the reaction of
exogenous NO and mitochondrial released O
2
.-
. (Fig.23)

Mitoplasts
.
NO consumption
Mitoplasts offer a fortuitous model for assessing the topological formation of ONOO
-

in the inter membrane space. Previous experiments show that at 0.2% digitonin
treatment, mitoplasts remained coupled, despite the loss of a portion of cytochrome c
(Han, Williams et al. 2001). Due to the negative charge on the O
2
.-
anion diffusion
into the matrix is unfavorable due to the limitation on its membrane permeability and

105
0 5 10 15 20 25 30 35
0
500
1000
1500
2000
2500
Control
20 μM SOD
Glutamate
SOD
Mitochondria
Current (pA)
Time (min)
Figure 23. Mitochondrial Consumption of Nitric Oxide. 0.67 mM of DETA-
NO (corresponding to a NO concentration of 1 μM) was added to the chamber of
the NO electrode and monitored using a WPI ISO-NO electrode and allowed to
stabilize for 15 min. 100 μg of intact brain mitochondria was added into the
reaction chamber and
.
NO consumption was monitored in the presence or absence
of 20 μM Cu,ZnSOD. Glutamate/malate and ADP was added to all samples.

106
Figure 24. Cu,Zn SOD sensitive consumption of NO in Mitoplasts. (A)
0.67 mM of DETA-NO (corresponding to an initial flux of
.
NO concentration
of 1 μM) was added to the chamber of the
.
NO electrode and monitored using a
clark type electrode (WPI ISO-NO) and allowed to stabilize for 15 min. 100
μg of intact brain mitochondria was added into the reaction chamber at time 15
min and
.
NO consumption was monitored without SOD to establish control
rate of
.
NO consumption by mitoplasts. Titration of membrane impermeable
Cu,ZnSOD to prevent to reaction of O
2
.-
and
.
NO by dismutation of O
2
.-
into
H
2
O
2
absence of 1,5 and 20 μM Cu,ZnSOD. Mitoplasts were supplemented
with 2.4 mM glutamate/malate at 25 min, denoted by glutamate on NO trace.
(B) Focused graph of the titer effects of Cu,ZnSOD on NO consumption by
mitoplasts.

22 24 26 28 30 32 34
1500
1750
2000
2250
2500
Control
20 μM SOD
5 μM SOD
1 μM SOD
Glutamate
Current (pA)
Time (min)
-5 0 5 10 15 20 25 30 35
0
500
1000
1500
2000
2500
3000
Control
20 μM SOD
5 μM SOD
1 μM SOD
Glutamate
SOD
Mitoplasts
Current (pA)
Time (min)
A
B

107
repulsion by the negative-inside membrane potential (Nicholls and Ferguson 2002;
Han, Antunes et al. 2003; Han, Canali et al. 2003). Hence, O
2
.-
formed at the Q
0
site
visceral to the inter membrane site is likely to either stay in the inter membrane space
or diffuse out through the VDAC. To determine if ONOO
-
formation occurs in the
inter membrane space, NO consumption was measured in mitoplasts. Addition of
mitoplasts resulted in a slight dip in steady state levels of NO that was transient,
returning to initial levels. Further consumption of
.
NO was observed upon the
addition of complex I substrates (Fig. 24A) which could be partially rescued by the
addition of Cu,ZnSOD. Sensitivity of
.
NO consumption to increasing concentrations
of Cu,ZnSOD (Fig. 24B), suggests that at least in the inter mitochondrial space, the
consumption of
.
NO and the subsequent formation of ONOO
-
is regulated by the
presence of O
2
.-
.

Total NO consumed by Mitoplasts
Measurements with NO electrode demonstrated that
.
NO was consumed by mitoplasts
at a rate of 12 nmol s
-1
mg
-1
, as calculated with a standard curve. Addition of
exogenous Cu,ZnSOD was found to inhibit NO consumption in a dose dependent
manner with a maximum inhibition at 45%, suggesting that NO is reacting with O
2
.-
at
a rate of 5.4 nmol s
-1
mg
-1
(data not shown). The reductive utilization of NO by
ubiquinol and cytochrome oxidase provides a minor pathway of NO catabolism (20%)
(Cadenas 2004), hence since exogenous SOD was able to inhibit a large percent of

108
0
2
4
6
8
10
12
14
20 μM SOD 5 μM SOD 1 μM SOD
Control
(nmol / s / mg of protein)
Rate of NO Consumption
Control
1 μM SOD
5 μM SOD
20 μM SOD
Figure 25. Rate of
.
NO consumed by Mitoplasts. Rate of
.
NO consumption
was calculated from a standard curve generated before each experiment with
calculated concentration of
.
NO vs. current (pA). Calculation of
.
NO
consumption was determined from the slope upon injection of 2.4 mM
glutamate/malate and the standard curve generated.

109
.
NO consumed (45%) to form ONOO
-
, our results indicate that a significant portion of
ONOO
-
generated in mitochondria is formed in the inter membrane space (Fig.25).
This is of particular importance considering that there is little or no SOD activity in
the inter membrane space, hence, favoring the formation of ONOO
-
.

Effect of NO on DCF Fluorescence in Brain Mitochondria
It has been demonstrated that application of
.
NO results in the generation of
O
2
.-
and H
2
O
2
from submitochondrial particles as well as mitochondria due to
inhibition of complex III electron transfer (Poderoso, Carreras et al. 1996). To confirm
that addition of NO results in a general increase in oxidant generation, the fluorogenic
compound DCFH-DA was used. In the presence of oxidants, the membrane
permeable DCFH-DA is converted to fluorescent DCF. Addition of NO resulted in a
1.5 fold increase in DCF fluorescence, indicating an overall increase in oxidant load in
mitochondria (Fig.26). In a cell free system, DCFH-DA does not react with NO,
hence the increase in DCF fluorescence is not due to a reaction between the dye and
NO(Myhre, Andersen et al. 2003), but a general increase in oxidant load within and
external of brain mitochondria. DCFH-DA has been demonstrated to react with
ONOO
-
in a rapid manner (Myhre, Andersen et al. 2003) more so than with O
2
.-
and
H
2
O
2
, and considering that NO is the only biomolecule that can out-compete
superoxide dismutase for O
2
.-
, increase in DCF fluorescence could be due to ONOO
-

formation.

110
0.0
0.5
1.0
1.5
Control
1 μM NO
SS
NO (1 μM
SS
) Control
DCF Fluorescence (A.U.)
Figure 26. Effect of
.
NO on DCF Fluorescence in Brain Mitochondria.
Intact isolated brain mitochondria was incubated with 2 µM of DCFH
supplemented with 2.4 mM glutamate/malate in the absence (control) or
presence of 1 µM steady state concentration of
.
NO.

111
ONOO
-
Formation by Mitoplasts
Addition of
.
NO and glutamate to mitoplasts resulted in low intensity EPR spectrum
characteristic of the DMPO-OH adduct (Fig 27A). The formation of the DMPO-OH
adduct could result from either the spontaneous decay of a DMPO-superoxide adduct
(DMPO-OOH) (Han, Williams et al. 2001) or the reaction of DMPO with the
hydroxyl radical (OH
.
) formed by homolysis of peroxynitrous acid, the protonated
form of ONOO
-
(Radi, Denicola et al. 2000) (ONOO
-
is in equilibrium with ONOOH,
pK
a
= 6.8) (Radi, Cassina et al. 2002). In the presence of high concentration of
Cu,ZnSOD and NO, the EPR signal was abolished suggesting that the formation of the
EPR signal is dependent upon the presence of O
2
.-
. The fact that Cu,ZnSOD is
membrane impermeable suggests that the origin of the DMPO-OH signal is
originating from the intermembrane space (Fig.27B). However, considering that the
rate of ONOO
-
formation is so rapid (k ~ 10
10
M
-1
s
-1
) (Radi, Denicola et al. 2000), it
is likely that the EPR signal observed in the presence of NO originates from ONOO
-
.
Addition of DMSO, a OH
.
scavenger(Koppenol, Moreno et al. 1992), resulted in a loss
of the EPR signal (Fig.27C) suggesting that the DMPO-OH adduct resulted from the
reaction of DMPO with the OH
.
formed by the homolysis of peroxynitrous acid.
Taken together, in the presence of NO, the formation of ONOO
-
occurs in the
intermembrane space.

112
3440 3460 3480 3500 3520
+ DMSO
+ 20 μM SOD
1 μM NO
SS
Magnetic Field (G)
Figure 27. Formation of ONOO
-
by mitoplasts. Under the assay conditions,
the reaction mixture contained mitoplasts (100 µg) in mitochondrial isolation
buffer supplemented with 160 mM DMPO,
.
NO at a steady state concentration
of 1 µM, and respiratory substrates (glutatmate/malate). A. mitoplasts + NO,
B. 20 µM Cu,ZnSOD, C. 1% DMSO.
A
B
C

113
Discussion
Devoid of the outer mitochondrial membrane barrier, mitoplasts offer a fortuitous and
useful model for assessing the formation of ONOO
-
in the inter membrane space. This
study showed that a significant proportion of
.
NO is consumed through the formation
of ONOO
-
, detected by EPR (Fig.26), in the inter membrane space. This is supported
by the sensitivity of
.
NO consumption of both mitoplasts and mitochondria to
membrane impermeable Cu,ZnSOD (Fig 22 and 23).
The ubisemiquinone (UQH
.
) component of the respiratory chain is the main
quantitative site of O
2
.-
production within the mitochondria (Cadenas 2004). The Q
cycle within complex III involves ubiquinol oxidation and ubiquinone reduction at the
outer (UQ
o
) and inner (UQ
i
) sites respectively. It has been established by our
laboratory that the majority of O
2
.-
generated upon ubisemiquinone autoxidation

is
released vectorially

into the matrix (70-80%) (Cadenas 2004) and a smaller portion
into the inter membrane space (20-30%) (Han, Canali et al. 2003; Cadenas 2004).
.
NO
elicits an antimycin like effect, supporting an increase generation of O
2
.-
through
inhibition of electron transfer at the ubiquinone-cytochrome b region of the
respiratory chain (Poderoso, Carreras et al. 1996), followed by ubisemiquinone
autoxidation (Poderoso, Carreras et al. 1999). Pivotal work done in 1994 by several
groups reported that
.
NO can bind to the binuclear center of Cytochrome oxidase
(COX), the terminal electron acceptor of the electron transport chain, and inhibit its

114
activity in a reversible and O
2
competitive manner (Brown and Cooper 1994; Cleeter,
Cooper et al. 1994). Inhibition of electron flow due to COX inhibition could then
lead to reduction of mitochondrial respiratory complexes leading to increase formation
of the ubiseimquinone radical and further generation of O
2
.-
into the matrix and inter
membrane space. The observation that further increase in
.
NO consumption in
mitochondria or mitoplasts occurred upon the addition of mitochondrial respiratory
substrates, indicates that generation of O
2
.-
into the intermembrane space is limiting in
the formation of ONOO
-
and metabolism of
.
NO (Fig.23 & 24). The formation of
ONOO
-
in the inter membrane space can also arise from the reduction of
.
NO by
ubiquinol yielding NO
-
(eq. 10-12) and the ubisemiquinone radical which undergo
secondary reactions to form more O
2
.-
and ONOO
-
(Poderoso, Lisdero et al. 1999;
Radi, Cassina et al. 2002). However, it is unclear the rate and the extent that these
reactions occur at.
.
NO + UQH
-
→ NO
-
+ UQ
.-
+ H
+
[10]
UQ
.-
+ O
2
→ UQ + O
2
.-
[11]
O
2
.-
+
.
NO → ONOO
-
[12]
The standard reduction potential for the conversion of O
2
to O
2
.-
is -0.16V. Given the
highly reducing environment of the mitochondria, many components of the respiratory
chain are thermodynamically capable of univalent reduction of oxygen. The
generation of O
2
.-
by complex I occur through the autoxidation of flavoproteins

115
(FMNH
2
/FMN) (Cadenas 2004) or the iron-sulfur cluster N2 (Genova, Ventura et al.
2001). However, the generation of O
2
.-
by complex I is likely to occur on the matrix
site. The inability of O
2
.-
to diffuse across membranes, makes complex I generation of
O
2
.-
an unlikely contributor to the formation of ONOO
-
in the inter membrane space.
Consistent with previous studies concerning the metabolism of
.
NO in the matrix
space, as defined by the use of submitochondrial particles, likewise the major route of
.
NO metabolism in the mitochondrial inner membrane space is attributed to the
formation of ONOO
-
through the expeditious reaction with O
2
.-
, a product of
ubisemiquinone autoxidation.
The formation of ONOO
-
in the inter membrane is likely to be regulated by (i)
the presence of Cu,ZnSOD (k =0.62 x 10
9
M
-1
s
-1
) (Okado-Matsumoto and Fridovich
2001) (ii) the reduction of cytochrome c by O
2
.-
(k= 2.5 x 10
5
M
-1
s
-1
)(Han, Antunes et
al. 2003) regenerating O
2
in the process (iii) a low pH in the inter membrane space
which facilitates spontaneous dismutation to H
2
O
2
(Turrens 2003) and (iv) diffusion of
O
2
.-
into the cytosol through VDAC(Han, Antunes et al. 2003). Although the presence
of Cu,ZnSOD in the mitochondrial inter membrane space of liver (Okado-Matsumoto
and Fridovich 2001) has been described, the concentrations and contribution of
Cu,ZnSOD catalyzed dismutation of O
2
.-
in the inter membrane space in brain
mitochondria is relatively unclear. Additionally, the modest rate constant of the
reaction of cytochrome c and O
2
.-
pales in comparison with the reaction rate between
NO and O
2
.-
which is 5 fold faster. The tight association of most of the cytochrome c

116
to the inner membrane limits the mobility of the cytochrome c pool further hinders its
ability as a O
2
.-
scavenger in the inter membrane space (Han, Antunes et al. 2003).
Hence, the formation of ONOO
-
in the inter membrane space as well as at the
cytosolic side would be regulated by the release of O
2
.-
through VDAC. The release of
O
2
.-
through VDAC were estimated at ~ 0.04 nmol/min/mg and increased 8 fold by the
complex III inhibitor antimycin. In the presence of
.
NO, which elicits an antimycin
like effect, we expect that the release of O
2
.-
into the cytosol might be increased. The
formation of extra mitochondrial ONOO
-
seems to suggest that even at 1.0 µM steady
state levels of NO, O
2
.-
production appears to occur in excess or the release through
VDAC could be faster where the site of O
2
.-
is close to VDAC such that the moment
O
2
.-
is generated, it is release through the VDAC into the cytosol. The rapid release of
O
2
.-
from mitochondria even in the presence of
.
NO supports the cytosolic formation of
ONOO
-
. The generation of ONOO
-
in the inter membrane space, especially during the
application of exogenous source of
.
NO such as aging (Lam and Cadenas 2007) or
inflammation, is therefore likely to be controlled by the steady state concentration of
O
2
.-
. The ability of ONOO
-
to diffuse directly across biological membranes has been
recently demonstrated (Denicola, Souza et al. 1998) and it may be possible that
ONOO
-
will diffuse out the mitochondria during prolonged formation of ONOO
-
such
as during chronic nitrosative or oxidative stress. The ability to recover only about
45% of total
.
NO consumed in mitoplasts upon the addition of Cu,ZnSOD (Fig.25)
suggests that approximately 55 % of
.
NO is consumed either by the ubiquinol pool or

117
interactions with cytochrome c oxidase albeit the contribution of the latter two
reactions have been estimated to account for about 20% of
.
NO consumed in the
mitochondria (Cadenas 2004). The percentage of ONOO
-
formed in the inter
membrane space might be a slight overestimate as some of the cytochrome c pool is
lost in the process of mitoplasts generation. This suggests that the rest of the
.
NO
(~35%) is likely to diffuse into the matrix and react with the matrix targets such as
glutathione or O
2
.-
generated by complex I and complex III. Hence it appears that a
significant portion of the initial ONOO
-
might be generated in the intermembrane
space.
The formation of a significant portion of ONOO
-
in the intermembrane space
proves exceptionally problematic due to the lack of an efficient antioxidant network
that is present in the matrix. The matrix capacity for the removal of ONOO
-
is
represented by NADH
2
, UQH
2
(Schopfer, Riobo et al. 2000) and glutathione (Cadenas
2004). At present, the inter membrane space is ill defined and the concentration of
these biomolecules and their existence in the inter membrane space is relatively
unclear. Other routes of metabolism of ONOO
-
may be more significant in the inter
membrane space such as the reaction of ONOO
-
with proteins or lipids in the inner
membrane. Due to the high protein content in the mitochondrial inner membrane, the
proteins on the inner membrane become a prime target for ONOO
-
formed in the inter
membrane space. ONOO
-
reacts predominantly with tyrosine and cysteine residues
leading to nitration and oxidation, respectively (Radi, Denicola et al. 2000). A

118
relevant pathway for the removal of ONOO
-
in the inter membrane space is likely the
reaction with carbon dioxide which lies in the millimolar range in mitochondria (k
=4.6 x 10
4
M
-1
s
-1
). The ONOO
-
/CO
2
pathway leads to the rapid formation of CO
3
.-

and
.
NO
2
(eq. 13) which can lead to the nitration of proteins (eq. 14 & 15).
ONOO
-
+ CO
2
→
.
NO
2
+ CO
3
.-
[13]
Tyrosine + CO
3
.-
→ Tyrosyl radical + HCO
3
-
[14]
Tyrosyl radical +
.
NO
2
→ 3 Nitrotyrosine [15]
Of particular importance is the nitration of mitochondrial complex I which leads to
irreversible inhibition in its activity (Riobo, Clementi et al. 2001; Murray, Taylor et al.
2003), especially in brain mitochondria that strictly channel reducing equivalents
(such as NADH) into site I. Nitration of complex I activity has been demonstrated to
play a critical role in the Parkinson’s disease model (Chinta and Andersen 2006). An
increase in nitration of mitochondrial proteins as a function of aging was demonstrated
in rat brain mitochondria. LC/MS/MS analysis of nitrated proteins identified F
1
-
ATPase as one of the proteins that was nitrated as a function of age. Associated with
the nitration of TYR 269 on the β- subunit, which faces the inter membrane
side(Abrahams, Leslie et al. 1994), was a 35% decrease in ATPase activity. Hence,
formation of ONOO
-
in the inter membrane space could in part facilitate the nitration
of F
1
-ATPase, regulating mitochondrial generation of ATP. Additionally, nitration of
cytochrome c (k= 2 x 10
5
M
-1
s
-1
) led to a gain in peroxidase like activity which

119
correlated to inhibition of mitochondrial respiration (Cassina, Hodara et al. 2000;
Batthyany, Souza et al. 2005). A similar concentration of ONOO
-
that was able to
inhibit components of the respiratory chain was able to inhibit the activity inner
membrane nicotinamide nucleotide transhydrogenase (NNT) which was not reversed
upon the addition of ubiquinol. Inhibition of the NNT led to an increase k
m
for both
NADH and NAD(P)H, thus affecting the generation of reducing equivalents in the
mitochondrial matrix (Forsmark-Andree, Persson et al. 1996). Although ONOO
-
and
ONOOH are in equilibrium, the pH close to the inter membrane space may be lower
and may facilitate an increase formation of ONOOH (eq. 8), a strong oxidizing
species. A small portion (< 1%) of ONOO
-
could undergo homolysis, forming OH
.

and NO
2
(eq.8). Although this reaction is relatively slow (k = 0.9 s
-1
), less routes of
ONOO
-
consumption might increase the latency of ONOO
-
and facilitate the formation
of OH
.
.
ONOO
-
+ H
+
↔ ONOOH → OH
.
+ NO
2
[16]

OH
.
radicals react with unsaturated fatty acids at near diffusion controlled rates (k=
10
9
M
-1
s
-1
) and
.
NO
2
formed is a lipophilic radical (k= 10
5
M
-1
s
-1
) capable of
initiating fatty acid oxidation as well (Radi, Denicola et al. 2000). Peroxidation of the
inner membrane results in a loss of cytochrome c and further impairment of
mitochondrial respiration rates (Lam and Cadenas 2007). OH
.
formed could also target
metal centers in components of the mitochondrial respiratory chain such as the iron

120
sulfur cluster in complex I. The formation of a significant portion of ONOO
-
in the
inter membrane space presents an additional mechanism through which the toxic
effects of
.
NO on mitochondrial function ensues. A lack of a competent and efficient
antioxidant network in the inter membrane space could favor a longer half life of the
species and enable diffusion of ONOO
-
into the matrix or into the cytosol (Fig.28).
Understanding the contribution of ONOO
-
formed in the inter membrane space on
cytosolic redox status and mitochondrial function would provide invaluable
information concerning mitochondrial function and metabolism of
.
NO with respect to
cellular function.

121
ONOO
-

I III IV
c
V
Q
Cytosol
VDAC
O
2
.-
O
2
O
2
.-
+
.
NO ONOO
-
O
2
O
2
.-
+
.
NO ONOO
-
.
NO
.
NO
~(45 %)
?
Inter membrane
space
Matrix
Oxidation of
mitochondrial
proteins
Figure 28. Topology ONOO
-
formation in the intermembrane space. The
mitochondrial physical topology might affect the formation of reactive oxygen
and nitrogen species during exogenous exposure to
.
NO. The ability for NO to
reach the mitochondrial matrix might be affect by the various reactions that it
undergoes within various compartments in the mitochondria.
CONCLUSION

The spectrum of cellular effects of
.
NO is reflected in the multitude of
pathways that are altered during
.
NO challenge. In this dissertation, we served to
address the hypothesis - nitric oxide generated during neuroinflammation leads to
changes in cellular redox status and protein post translational modification which
play a role in energy impairment leading to neuronal injury. This hypothesis was
achieved through four experimental approaches designed to address (i) the modulation
of cellular redox status, (ii) the effect of an altered GSH redox status modulated by
.
NO on protein post translational modifications and (iii) the effect of cytosolic events
modulated by
.
NO on mitochondrial function. and (iv) the role of
.
NO as a direct
modulator of mitochondrial generation of reactive oxygen and nitrogen species,
The data presented in this dissertation demonstrates that alteration of cellular
redox status correlated with decreased cell viability.
.
NO modulation of the cellular
redox status by
.
NO occurs by (i) direct nitrosation/transnitrosation reaction between
NO and GSH, forming GSNO and the subsequent formation of GSSG through GSNO
reductase activity, and (ii) NO mediated effects on mitochondrial generation of
reactive oxygen and nitrogen species.
.
NO inhibition of mitochondrial respiration resulted in a substantial formation
of ONOO
-
(~ 45% of
.
NO consumed) in the intermembrane space due to increase
formation of O
2
.-
through autoxidation of components of the electron transport chain.

122

123
Formation of ONOO
-
in the inter membrane space may have a longer half life due to a
lower/absence of antioxidant enzymes typically present in the matrix space, therefore
potentially promoting nitration of proteins such as cytochrome c. Studies using intact
isolated mitochondria demonstrated that despite ONOO
-
formation in the
intermembrane space, O
2
.-
was released from mitochondria and resulted in extra
mitochondrial formation of ONOO
-
. Oxidation of ONOO
-
by GSH leads to GSSG
formation, potentially altering both mitochondrial and cytosolic redox status.
Exposure of primary cortical neurons and astrocytes to extracellular
.
NO led to
alterations of the cellular redox status through the increase formation of GSNO, GSSG
and the concomitant decrease in GSH. Changes in GSNO, GSSG and GSH levels lead
to oxidation of the redox environment (increase E
hc
) which correlated with decreased
cellular viability. The importance of GSH homeostasis was highlighted using
astrocytes as a comparative cell model which have been previously demonstrated to
have a higher tolerance to oxidative and nitrosative stress. As compared to neurons,
astrocytes had a higher GSNO and GSSG reductase activity and absolute
concentration of GSH. This translated into a higher redox buffering capacity against
.
NO challenge. Alteration of the cellular redox environment resulted in S-
glutathionylation of a specific subset of proteins, albeit differentially in both cell
types. Glutathionylation of GAPDH activity and the consequential loss in its activity
occurred due to NO mediated alterations in cellular redox status. GAPDH
glutathionylation was discovered to occur in an age dependent manner in a triple

124
transgenic AD mice model. As observed in the primary cortical model,
glutathionylation of GAPDH correlated with decreased activity and occurred at earlier
stages of the disease pathology (6 months). This suggests that glutathionylation of
proteins occur relatively early in the progression of AD and is likely that these
changes are a result of altered cellular redox environment. Reversible S-
glutathionylation of redox sensitive proteins such as GAPDH might serve to initially
protect thiolate anions from irreversible oxidation. However, a consequence of this
protection is the loss of enzymatic activity of important enzymes such as GAPDH.
Oxidation of the cellular redox environment, and the subsequent inhibition of
the activity of GAPDH, an important enzyme in glycolysis disrupts the provision of
pyruvate to mitochondria. Addition of mitochondrial energy substrates, pyruvate,
malate and β-hydroxybutyrate, lead to attenuation of NO mediated neurotoxicity in
primary cortical neurons, but did not reverse
.
NO mediated loss of cellular ATP levels.
Rather, the protective effects were mediated through the mitochondrial redox couples
NAD(P)
+
/NAD(P)H. Mitochondrial substrate supplementation of isolated brain
mitochondria resulted in an increase in NAD(P)H and GSH levels. Mitochondrial
substrate supplementation resulted in an increase in mitochondrial GSH through the
release of protein bound GSH from S-glutathionylated proteins. The release of protein
bound GSH does not require flow of electrons through the mitochondrial electron
transport chain but rather, most likely the through the generation of NADPH.

125
FUTURE PERSPECTIVES

The insidious nature of chronic idiopathic neurodegenerative diseases such as
Alzheimer’s and Parkinson’s diseases provides a difficult situation for clinicians as
well as for individuals with respect to diagnosis, management and treatment of the
diseases. The sequential genetic and molecular events that ultimately lead to the
manifestation of the disease are complex and varied. As such, the major obstacle that
faces the treatment and design of efficacious treatment modules remains the
incomplete understanding of the disease pathology. The data presented in this thesis
provides the foundation for future research that is outlined below:
1. The relationship between normal and pathological aging. Chronic
neurodegenerative disorders are often viewed as accelerated aging, as such
several factors between normal brain aging and pathological aging overlap
(Yankner, Lu et al. 2007). As we have demonstrated, inhibition of GAPDH due
to glutathionylation occurred as a function of age in the triple transgenic mice
model. Of interest is to compare the effect of age related changes in GAPDH
activity in normal brain aging as a function of GAPDH changes in pathological
aging. This would provide further relevance to the loss of GAPDH activity
during pathological aging. Leonard Hayflick is the biggest proponent of the idea
that if we understand the fundamental changes that occur during aging, then we
will gain further insight into the development of age related diseases

126
(Hayflick 2007). Perhaps not so surprising is the efficacy of caloric restriction
on aging as well as AD. Caloric restriction, which is the only proven regiment to
extend lifespan, and prevented age related deficits in cognitive function in AD
(Halagappa, Guo et al. 2007). Understanding and identifying common, as well as
deviant factors between normal and pathological aging would further the
understanding of the development of pathological aging. This has lead to an
interest in understanding sirtuins, a major class of histone deacetylase up
regulated during caloric restriction (Anekonda and Reddy 2006), with respect to
AD. Hence, a closer look at aging and aging related regiments, such as acetyl-L-
carnitine, might give novel targets for further drug development.
2. Development of Risk Assessment Analysis Profile. Alterations in energy
utilization are best correlated with dementia and are the earliest, consistent
changes that occur in AD. The loss in the ability to sufficiently supply ATP for
cellular needs due to impairment in glycolysis and mitochondrial function
ultimately leads to the demise of neurons. We have shown that addition of
mitochondrial energy substrates can ameliorate NO mediated neurotoxicity as
such, it would be beneficial to further understand the role of mitochondrial
energy substrates in modulating cellular viability, particularly, looking at overall
changes in the pyridine nucleotide. Initial work done in this dissertation has laid
a ground work showing potential therapeutic effects of mitochondrial energy
substrates in a neuoroinflammatory model that needs to be further expanded upon

127
by doing temporal analysis of the effects or risk assessments of mitochondrial energy
substrates with respect to changes in Nernst Potential, a component of cellular
viability. This idea is illustrated in the figure in the next page (Fig. 29).
As they say, “all roads lead to Rome”. Whether mitochondrial impairment is a
proximate cause for aging or age related degenerative diseases, it is irrefutable that
proper mitochondrial function must be maintained in order for the cell to survive and
effectively function. Manipulating pathways to maintain proper mitochondrial
function and cellular redox status still remains an ideal approach. This is reflected by
the fact that when glycolysis was impaired through glutathionylation of GAPDH,
manipulation by addition of mitochondrial energy substrates led to modulation of
mitochondrial redox status and attenuation of
.
NO mediated neurotoxicity.

Figure 29. Conceptualization of Risk Assesment profile of the therapeutic
values of mitochondrial energy substrates in
.
NO mediated neurotoxicity.
.
NO
128

129
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Abstract (if available)

Abstract The nefarious role of NO in neurodegenerative diseases such as Alzheimer's disease stems from the excessive production and labile nature of NO. The mechanisms of NO redox signaling through protein post translational modification is ill-defined in the neurodegenerative model. S-nitrosylation, which can be an intermediary step leading S-glutathionylation of proteins has emerged as a well characterized mechanism through which NO reversibly regulates cell function. However, there is little evidence concerning the regulation of S-glutathionylation of proteins by NO, which proteins are glutathionylated in neurons during nitrosative stress, and the consequence of glutathionylation of proteins on overall cellular and mitochondrial function. Therefore, the hypothesis to be tested is that nitric oxide generated during neuroinflammation leads to changes in cellular redox status and protein post translational modification which play a role in energy impairment leading to neuronal injury.

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