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Role of neuronal nitric oxide synthase in aging and neurodegeneration
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Role of neuronal nitric oxide synthase in aging and neurodegeneration
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Content
ROLE OF NEURONAL NITRIC OXIDE SYNTHASE IN AGING AND
NEURODEGENERATION
by
Yeung Lam
_____________________________________________________________________
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)
December 2007
Copyright 2007 Yeung Lam
ii
DEDICATION
To my parents, brother and my girlfriend for their unconditional love and support.
“Tough times never last, but tough people do.
When the going gets tough, the tough get going.
Change what you can and accept what you can't change.”
iii
ACKNOWLEDGEMENTS
I am indebt to my mentor Dr. Enrique Cadenas for his patient guidance and invaluable
suggestions throughout my study. I am so grateful for his support and shelter during
the toughest time of my Ph.D. Thank you for granting the responsibility that I can
handle; the freedom that I need to develop my own idea; and the environment in which
I can enjoy my work. I would also like to thank all my committee members: Dr.
Ronald Alkana and Dr. Howard Hodis for their time to examine my dissertation.
I am really thankful to all the former and current laboratory colleagues (Dr. Allen
Chang, Dr. Jerome Garcia, Dr. Derick Han, Dr. Juliana Huang, Dr. Qiongqiong Zhou,
Ryan Hamilton, Joanne Lee, Lulu Tang, William Tsoi, Li-Peng Yap and Fei Yin) for
their support. They make the laboratory a harmonic environment so that I can enjoy
and concentrate on my work.
Last but not least, I have to express my deepest appreciation to my friends in Hong
Kong (Sharon Chung, Charis Liu, Danny To). Although they are apart from me, they
are always my spiritual support. They support me in bad times, honor me for success,
strengthen me in challenge, and relief me from sadness. May our friendships last
forever.
iv
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF FIGURES vi
ABSTRACT viii
GENERAL INTRODUCTION 1
1. Brain aging 1
2. Nitric oxide: role in brain function and pathology 2
a. Nitric oxide (
.
NO) and nitric oxide synthase (NOS) 2
b.
.
NO mediated protein post-translational modifications 5
c.
.
NO mediated apoptosis 9
3. The aged proteasome system and its role in neurodegenerative disease 11
4. Case study: neuronal NOS and its role in inherent and environmental
toxin induced Parkinson’s disease (PD) 14
SIGNIFICANCE 16
HYPOTHESIS & SPECIFIC AIMS 19
CHAPTER I: 22
CORRELATION OF ELEVATED NEURONAL NITRIC OXIDE
SYNTHASE LEVEL DURING AGING WITH COMPROMISED
ENERGY PRODUCTION
Introduction 22
Materials and methods 26
Results 29
Discussion 42
CHAPTER II: 47
COMPROMISED PROTEASOME DEGRADATION SYSTEM
ELEVATES NEURONAL NITRIC OXIDE SYNTHASE LEVEL
AND INDUCES NEURONAL CELL DEATH
Introduction 47
v
Materials and methods 50
Results 53
Discussion 64
CHAPTER III: 68
NEURONAL NITRIC OXIDE SYNTHASE: IMPLICATION FOR
ENVIRONMENTAL TOXIN INDUCED APOPTOSIS IN
DOPAMINERGIC NEURONS
Introduction 68
Materials and methods 73
Results 76
Discussion 85
SUMMARY 88
BIBLIOGRAPHY 92
vi
LIST OF FIGURES
Fig. 1: Role of
.
NO at different cellular concentrations 3
Fig. 2: Overview of specific aims 20
Fig. 3: nNOS protein content in rat brain increases during aging. 31
Fig. 4: Nitrosylation and nitration of rat brain cytosolic and mitochondrial
proteins increase during aging. al proteins during aging. 32
Fig. 5: SCOT and F
1
-ATPase beta subunit are identified as nitration targets
by LC/MS/MS during aging. 34
Fig. 6: SCOT and F
1
-ATPase enzymatic activities reduce during aging 38
Fig. 7: Mitochondrial respiratory decreases with age 40
Fig. 8: Mitochondrial cytochrome c release increases with age 41
Fig. 9: Inhibition of proteasome-ubitquitin degradation system leads to
increase in nNOS content 54
Fig. 10: Increase in nNOS content by inhibiting proteasome is correlated to
increase in cytotoxicity and decrease in neuronal viability, which
correlated to the inhibition of proteasome 55
Fig. 11: Enhanced nNOS causes increase in
.
NO and thus enhances ONOO
-
formation 58
Fig. 12: Reduction of cell viability by MG132 is
.
NO and JNK mediate 60
Fig. 13: MG132 induced
.
NO production causes caspase 9 and 3 activation
which can be reversed by NOS inhibitor (L-NAME and 7-NI) 62
Fig. 14: Paraquat has a similiar structure to MPP
+
69
Fig. 15: Paraquat dose-dependently decreases cell viability 76
vii
Fig. 16: Paraquat dose-dependently increases LDH release 77
Fig. 17: DPI has complete protecting effect against paraqaut-induced
LDH releases 78
Fig. 18: PQ radical generation is NOS dependent and does not require
uncoupling of NOS 80
Fig. 19: L-NAME dose-dependently relieves paraquat toxicity 81
Fig. 20: nNOS induces paraquat formation 83
Fig. 21: Paraquat dependent NADPH consumption increases with age
implicating enhanced paraquat radical formation 84
Fig. 22: Schematic diagram shows the proposed relationships between
aging induced neurodegeneration due to upregulation of nNOS
caused by proteasomal dysfunction. 90
viii
ABSTRACT
Nitric oxide synthase (NOS) is a flavin- and heme- containing enzyme that catalyzes
the metabolism of L-arginine to L-citrulline and nitric oxide (
.
NO) in the presence of
O
2
and NADPH. Neuronal NOS (nNOS) is a Ca
2+
-calmodulin-dependent isoform of
NOS that is constitutively expressed in neuronal cells. The cellular level of nNOS is
regulatory by its turnover through degradation by the proteasome. Various studies
have demonstrated that proteasome activity declines with age. Furthermore,
dysfunction proteasome is implicated in Parkinson’s disease (PD) in which the
formation of Lewy bodies and a progressive degeneration of dopaminergic neurons are
observed. However, to date, there has been no extensive study undertaken to
investigate the role of proteasome function, aging, and nNOS level with respect to
viability of dopaminergic neuron. Here, we propose that aging-induced dysfunction of
proteasome leads to accumulation of nNOS protein, thereby increasing the production
of reactive nitrogen species (RNS), such as
.
NO and peroxynitrite (ONOO
-
), and thus
results in neuronal death due to increased nitrative / nitrosative stress. In this
dissertation, by using brains from rats of different age and a PC12 dopaminergic cell
model, it is demonstrated that nNOS protein levels increased with age and this
correlated with an increase in both nitrosative and nitrative stress. Under conditions of
elevated nNOS expression, two important mitochondrial enzymes involved in
mitochondria bioenergetics, succinyl-CoA:3-oxoacid CoA-transferase (SCOT) and F
1
-
ATPase, were found to be nitrated. Nitration of SCOT and F
1
-ATPase lead to a
ix
decrease in their activities, thus suggesting a compromised energy production at the
level of reducing-equivalent generation and oxidative phosphorylation, respectively.
The consequences of declined energy production were linked to increased apoptosis as
shown by enhanced cytochrome c release in aged brain. Data from the PC12 model
suggests that aging-induced dysfunction or impairment of the proteasome system leads
to enhanced expression of nNOS which concomitant
.
NO production and ONOO
-
formation. The latter activated c-Jun N-terminal Kinase (JNK). JNK induces
phosphorylation of Bcl
XL
(inhibition) eventually triggered the activation of
downstream apoptosis cascade that included the commitment (caspase-9) and
execution (capase-3) phase. The cytotoxic consequences of an enhanced nNOS
activity was further supported by an enhanced activation of paraquat (a potent
herbicide) in rat brain homogenates and PC12 cell lysates, leading to decreased cell
viability. Inhibition of nNOS activity abolished the formation of paraquat radical, thus
suggesting the important role of nNOS in environmental toxin-induced sporadic
Parkinsonism. Data presented herein this dissertation strongly supports the notion that
the age-related elevation of nNOS may contribute to the increased nitric oxide-
mediated neuronal cell death, which is inherent in to progression of the
pathophysiology of PD.
1
GENERAL INTRODUCTION
1. Brain aging
Aging is a process characterized by a general decline in physiological functions,
including a marked effect on brain activities, such as neuromuscular coordination,
cognitive performance, and environmental awareness (Swerdlow 2007). These
activities involve receiving, interpreting and transmitting information by the brain and
require the generation of action potential, thus the brain has a high metabolic rate and
demands significantly larger amounts of energy (10% of total energy consumption)
compared to other organs. This characteristic renders the brain particularly susceptible
to an energy crisis. Mitochondrion is the power house providing energy for the above
mentioned activities. One of the most prominent metabolic processes carried out by
mitochondria is oxidative phosphorylation (OXPHOS). OXPHOS generates ATP,
which is considered as the universal energy currency, by utilizing reducing equivalents
formed from the TCA cycle. Beside OXPHOS, mitochondria are also the sites of
many different types of anabolic and catabolic pathways including oxidation of amino
acids, fatty acids, and pyruvic acid. Aging is a risk factor that increases the brain’s
susceptibility to disease, as aged organisms usually show a decreased capacity to carry
out OXPHOS and disturbed cell metabolism due to mitochondrial dysfunction. Also,
cellular composition of brain consists mainly of terminally differentiated neurons
hence, the regenerative capacity of the brain in forms of cellular regeneration is
2
relatively impaired/reduced as compared with other organs such as liver, which can
regenerate itself (Andersen, 2004). Taken together, the brain is highly susceptible to
neuronal loss due to decrease in energy production which is often caused by age
associated mitochondrial dysfunction.
2. Nitric oxide: role in brain function and pathology
a) Nitric oxide (
.
NO) and nitric oxide synthase (NOS)
In the brain, nitric oxide (
.
NO) is one of the most important biological messengers that
play important roles in neurotransmission. It is responsible for synaptic plasticity and
long term potentiation (LTP) by acting as a retrograde signaling molecule (Vincent
1994).
.
NO is a small and hydrophobic gas molecule produced enzymatically by a
flavin and heme containing enzyme, nitric oxide synthase (NOS). It consists of single
oxygen bonded to one nitrogen atom thus it is a free radical. However, it is fairly non-
reactive when comparing to other free radical molecule such as superoxide (O
2
.-
) thus
it can diffuse several cell diameters before reacting with other molecules. It can freely
permeate biological membranes due to its non-polar nature. Owing to its ability to
diffuse across biological membrane and travels relatively long distance (~10 μm/sec),
hence it is capable to act as a key biological messenger.
3
At physiologically low concentrations,
.
NO is usually anti-apoptotic, acting as a
messenger for numerous intra- and intercellular physiological roles such as
neurotransmission. However, when the cellular
.
NO concentration increases, it
becomes cytotoxic through further oxidation to form reactive nitrogen species (RNS).
High level of
.
NO can induce neuronal cell death by damaging DNA, lipid and protein
functions (Fig. 1) (Davis, Martin et al. 2001). The cytotoxic effects of
.
NO on the
brain can be manifested through two major mechanisms: 1) RNS mediated protein
post-translational modification and 2) apoptosis.
Fig. 1 Role of .NO at different cellular concentrations.
Figure adopted from Davis, Martin et.al. Annu Rev
Pharmacol Toxicol. 2001;41:203-36.
4
.
NO is formed by the metabolism of L-arginine (L-Arg) to L-citrulline (L-Cit) in the
presence of O
2
and NADPH by NOS, which is a flavin- and heme- containing enzyme.
There are three isoforms of NOS: 1) neuronal NOS (nNOS); 2) endothelial NOS
(eNOS) and 3) inducible NOS (iNOS) (Palacios, Knowles et al. 1989; Bredt and
Snyder 1990). These three enzymes share a high degree of similarity in that they all
contain an N-terminal oxygenase and a C-terminal reductase domains. They all require
several cofactors including heme, tetrahydrobiopterin, calcium, calmodulin, FAD and
FMN in order to function. Dimerization of NOS in a head to tail formation leads to an
active NOS. The reductase domain serves to transfer the electron from NADPH to the
oxygenase domain. By utilizing the electron, the oxygenase domain converts O
2
and
L-Arg into
.
NO and L-Cit. Although they share a high degree of structural similarity;
these three isoforms differ in their expression pattern, cellular location and functions.
nNOS and eNOS are constitutively expressed and play important physiological
signaling roles. Their activities are regulated by Ca
2+
signaling since binding of Ca
2+
to
the cofactor calmodulin stabilizes the dimmer and thus stimulates their respective
activities. One example is the activation of NMDA (N-methyl d-aspartate) receptor
that increases intracellular Ca
2+
level which leads to activation of the enzyme. The
turnover rate of nNOS and eNOS also plays an important role in regulating their
activity since they are constitutively expressed. Whereas iNOS is cytokine-inducible
and regulated by gene transcription. It functions as the effector and regulator of
immune response (Forstermann, Schmidt et al. 1992). The association of calmodulin
5
to iNOS is rather tight hence Ca
2+
level has relatively no effect on iNOS activity. Thus
the regulation of iNOS is at transcriptional level rather than through Ca
2+
signaling.
b)
.
NO mediated protein post-translational modifications
.
NO can induce protein post-translation modifications by addition of NO / NO
2
group
to specific residue(s) of the protein. The two major post-translation modifications
induced by
.
NO are: 1) S-nitrosylation (adduction of NO
+
) and 2) Tyrosine nitration
(adduction of NO
2
).
S-nitrosylation is a reversible post-translational modification of the cysteine thiol side
chain by RNS to form the generic nitrosothiol structure (R-S-N=O). In order to form
protein nitrosothiol, either 1)
.
NO needs to be activated to form a nitrosonium (NO
+
)
carrying RNS or 2) thiol needs to be “activated” through oxidation to form a thiyl
radical to allow nitrosylation reaction to take place. Then NO
+
can react with protein
thiolate (RS-) to form RSNO via nucleophilic substitution based on Stamler’s acid-
base motif theory; the pKa of cysteinyl thiol can be affected by neighboring acidic and
basic amino acid residues in the 3D structure, making this particular thiol easier to lose
a proton, which leads to the formation of thiolate anion (Pr-S
-
) and become a target of
NO
+
. Thus, S-nitrosylation is actually a transfer of NO
+
instead of
.
NO. Since S-
nitrosylation is a reversible protein modification, it can act as a switch for modulating
protein function which means it can either cause an increase or decrease in activity of
the protein. A plethora of proteins such as caspases 3 and 9 have been shown to be
6
nitrosylated (Mannick, Schonhoff et al. 2001; Torok, Higuchi et al. 2002),
demonstrating that nitrosylation plays role in
.
NO mediated modulation of cell
functions.
Tyrosine nitration is an irreversible covalent protein post-translational modification by
addition of NO
2
group to the aromatic ring of tyrosine residue by ONOO
-
. O
2
• −
and
•
NO are the stoichiometric precursors of ONOO
-
and react at diffusion limited rates to
form ONOO
-
. This modification usually leads to a decrease in protein function. Two
major pathways are responsible for tyrosine nitration in vivo: 1) chemical nitration by
peroxynitrite (ONOO
-
) (Beckmann, Ye et al. 1994) or 2) catalytic action of heme
peroxidases using hydrogen peroxide and nitrite (Eiserich, Hristova et al. 1998).
Nevertheless, both pathways require exposure of the biological system to
.
NO. Briefly,
nitration encompasses the activation of tyrosyl radical (Tyr
.
) by one-electron oxidants
(CO
3
. −
and
.
NO
2
) that are yielded from the reaction between ONOO
-
and CO
2
. Then
.
NO
2
derived either after 1) the homolysis of ONOO
−
or 2) Oxidation of NO
2
−
which
is derived from the decomposition of
.
NO or ONOO
−
adds to the Tyr
.
in a fast radical–
radical termination reaction to yield 3-NO
2
-Tyr (3-nitro-L-tyrosine). Hence, nitration
is a hallmark of ONOO
-
formation.
In this dissertation, tyrosine nitration of specific enzymes involved in brain energy
metabolism will be emphasized and discussed since it is a permanent modification and
is highly relevant to neurodegenerative diseases. The occurrence of biological tyrosine
7
nitration is relatively low (100-500 μmol/mol of tyrosine in case of inflammation)
(Brennan, Wu et al. 2002; Aslan, Ryan et al. 2003) suggesting that tyrosine nitration is
a selective process targeting a few tyrosine residues in a limited number of proteins.
There is increasing evidence that protein function can be modulated by tyrosine
residue(s) nitration. There are numerous reports showing that nitration leads to loss of
protein function, however, nitration mediated activation of protein function has also
been described (Ji, Neverova et al. 2006) which suggests the ultimate functional
consequence of nitration depends on the sites of modification of the enzyme.
This dissertation will discuss about two important enzymes in the energy metabolism
pathways that were found to be nitrated during aging: 1) Succinyl-CoA:3-oxoacid
CoA-transferase (SCOT) and 2) F
1
-ATPase (Complex V).
SCOT is the key-limiting enzyme in ketolysis, which is a process that converts ketone
bodies such as acetoacetate to acetyl-CoA, which feeds into the TCA cycle. Under
carbohydrate depletion or when carbohydrate cannot be utilized efficiently such as in
diabetic patients, ketolysis sustains energy production by enabling fat-derived energy
to be used during oxidative phosphorylation. Ketolysis occurs in the mitochondria
matrix in many extra-hepatic organs and is of particular importance as ketone bodies
as the only alternative source of energy for the brain during glucose deficiency
(Grinblat, Pacheco Bolanos et al. 1986).
8
ATPase is a critical transmembrane protein complex found on mitochondrial inner
membrane and is the last complex located in the electron transport chain for
generating ATP. It is compromised of a proton-conducting F
0
unit and a catalytic F
1
unit. F-ATPase generates ATP through the proton motive force generated during
OXPHOS. The driving force of the proton gradient allows the passive re-entry of
protons through the F
1
-ATPase. The energy released by the transport reaction
synthesizes ATP from ADP and inorganic phosphate.
Since both of the enzymes are critical components in energy metabolism. It suggests
that the post-translation modification (nitration) links the change in redox status
(increased nitrative stress) to the production of energy (generation of reducing
equivalent and ATP). The physiological consequence (neuronal cell death) caused by
nitration induced impairment of SCOT and F
1
-ATPase activities during aging will be
presented later in this dissertation.
Formation of 3-nitro-L-tyrosine is considered as a biological marker of the production
of
.
NO derived ONOO
-
(Greenacre and Ischiropoulos 2001). Nitration of proteins,
more specifically the accumulation of 3-nitro-L-tyrosine has been implicated in PD
and various other neurodegenerative diseases suggesting that tyrosine nitration plays a
pathological role in the development of the diseases, which can be due to the functions
of nitrated protein being modified. It has been shown that nitration inhibits complex I
activity, which precedes changes of redox status (glutathione loss) and apoptosis in PD
9
(Bharath and Andersen 2005). Nitration can also potentially compromising cellular
bioenergetics, since most of the modified protein are mitochondrial proteins and
belong to the energy production pathways (Lacza, Snipes et al. 2003). Nitration of
complex I results in enhanced ROS and RNS production (Murray, Taylor et al. 2003),
mainly by enhancing O
2
.-
generation through the autoxidation of ubisemiquinone
(Han, Williams et al. 2001), hence further increasing the level of oxidative and
nitrative stress that further pushing the redox status to a more oxidized status that is
harmful to the cell.
C)
.
NO mediated apoptosis
Aside from modulating protein post-translational modification,
.
NO is emerging as a
predominant factor of neurodegeneration (Bobba, Atlante et al. 2007; Malinski 2007;
Singh and Dikshit 2007). Apoptotic cell death has been suggested to be the common
pathway for neuronal cell loss in neurodegenerative diseases (Cotman and Anderson
1995; Thompson 1995). The mechanisms underlying apoptosis have been extensively
studied (Cotman and Anderson 1995; Thompson 1995). It is generally believed that
mitochondria play an central role in the execution of apoptosis, mainly by 1) the
release of cytochrome c; 2) release of cytochrome c activates downstream caspases, 3)
initiation of the irreversible phase of apoptosis through the formation of the
apoptosome which is regulated by molecules of the Bcl-2 family: Bcl-
2
and Bcl-
XL
of
the outer mitochondrial membrane and 4) generation of ATP as apoptosis is ATP
10
dependent. As mentioned earlier,
.
NO plays a dual role in apoptosis. It can be either
anti-apoptotic or pro-apoptotic. The different effects of
.
NO on apoptosis may be
explained by the difference in cellular concentration of
.
NO, types of modification and
its reaction targets.
.
NO prevents apoptosis by S-nitrosylation of the essential cystiene
in the active center of caspases which renders the caspases completely inactive (Li,
Billiar et al. 1997), or nitration of specific tyrosine residues of cytochrome c by
ONOO
-
formation and attenuates cytochrome c -induced caspase-9 activation
(Nakagawa, Komai et al. 2007). On the other hand,
.
NO promotes apoptosis by
inhibiting cytochrome oxidase (complex IV) of the mitochondrial electron transport
chain, which causes inhibition of oxygen consumption, lowering of mitochondrial
membrane potential and causing cytochrome c release by activation of mitochondrial
permeability transition pore. Release of cytochrome c will trigger downstream
caspases leading to apoptosis.
The cytotoxic effect of
.
NO has also been postulated to be modulated in part by
ONOO
-
.
Autoxidation of ubisemiquinone of the mitochondrial electron transport chain is the
major source of O
2
.-
production (Cadenas, Boveris et al. 1977). Reaction between
.
NO
diffused from the cytosol with mitochondrial generated O
2
.-
leading to formation of
ONOO
-
within the mitochondria (reaction k
2
= 1.9 x 10
10
M
-1
s
-1
). This reaction takes
precedent over the reduction of O
2
.-
to hydrogen peroxide by superoxide dismutase
(SOD), as the reaction occurs at ~10-fold slower rate (reaction k
2
= 2.3 x 10
9
M
-1
s
-1
).
11
ONOO
-
can directly damage various cellular components such as mitochondrial DNA,
lipids, and proteins. The direct damage on DNA causes upregulation of proapoptotic
protein Bax that eventually activates apoptosis. Recently, it has been reported that
ONOO
-
can activate the c-Jun N-terminal Kinase (JNK) pathway and induced
apoptosis (Shrivastava, Pantano et al. 2004). JNK phosphorylates a variety of
cytosolic proteins, such as p53, cytoskeleton proteins, and the glucocorticoid receptor
(Giasson and Mushynski 1997; Maundrell, Antonsson et al. 1997; Fuchs, Adler et al.
1998). JNK can also modulate the mitochondrion-driven apoptotic pathway through its
translocation to mitochondria and subsequent phosphorylation of Bcl-2 family
members such as Bcl-2, Bcl-xL (Schroeter, Boyd et al. 2003) and then leads to release
of cytochrome c.
3. The aged proteasome system and its role in neurodegenerative disease
As mentioned earlier,
.
NO becomes cytotoxic at high cellular concentrations, thus
regulation of NOS activity is very important for maintaining
.
NO at a normal
physiologically low concentration. Constitutively expressed NOS (nNOS and eNOS)
are regulated by their turnover rate through degradation by the proteasome system.
The proteasome is a large intracellular multicatalytic protease that is ubiquitously
expressed in all cell types in the central nervous system (CNS) (Keller, Gee et al.
2002). It is responsible for the majority of intracellular proteolysis of unwanted,
12
oxidized, misfolded and aggregated proteins (Rock, Gramm et al. 1994; Goldberg,
Akopian et al. 1997; Tanaka 1998). These abnormal proteins are first ubiquitinated
and then targeted for degradation. However, in several neurodegenerative diseases,
these ubiquitinated proteins start to accumulate in neurons (Mayer, Lowe et al. 1991;
Halliwell and Jenner 1998). Proteasome itself is susceptible to free radical attack due
to oxidative modification of proteasome. Cellular components damaged by free radical
such as 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation which
covalently binds to proteasomes may as well impair proteasome function (Friguet and
Szweda 1997; Reinheckel, Sitte et al. 1998; Okada, Wangpoengtrakul et al. 1999).
Recent studies also indicate that proteasome activity may be impaired during, and very
likely contribute to, the aging process (Conconi, Szweda et al. 1996; Petropoulos,
Conconi et al. 2000; Keller, Gee et al. 2002; Zeng, Medhurst et al. 2005).
Compromised proteasome function is also related to decrease in the level of
glutathione, which is a major antioxidant in the cell, and increase in oxidation of key
proteins those have been implicated in various neurodegenerative diseases
(Markesbery 1997; Butterfield, Howard et al. 1999; Butterfield and Kanski 2001).
Taken together, impairment of proteasome function can lead to the accumulation /
aggregation of abnormal proteins, change of cellular redox (decrease in glutathione
level which decreases cellular anti-oxidation capacity), and affects cellular physiology.
Neuronal specific NOS (nNOS) has been suggested to play an important role in the
development of PD. For example, studies have shown that the overexpression of
13
neutrophil nNOS in PD patients (Gatto, Riobo et al. 2000); Lo et al has identified a 5’-
flanking region polymorphism of the nNOS gene, which causes an increased NOS
activity, associated with PD (Lo, Hogan et al. 2002). In general, it is widely accepted
that nNOS-mediated neurodegeneration is mainly due to upregulation of its activity
through producing large amount of
.
NO that are in excess of the physiological level.
Thus, regulation of nNOS activity is very important and one of the major regulatory
mechanisms is by controlling the turnover rate of the protein through the proteasome
degradation system. However, proteasome impairment has been implicated in PD. It is
manifested by the formation of Lewy bodies (LBs), which are the pathological
hallmark of the disease. LBs are usually ubiquitin-immuoreactive that implicates an
impairment of the ubiquitin- proteasome system. Surprisingly, proteins accumulated in
LBs are often found to be post-translationally modified by
.
NO, i.e. nitrated. However,
mechanism leading to increased amount of nitrated proteins in Lewy bodies has not
been extensively studied. Taken together, it may be surmised that accumulation of
nitrated proteins in PD might be a consequence of age dependent impairment of
proteasome function and subsequent upregulation of nNOS level/activity.
14
4. Case study: nNOS and its role in inherent and environmental toxin induced
neurodegenerative disease – implication in Parkinson’s disease (PD)
PD is a progressive neurodegenerative disorder that is clinically characterized by
resting tremor, muscle rigidity, impaired cognitive function, and depression (Jankovic
1992). PD affects about 1% of the senior population aged 65 and older (Schoenberg
1987; Youdim and Riederer 1997). Over 1 million Americans are affected by this
disease, and about 50,000 new cases are diagnosed each year (Fahn and Przedborski
2000). PD is pathophysiologically manifested as the depletion of the neurotransmitter
dopamine in the striatum due to the gradual loss of dopaminergic neurons in the region
of substantia nigra pars compacta (SNpc) of the mid-brain (Burke 1998). Exisiting
published data demonstrated that mitochondrion-mediated apoptosis may be involved
in the selective neuronal loss observed in neurodegeneration (Andersen 2001).
Although the etiology of PD remains elusive, it is generally accepted that both genetic
and environmental factors could contribute to the development of the disease. An
epidemiologic study of nearly 20,000 twins dismissed the likelihood of a genetic
component in the etiology of PD when it occurred at or after 50 years of age (Tanner,
Ottman et al. 1999). Genetic factors only became evident in individuals with family
history and early on-set of Parkinsonism at or before 50 years of age. Therefore, only
~10% of PD cases, which often begin early in life, could be traced to genetic
mutations; while the majority (~90%) of PD cases are sporadic. The information
15
presented above suggests that aging and environmental factors are the most common
risk factors for developing PD.
As discussed in the previous paragraphs, emerging evidences demonstrate that
.
NO
cytotoxicity is highly related to neuronal cell death and the development of PD. It is
very likely that activity of nNOS is upregulated by aging due to aging mediated
impairment of proteasome function. The changes in these cellular components are
very likely the mechanisms that contribute to the alternation of cellular redox
environment and leads to the development of PD through
.
NO-mediated cytotoxicity.
16
SIGNIFICANCE
PD is an aging-associated neurodegenerative disease that affects a large number of
people (1 million) over the age 65 and number of PD cases is continuously increasing
(+50,000 per year). There is existing information that the development of PD is related
to 1) aging, 2) proteasome function, 3) nNOS function or 4)
.
NO toxicity, however,
there are no studies concerned with the relationship among these four components.
The work presented here, through establishing relationships between the four
components, provides several important novel findings that can help to better
understand the mechanisms underlying the onset of PD. The findings herein
potentially contribute to the development of effective preventive and therapeutic
intervention therapies.
a) Aging induced
.
NO toxicity – a novel
.
NO toxicity model
.
NO plays an important physiological and pathological role in brain depending on its
concentration. There are two existing models of
.
NO toxicity: 1) Inflammatory
(Murphy, Simmons et al. 1993; Simmons and Murphy 1993) and 2) NMDA
excitotoxicity models (Ayata, Ayata et al. 1997). The work presented in this
dissertation demonstrates for the first time a novel model by which nNOS is
upregulated during aging and induces nitric oxide toxicity. By identifying the targets
and pathways that are affected by aging induced
.
NO toxicity and the physiological
consequences caused by the change will provide valuable and novel information about
17
how does aging contribute to the change of cellular
.
NO level and the fundamental
mechanisms underlying the development of PD
b) Protein post-translational modification - a linker between redox status and
bioenergetics
Brain mitochondria are fragile and have a low turnover rate thus they are extremely
prone to damage, i.e. mitochondria dysfunction. Damage to specific mitochondrial
enzymes by RNS-induced protein post-translational modification in the bioenergetics
pathway during aging indicates that protein post-translational modification links the
change of redox status to energy metabolism. Identification of specific modified
proteins during aging provides possible targets for therapeutic intervention therapies.
This study demonstrates the first time that SCOT and F
1
-ATPase are increasingly
nitrated and inhibited with respect to age indicating critical biogenergetics components
are affected by
.
NO cytotoxicity during aging and suggesting possible mechanism that
leads to aging associated mitochondrial dysfunction.
c) Establishing inherent role of nNOS in environmental toxin induced PD
~90% of PD are sporadic cases suggests important role of environmental factor in the
development of the disease. Paraquat is the most commonly used herbicide and has
been shown to induce neuronal loss. This dissertation addressed the role of nNOS in
paraquat induced neurodegeneration by demonstrating the critical role of nNOS as a
paraquat diaphorase in the induction of paraquat cytotoxicitiy during aging process.
18
This finding provides valuable information for developing possible approaches to
prevent the onset of PD.
19
HYPOTHESIS AND SPECIFIC AIMS
As reviewed in the introduction section, aging compromises the proteasome
degradation system, which is often observed in PD and very likely induces the
accumulation of NOS which causes elevated nitrative stress in the cells by
overproducing
.
NO. Dysfunction of mitochondria is also related to aging, which
lowers the ability of energy production, rendering the cell more susceptible to energy
crisis and lowering its viability. Taken together, increased
.
NO production and
dysfunction of mitochondria are both induced by aging and renders the brain more
vulnerable to insults and cell death. Thus, aging can be a risk factor for developing
neurodegenerative disease. However, there is no study directly relating aging,
proteasome function and nNOS level to the viability of dopaminergic neuron and its
implication in neurodegenerative disease.
Based on the information described in the previous section, we hypothesize that nNOS
accumulates in aging causing increased nitrosative and nitrative stress due to
elevated
.
NO production, which compromises mitochondria function and triggers
mitochondrial caspase induced apoptosis.
The hypothesis was tested through 3 specific aims (Fig. 2):
1) Determine the effect of aging on brain nNOS, nitrative and nitrosative stress levels
and their effects on mitochondrial function during aging by: a) assessing nNOS
20
protein, nitrosocystiene and nitrotyrosine levels in rat brain homogenate at different
ages, b) identifying nitrated mitochondrial proteins by utilizing LC-MS/MS, c)
employing specific experiments to determine the change of individual protein function
after nitration, d) assessing physiological consequence of alternated mitochondrial
protein functions.
2) Determine the mechanisms underlying aging related
.
NO-induced neuronal loss by
a) assessing the change of cell viability due to proteasome inhibition induced nNOS
upregulation, b) measuring the change of
.
NO production, c) assessing whether JNK
pathway is involved in
.
NO mediated apoptosis through measuring JNK and Bcl
XL
phosphorylation and d) assessing the activation of capases 9 and 3.
3) Determine the role nNOS in environmental toxin induced Parkinsonism by a)
assessing effect of herbicide (paraquat) on PC12 cells viability and cytoxicity, b)
assessing effect of NOS inhibition on paraquat toxcitiy and c) identifying nNOS as
paraquat diasphorase by utilizing clear native - poly acrylamide gel electrophoresis
(CN-PAGE) and d) assessing paraquat activation in relation to nNOS level during
aging.
21
Fig. 2 Overview of specific aims
22
CHAPTER I
Correlation of elevated neuronal nitric oxide synthase level
during aging with compromised energy production
1. Introduction
Aging is a risk factor for many diseases, in particular, neurodegenerative diseases such
as Alzheimer’s, Huntington’s and Parkinson’s diseases. Due to its high metabolic rate
and limited ability for cellular regeneration as compared to other organs, the brain
becomes more susceptible to change in energy production. The cumulative damage to
cellular components caused by free radicals have been proposed as the major
mechanism underlying age induced neurodegeneration (Lin and Beal 2006). The
proteasome is a large intracellular mulicatalytic protease that is responsible for
degrading damaged cellular components that are oxidized, misfolded and aggregated
proteins (Goldberg, Akopian et al. 1997; Davies 2001; Ding, Lewis et al. 2002).
Various studies have demonstrated that proteasome activity declines with age (Friguet,
Bulteau et al. 2000; Ferrington, Husom et al. 2005; Zeng, Medhurst et al. 2005), which
may contribute to the elevations in protein oxidation and aggregation. Accumulation
of damaged protein may lead to cellular dysfunction and consequently cell death
inherent in the neurodegenerative process.
23
The cellular turnover of the constitutively expressed neuronal nitric oxide synthase
(nNOS), which is the predominant isoform expressed in the brain, is degraded by the
proteasome (Noguchi, Jianmongkol et al. 2000; Dunbar, Kamada et al. 2004). nNOS
is a flavin- and heme- containing Ca
2+
-calmodulin-dependent enzyme that is
constitutively expressed in neuronal cells. It catalyzes the metabolism of L-arginine to
L-citrulline and
.
NO in the presence of O
2
and NADPH.
.
NO is a gaseous biological
signaling molecule which plays numerous physiological roles including neuronal
signal transmission (Zhuo and Hawkins 1995; Bon and Garthwaite 2001), vascular
tone (Lee 2000), and immune host defense (Moncada, Palmer et al. 1991). It is also a
free radical which plays a role in aging process (McCann 1997; Calabrese, Bates et al.
2000; Floyd and Hensley 2000). Elevated NO production has been implicated on
neuronal loss and increased neuronal apoptosis (Huang, Huang et al. 1994; Estevez,
Spear et al. 1998; Vernet, Bonavera et al. 1998; Lau, Petroske et al. 2003). Thus,
regulation of
.
NO levels within neurons is of paramount importance due to its
neurotoxic consequences.
.
NO rapidly reacts with O
2
.-
, which is mainly generated in the mitochondria through
autoxidation of ubisemiquinone (Cadenas, Boveris et al. 1977), at diffusion-limited
rates to form ONOO
-
locally within the mitochondrion (Beckman, Beckman et al.
1990; Estevez, Spear et al. 1998). This ONOO
-
is a powerful oxidant capable of
attacking protein amino acid residues such as nitration, cysteine oxidation and
modifying their respective functions. Numerous proteins such as Mn-SOD, Apo B and
24
COX-1 have been demonstrated to be nitrated during pathological circumstances,
indicating that nitration of protein is important in the modulation of cellular functions
(Beckman, Beckman et al. 1990; Leeuwenburgh, Hardy et al. 1997; Yamakura, Taka
et al. 1998; Deeb, Resnick et al. 2002). Levels of 3-nitrotyrosine, a biomarker of
nitrative stress cause by attack from ONOO
-
and other reactive nitrogen species
(RNS), have been reported to be elevated in several neurodegenerative diseases such
as Parkinson’s and Alzheimer’s diseases (Beal, Ferrante et al. 1997; Halliwell 1997;
Greenacre and Ischiropoulos 2001). ONOO
-
is a highly reactive oxidizing species and
has been shown to induce mitochondrial membrane depolarization (Li, Trudel et al.
2002), lipid peroxidation (Radi, Beckman et al. 1991) and inactivation of important
mitochondrial proteins such as ATP synthase, aconitase, α-ketoglutarate
dehydrognease and creatine kinase (Konorev, Hogg et al. 1998; Han, Williams et al.
2001; Brookes, Levonen et al. 2002; Nulton-Persson, Starke et al. 2003).
.
NO can also
directly affect mitochondrial respiration by reversible inhibition of complex IV.
Inhibition of mitochondria respiration leads to a decrease of inner mitochondrial
membrane potential, induction of the mitochondrial permeability transition, release of
cytochrome c into the cytosol and the activation of caspase 9 and other downstream
caspases that ultimately leading to apoptosis (Clementi, Brown et al. 1998; Yabuki,
Tsutsui et al. 2000; Cooper, Davies et al. 2003; Stewart and Heales 2003).
Furthermore,
.
NO has been implicated in excitotoxic phenomenon which involves
excessive activation of NMDA receptor mediated glutamate release and is related to
neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease
25
(Dawson, Dawson et al. 1991; Coyle and Puttfarcken 1993; Dawson and Dawson
1998).
It maybe surmised that the decrease in proteasome functions during aging would lead
to accumulation of nNOS which causes increase
.
NO production and nitrative stress.
This study was aimed at assessing the role of aging in relation to the changes in the
level of nNOS and the functional consequences of increased nitrative stress to
mitochondrial functions in brain aging. We demonstrated that nNOS protein level in
the brain was elevated during aging. This elevation of nNOS protein level is correlated
to increased nitrosylation and nitration of brain proteins. Succinyl-CoA:3-oxoacid
CoA-transferase (SCOT; E.C.2.8.3.5), which is the key mitochondrial matrix enzyme
for ketolysis that provides the only alternative energy for brain during glucose
deficiency; and F
1
-ATPase (Complex V) of the mitochondrial electron transfer chain
were found to be increasingly nitrated with respect to aging in vivo. Their respective
activities both declined and accompanied a decreased in oxygen consumption rate
observed during aging. This suggests that protein post-translational modification of
mitochondrial proteins lead to compromised energy production capacity.
Mitochondrial cytochrome c content also declined during aging suggesting enhanced
neuronal apoptosis in aging. Taken together, these data raises the possibility that the
age-related elevation of nNOS may contribute to the increased
.
NO related energy
metabolism deficiency and neuronal cell death.
26
2. Materials and Methods
2.1 Animal
Fisher 334 rat at different ages (6, 14 and 24 months) were purchased from the
National Institute of Aging (NIA). Each rat was individually housed in the animal
facility under standard conditions (12/12 light-dark cycle, humidity at 50±15%,
temperature 22±2
o
C and 12 air changes/hr). All procedures were approved by the local
Animal and Care and Use Committee.
2.2 Isolation of rat brain mitochondria
Rat brain mitochondria were isolated from adult male Fisher rats by differential
centrifugation as described previously (Han, Williams et al. 2001). Rat brain were
excised, chopped into fine pieces, washed with 0.25 M sucrose and homogenized in
isolation buffer containing 210 mM mannitol, 70 mM sucrose and 2 mM Hepes, pH
7.4, plus 0.05% (w/v) BSA. The homogenate was centrifuged at 800 g for 8 min, the
pellet was removed, and the centrifugation process was repeated. The supernatant was
centrifuged at 8000 g for 10 min, the pellet was washed with the isolation buffer, and
the centrifugation was repeated. The pellet containing a mixture of organelles was
further fractionated by centrifugation at 8500 g for 10 min in a Percoll gradient
[consisting of three layers of 18, 30 and 60% (w/v) Percoll in sucrose/Tris buffer (0.25
M sucrose, 1 mM EDTA and 50 mM Tris/HCl), pH 7.4]. Mitochondria were collected
from the interface of 30% and 60% Percoll and washed with the sucrose/Tris buffer.
27
Mitochondrial protein concentration was determined using protein assay reagent (Bio-
rad).
2.3. Detection of nNOS, nitrosylation and nitration
100 μg of mitochondrial protein were separated by 10% SDS–PAGE, electro-
transferred to polyvinylidene fluoride (PVDF) membrane and immunostained by
standard methods. α-nNOS (Upstate), α-Cytochrome c (Santa cruze) α-
nitrosocysteine (A.G. Scientific) and α-nitrotyrosine (Upstate) were used to detect the
level of nNOS protein, nitroslyation and nitration, respectively.
2.4 Immunoprecipitation
Mitochondria were lysed by freeze-thaw method in RIPA buffer containing
proteasome inhibitor cocktail (Roche). 1 mg/ml of total mitochondrial protein was
incubated with 10 μg of anti-nitrotyrosine antibody (Upstate) overnight at 4 °C.
Immune complexes were collected by incubation with protein G-agarose (Santa Cruz)
for 4 hr at 4 °C. Immunoprecipitated proteins were released from agarose by boling in
non-reducting sample buffer (Pierce) and separated on 10 % SDS-PAGE gel then
sequenced by LC/MS/MS at the proteomic core factility at School of Pharmacy,
University of Southern California .
28
2.5 Measurement of SCOT catalytic activity
SCOT activity was measured using procedure describled by Williamson et al.
(Williamson, Bates et al. 1971). Briefly, mitochondria were disrupted by sonication
and then centrifuged at 100,000 g for 20 min. Supernatant were incubate with 50 mM
Tris-HCl, pH 8.0, 5 mM MgCl
2
, 4 mM iodoacetamid, 0.2 mM acetoacetate and 0.1
mM succinyl-CoA. Catalytic activity was measured spectrophotometrically at 313 nm.
2.6 Measurement of complex IV activity
Complex IV activity was measured using MitoProfile Assay Kit (Mitosciences)
according to manufacturer’s instructions. Briefly, complex IV was immunocaptured
and incubate with cytochrome c. The activity is measured by the oxidation of
cytochome C at 550 nm.
2.7 Measurement of F
1
-ATPase activity
ATP synthase activity was measured in purified mitochondria from rat brain at
different age. It was measured in the direction of ATP hydrolysis using continuous
spectrophotometric assay. 50 μg of broken mitochondria were added to 1ml reaction
solution contains 250 mM sucrose, 50mM HEPES, pH 8.0, 5 mM MgSO4, 2.5 mM
sodium phosphoenopyruvate, 2 μg antimycin, 5 ml of PK/LDH mixture and 2.5 mM
ATP. Reaction was initiated by adding 0.35 mM NADH and the linear reaction was
measured for 5 min at 340 nm at 25
o
C (ε
340
=6.22mM
-1
cm
-1
).
29
2.8 Measurement of oxygen consumption
Oxygen consumption was measured amperometrically with a Clark-type electrode
(Hansatech, UK) assembled to a thermostatic water jacket. Solution, containing 25
mM Sucrose, 75 mM mannitol, 5 mM KH
2
PO
4
, 100 mM KCl, 0.05 mM EDTA, 20
mM HEPES and 5 mM MgCl
2,
in the electrode chamber were maintained under
continuous stirring with a magnetic agitator. State 4 respiration was initiated by adding
2.5 mM pyruvate + 2.5 mM malate and State 3 respiration was initiated by adding 350
μM ADP. Respiratory control ratio (RCR) was measured as ratio of State 3/State 4
respiratory rates.
2.9 Statistical analysis
Data are reported as means ± S.D. of at least six independent experiments. Significant
differences between mean values were determined by student t-test. Means were
considered to be statistically distinct if P < 0.05.
3. Results
3.1 Rat brain nNOS protein level elevated during aging
nNOS content in rat brain homogenate increased during aging. The level of total
nNOS content in rat brain at different ages was detected by western blot. After
30
normalizing with β-actin, the level of nNOS was found to be elevated approximately
2-fold in 24 month-old rat brain as compared to 6 month-old rat (Fig. 3).
3.2 Increased total nitrosylation and mitochondrial nitration level during aging
To ascertain if elevated nNOS leves observed during aging is related to increase of
nitrosative / nitrative stress level, endogenerous nitrosalytion and nitration of cytosolic
and mitochondrial fractions were analyzed by immunoblotting. The amount of S-
nitrosocysteines was increased as a function of age as detected by western blot using a
specific antibody against nitrosocysteines (Fig. 4A). Specificity of the α-
nitrosocysteines antibody was determined by treating cytosolic sample with 1mM
mercuric chloride, which induces the cleavage of RSNOs to form nitrite (Feelisch,
Rassaf et al. 2002), as shown in Fig. 4A. Specific nitrosocysteine bands are indicated
by the arrows. Nitration was only detected in mitochondrial fraction, but not cytosolic
fraction. No band was detected after incubating the same membrane with 1mM
dithionite, which reduces nitrotyrosine to aminotyrosine, confirming the specificity of
the anti-nitrotyrosine antibody (data not shown). Nitration of mitochondrial proteins
was increased during aging (Fig. 4B). Several bands were detected by α-
nitrosocysteine and α-nitrotyrosine antibodies suggesting that 1) nitrosylation and
nitration occur in different cellular compartments and 2) nitrosylation and nitration of
specific cytosolic and mitochondrial proteins, implicating specific regulation of these
proteins by RNS.
31
Fig. 3 nNOS protein content in rat brain increases during aging.
Representative western blot of total brain homogenate obtained from
Fisher 344 rat at 6, 14 and 24 months. The level of nNOS protein content in the
total cell homogenate was detected by western blot using α-nNOS antibody. The
intensity of nNOS band was quantified and calculated against β-actin level using
Versa Doc imaging system (Bio-rad). n=9, *P < 0.05
32
Fig. 4 Nitrosylation and nitration of rat brain cytosolic and mitochondrial proteins increase
during aging.
Rat brains at 6, 14 and 24 months were immunoblotted for nitrosylation and nitration
level. A) Total nitrosocysteine in the cytosolic fraction was detected by α-nitrosocysteine
antibody. Negative control (-ve) was created by treating 24 months sample with 0.5 mM
mecuric chloride for 10min at RT under light. B) Total nitrotyrosine in the mitochondrial
fraction was detected by α-nitrotyrosine antibody.
A)
B)
33
3.3 SCOT and F
1
-ATPase were nitrated during aging
Since increased protein nitration was detected in the mitochondria, the nitrotyrosine
immunoreactive, we aimed to identify the proteins that showed increase nitration with
respect to age. 58kDa proteins were immunoprecipitated by anti-nitrotyrosine
antibody and subjected to LC MS/MS analysis. Mitochondrial enzyme Succinyl-Co A
Transferase (SCOT) and F
1
-ATPase were identified as the nitration target (Fig. 5A).
The site of nitration of F
1
-ATPase was identified as Tyr269 (Fig. 5B). The protein
level of both SCOT and F
1
-ATPase remained unchanged during aging (Fig. 5C). 3D
mapping of Tyr269 was based on the crystal structure of bovine F
1
-ATPase, which
shared high sequences similarity that was enough for comparison purposes. The
nitrated tyrosine seems to be at the periphery of the active site (Fig. 5D). The carbon
of tyrosine that is nitrated is relatively close (10.7 Ǻ) to magnesium atoms associated
with the structure. The nitro group of the nitrated Tyr269 is also at close proximity to
Asp256 (9.85 Ǻ), which belongs to the active site of the beta chain (Abrahams, Leslie
et al. 1994) (Fig. 5E). This information suggests that SCOT and F
1
-ATPase were
susceptible to ONOO
-
induced post-translation modification and nitration may lead to
alternations in their catalytic activity.
34
A)
B)
Fig. 5
35
Fig. 5: Continued
C)
D)
36
Fig. 5 SCOT and F
1
-ATPase beta subunit are identified as nitration targets by LC/MS/MS
during aging.
A) SCOT and F
1
-ATPase were identified by LC/MS/MS as nitration targets during
aging. B) Nitration of Tyr269 of ATP synthase beta subunit was identified by LC/MS/MS as
mentioned in the method section. C) A representative image showing that SCOT and ATP
synthetase protein level remained unchanged during aging as detected by α-SCOT and α-
ATP synthase antibodies, respectively. COX IV staining was included as the loading control.
SCOT activity is significantly reduced during aging (n=9). D) 3D mapping of Tyr269 based
on crystal structure of bovine F
1
-ATPase. Important components and their corresponding
colors: Alpha chains (red); beta chains (blue); gamma chain (purple); F
1
alpha active site
residues (red); F
1
beta active site residues (blue); Mg
2+
(green); ANP (violet); ADP
(turquoise) and nitrotyrosine 269 (yellow, 219 in crystal structure). E) Close up of the
nitration site showed that nitro group (red stick) of Tyr269 (yellow stick) is at close proximity
to the Mg
2+
(green, 10.71 Ǻ) and Asp256 (blue, 9.85 Ǻ) of the active site.
E)
Fig. 5: Continued
37
3.4 Nitration compromised SCOT and F
1
-ATPase activities
To determine the functional consequences of nitration on SCOT and F
1
-ATPase, their
respective catalytic activities were measured. Catalytic activities of nitrated SCOT and
F
1
-ATPase were both significantly decreased during aging (Fig. 6A, B). The activity
of cytochrome oxidase (COX IV) with respect to age was also measured. COX IV is
the terminal electron acceptor of mitochondria electron transfer chain and controls the
rate of mitochondria respiration thus it is widely accepted that COX is a good
indicator of mitochondria function. Our data also showed a slight, age-depend
decrease in its activity (Fig. 6C). The rate of decline in cytochrome c oxidase activity
is less significant then that of SCOT suggesting that, in case of aged-related nitrative
inhibition, SCOT and F
1
-ATPase are potential key limiting factors affecting energy
metabolism rather than cytochrome c oxidase (Fig. 6C). The decline in SCOT activity
also suggests that aged animal becomes less efficient in utilizing ketones as an
alternative source of energy causing the brain are more vulnerable to an energy crisis
in the event of glucose depletion or deficiency in glucose utilization, i.e. diabetes .
38
Fig. 6
A)
B)
39
Fig. 6 SCOT and F
1
-ATPase enzymatic activities reduce during aging.
Enzymatic activity of A) SCOT and B) F
1
-ATPase were significantly decreased
during aging. C) A slight decline of cytochrome c oxidase enzymatic activity was observed
during aging. D) Comparison of the degree of decline of enzymatic activity between SCOT
( ■, solid line); F
1
-ATPase ( ●, broken line) and cytochrome c oxidase ( ▲, broken line). The
reduction of SCOT and ATP synthethase activities were more drastic than cytochrome c
oxidase suggesting SCOT and F
1
-ATPase are more sensitive to nitration inhibition.
Fig. 6: Continued
C)
D)
40
3.5 Decreased respiratory control ratio (RCR)
Since nitration caused a decrease in enzymatic activities of two critical enzymes
(SCOT and F
1
-ATPase) in the energy metabolism pathways, the function consequence
of the inhibition of their activities on mitochondria function was measured by the
oxygen consumption rate. Calculated RCR ratios with glutamate and malate at 6
month were 6.83 ± 0.31; 14 months was 5.91 ± 1.52 and 26 month was 5.12 ± 0.29,
indicating a significant decrease (~25%) of oxygen consumption during aging. (Fig.
7). The lower RCRs were caused primarily by increases in state 4 respiration. There
was no significant change in state 3.
Fig. 7 Mitochondrial respiratory decreases with age.
Oxygen consumption was measured by a Clark-type O
2
electrode. Respiratory
control ratio (RCR, State 3 / State 4) of rat brain mitochondria decreased with age. State 4
respiration was initiated by addition of glutamate + malate and state 3 respiration was
initiated by addition of ADP.
41
3.6 Compromised energy production enhanced cytochrome c release
The decrease in RCR ratio implicated a change in structural integrity of the
mitochondrial membranes. Physiological consequence of increased ONOO- formation
(increased nitrative stress) induced change in membrane integrity was thus assessed by
the degree of cytochrome c release. Level of cytochrome c inside mitochondria
decreased with age and a corresponding increase in cytosolic cytochrome c level was
observed (Fig. 8). This data suggested an enhanced cytochrome c release to cytosol
during brain aging.
Fig. 8 Mitochondrial cytochrome c release increases with age.
Mitochondrial and cytosolic fractions of rat brain at different ages were
collected and subjected to immunoblotting using α-cytochrome c antibody.
Mitochondrial cytochrome c level decreased with age and a corresponding increase in
cytosolic cytochrome c level was observed. COX IV staining was included as the
loading control (n=9).
42
4. Discussion
It has been reported that the efficiency of the proteasome degradation system declines
with respect to age. In this study, protein level of nNOS, which is a well known
proteasome target, was found to be increased with age. The increase of nNOS
positively correlated with the increase in cytosolic nitrosylation and mitochondrial
nitration of proteins suggesting that impairment of proteasome system leads to an
increase in nNOS protein levels, enhanced
.
NO
generation and ONOO
-
formation that
contributed to elevated nitrosative and nitrative stress and post-translational
modification of specific proteins.
The physiological consequences brought by the permanent protein post-translational
modification, i.e. nitration, during aging is relatively unclear. Nitration is a commonly
studied covalent modification of proteins attributed to
.
NO, which leads to the
formation of 3-nitrotyrosine (NO
2
Tyr), and can be detected immunochemically using
anti-nitrotyrsosine antibody. In the current study, nitration was not detected in
cytosolic proteins (data not shown) but only detected in mitochondrial proteins by
immunoblotting. This may be caused by the localized formation of nitrating agent
ONOO
-
due to the generation of O
2
.-
in the mitochondria. A major source of O
2
.-
is the
mitochondrial respiratory chain where the non-enzymatic production occurs as a result
of single electron transfer to O
2
by reduced prosthetic groups during electron leakage.
Since half-life of ONOO
-
is extremely short due to its high reactivity, ONOO
-
reacts
43
rapidly with proteins at the site of its formation, it becomes unlikely to nitrate proteins
localized far away from mitochondria. Even though certain cytosolic proteins may be
nitrated but levels of individual modified proteins could be too low to be detected by
the current experimental approach. In addition, our findings are further supported by
work done by Viner et al., in which they were also unable to detect cytosolic protein
nitration (Viner, Ferrington et al. 1999). One may argue that superoxide dismutases
(SOD) (Cu/ZnSOD is the cytosolic isoform, whereas MnSOD is the mitochondrial
isoform) catalyze the enzymatic dismutation of O
2
.-
to H
2
O
2
may prevent the
formation of ONOO
-
and hence prevent ONOO
-
mediated mitochondrial protein
nitration. However, it has been shown that
.
NO efficiently competes with SOD for O
2
.-
at a rate 10 times faster than SOD dismutation of O
2
.-
(Beckman and Koppenol 1996).
Therefore, under circumstances of increased NO
.
and O
2
.-
production such as aging
(Navarro and Boveris 2004),
.
NO can out compete SOD for O
2
.-
and still resulting in
ONOO
−
formation and eventually increase nitration of mitochondrial proteins.
Nitration of Brain SCOT and F
1
-ATPase leads to decrease in their respective catalytic
activity during aging. SCOT mediates the rate-determining step of ketolysis in
extrahepatic tissues by converting ketone bodies into acetoacetyl CoA, which
subsequently enters the citric acid cycle for the generation of reducing equivalents. In
brain, ketone bodies are the only alternative energy source during the periods of
glucose deficiency. Ketone bodies have been suggested to be useful in rescuing
mitochondrial respiration and for treatment of PD by increasing succinate generation
44
through SCOT that feeds into complex II so bypassing the presumed complex I defect
and maintaining mitochondrial function and ATP production (Kashiwaya, Takeshima
et al. 2000; Tieu, Perier et al. 2003; Vanitallie, Nonas et al. 2005). Thus, nitration of
SCOT leads to further impairment of mitochondria energy generation due to loss of
the ability to utilize ketone bodies during aging. This loss is further compounded by
the age dependent loss of pyruvate dehydrogenase (PDH) function, which is the key
enzyme that regulates the entry to pyruvate into to the TCA cycle (Zhou, Lam et al.
2007).
This is the first study showing the F
1
-ATPase is being nitrated at Tyr269 during aging
casuing a compromised catalytic activity. 3D mapping of Tyr269 in F
1
-ATPase puts
into perspective where the nitration damaged tyrosine is situated in terms of the 3-D
structure of the head group and the active site/substrate binding site (Fig. 5E). The
close proximity of the nitro group to the magnesium and Asp256 in the active site
(Abrahams, Leslie et al. 1994), suggests that nitration alters the substrate binding site
of F
1
-ATPase, probably by pushing the Asp256 and magnesium iron. This results in
declined efficiency for substrate binding to the active site and thus lowering the ATP
production rate. It has been well established that mitochondria respiration decreases
during aging. The slight decrease in COX activity was not significant, rather the
decrease in SCOT and F
1
-ATPase activities were significant suggesting that nitration
induced inhibition of SCOT and F
1
-ATPase activities might contribute to the decrease
in mitochondria respiration during aging.
45
It may be surmised that the NO mediated decrease in SCOT, F
1
-ATPase and
cytochrome c oxidase catalytic activity occurs through ONOO
-
generation. Nitration
of SCOT and F
1
-ATPase leads to decrease in energy generation with corresponding
increase in free radical generation. This is supported by an increase in the state 4
respiration suggesting that the aged mitochondria are more “uncoupled”, and leads to
increase in mitochondrial O
2
.-
release (Cadenas and Davies 2000). This further leads to
increase in ONOO
-
formation. It is well documented that ONOO
-
can cause lipid
peroxidation which affects mitochondrial membrane integrity. Also, ONOO
-
has been
documented to induce both necrotic and apoptotic cell death which eventually lead to
neurodegeneration (Heales, Bolanos et al. 1999). To assess the physiological
consequences due to declined energy production caused by nitration, the degree of
cytochrome c release into the cytosol with respect to aging was assessed. Our result
showed that there was an increase in cytochrome c release to the cytosol which is
positively correlated to an increase in state 4 respiration, suggesting that increase NO
generation and subsequent nitration of proteins may lead to uncoupling of
mitochondria. Increased ONOO
-
formation, as indicated by increased nitration, can
also contribute to the increase in state 4 respiration through inducing lipid
peroxidation of mitochondrial membrane lipids.
Taken together age dependent declined in proteasome function leads to accumulation
of nNOS protein and contributes to the increased production of
.
NO and ONOO
-
,
46
which leads to increased level of nitrosative and nitrative stress as a function of aging.
The increase in nitrative stress compromises energy production by inhibiting critical
enzymes at different stages of the energy metabolism pathways by protein nitration.
This eventually renders the brain more susceptible to energy crisis and cell death.
This finding bears significance to the pathological development of neurodegeneration
diseases such as Alzheimer’s or PD where increased
.
NO production and nitration
level have been documented.
47
CHAPTER II
Compromised proteasome degradation system elevates
neuronal nitric oxide synthase level and induces neuronal
cell death
1. Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by
resting tremor, bradykinesia, muscle rigidity, impaired cognitive function and
depression. PD is pathophysiologically manifested by two phenomena: 1) Progressive
and selective degeneration of dopaminergic neurons, which causing depletion of the
neurotransmitter dopamine in the region of substantia nigra pars compacta (SNpc) of
the mid-brain. 2) Formation of ubiquitinated inclusions of misfolded proteins known
as Lewy bodies, which implicates a compromised proteasome system. The proteasome
is a large intracellular multicatalytic protease that presents in all cell types in the
central nervous system (CNS) (Keller, Gee et al. 2002). It is responsible for majority
of intracellular proteolysis of oxidized, misfolded and aggregated proteins (Rock,
Gramm et al. 1994; Goldberg, Akopian et al. 1997; Tanaka 1998). These abnormal
proteins are first ubiquitinated and then targeted for degradation. However, in PD,
these ubiquitinated proteins begins to accumulate within neurons (Mayer, Lowe et al.
48
1991; Halliwell and Jenner 1998). The presence of ubiquitinated proteins implies a
compromised proteasome degradation system. Aging compromises proteasome
function has been showed in various study (Conconi, Szweda et al. 1996; Petropoulos,
Conconi et al. 2000; Keller, Gee et al. 2002; Zeng, Medhurst et al. 2005). Proteasome
itself is susceptible to free radical attack and some proteins damaged by free radical
may impair the function of proteasome as well (Friguet and Szweda 1997; Reinheckel,
Sitte et al. 1998; Okada, Wangpoengtrakul et al. 1999). Impairment of proteasome
also leads to decrease in GSH level, and increase in protein oxidation that has been
implicated in various neurodegenerative diseases such as Parkinson’s and Alzheimer’s
diseases (Markesbery 1997; Butterfield, Howard et al. 1999; Butterfield and Kanski
2001).
NOS is a degradation target of the proteasome (Friguet, Bulteau et al. 2000; Dunbar,
Kamada et al. 2004; Ferrington, Husom et al. 2005; Zeng, Medhurst et al. 2005). It is a
flavin- and heme- containing enzyme that catalyzes the metabolism of L-arginine to L-
citrulline and
.
NO in the presence of O
2
and NADPH (Palacios, Knowles et al. 1989;
Bredt and Snyder 1990). It has been demonstrated in Chapter I that the elevation of
nNOS level during aging affects mitochondrial functions, e.g. decrease in
mitochondrial respiratory and enhanced cytochrome c release. Additionally, the
increase of RNS, i.e. ONOO- formation, has been demonstrated through the increase
of mitochondrial protein nitration during aging. However, the signaling pathway that
leads to modulation of mitochondrial functions and neurodegeneration by
.
NO has not
49
been explored. Furthermore, up-to-date there are very few studies trying to investigate
the mechanistic roles of nNOS induced
.
NO production due to proteasome
impairment, so their relationship remains poorly understood.
It has been reported that c-Jun N-terminal Kinase (JNK) activation plays a role in
reactive nitrogen species, such as ONOO
-
, induced apoptosis (Shrivastava, Pantano et
al. 2004). JNK phosphorylates a variety of cytosolic proteins, such as p53, Bcl-2
family members, cytoskeleton proteins, and the glucocorticoid receptor (Giasson and
Mushynski 1997; Maundrell, Antonsson et al. 1997; Fuchs, Adler et al. 1998). JNK
can also modulate the mitochondrion-driven apoptotic pathway through its
translocation to mitochondria and subsequent phosphorylation of mitochondrial
proteins such as Bcl-2, Bcl
XL
(Schroeter, Boyd et al. 2003). Phosphorylation of Bcl
XL
leads to release of cytochrome c which in turns activate caspase 9 and 3 thus resulting
in apoptosis (Hengartner 2000).
We propose that dysfunction of proteasome leads to accumulation of nNOS protein
and thereby increase the production of reactive nitrogen species (RNS), e.g.
.
NO,
ONOO
-
, and results in JNK mediated neuronal death due to increased nitrative stress.
This study was aimed at assessing the role of decreased proteasome degradation,
which resembles the condition in PD or aging brain, in
.
NO induced neuronal cell
death. Differentiated PC12 cell line, which is a dopaminergic neuronal model, was
50
exposed to MG132 (a general proteasome inhibitor) and the underlying mechanisms
by which
.
NO mediated apoptosis due to elevated nNOS level were studied.
2. Materials and Methods
2.1 Materials
Antibodies against nNOS was purchased from Sigma (St Louis, MO); JNK1, pJNK
and Caspase 9 were purchased from Santa Cruz Biotech (Santa Cruz, CA); β-actin
was from Chemicon (Temecula, CA); p-BclXL was from Upstate (Lake Placid, NY).
The phosphatase inhibitor cocktail (catalog N° 524624; Calbiochem, La Jolla, CA)
contained (-)-p-bromotetramisole oxalate, cantharidin, and microcystin LR. Protease
inhibitor cocktail (catalog N° P8340) and all other chemicals or reagents were
obtained from Sigma (St Louis, MO).
2.2 Rat pheochromocytoma cells (PC12)
PC12 cells were purchased from ATCC and maintained in growth medium (RMPI +
10% donor horse serum + 5% fetal bovine serum). Cells differentiation was done in
differentiation medium (RPMI medium + 1% donor horse serum + 100 ng/ml nerve
growth factor + 50 ng/ml cyclic AMP) for 5 days.
51
2.3 Cell viability and cytotoxicitiy measurement
Differentiated PC12 cells were cultured in 6-well plates at 7 X 10
6
cells/well and
treated with MG132 (0 to 10 μM) in differentiation medium for 24 or 48 hours and
then subject to MTT or LDH assay. For rescue experiments, cells were pretreated
with 3mM L-NAME for 30 min prior to MG132 treatment.
Cell viability was assessed by incubating treated cells with MTT reagent (5 mg/ml)
(Sigma) for 30 min at 37
o
C in a humidified 5% CO
2
incubator. The reduced
intracellular formazan product was dissolved by replacing 2 ml of differentiation
medium with the same volume of DMSO. The absorbance at 490 nm was measured
with a microplate reader. Data are expressed as the percentages of cell viability
calculated by the following equation:
Cell viability=(OD
treatment group
/OD
control group
)×100%
For Cytotoxicity measurement, Lactate dehydrogenase (LDH) release assay kit
(Promega) was used following manufacturer’s instructions. The release of LDH from
cells represents an index of cytotoxicity or necrosis.
2.4 Proteasome inhibition assay
PC12 cells were disrupted by sonication in buffer containing: 10 mM Hepes, 137 mM
NaCl, 4.6 mM KCl, 1.1 mM KH
2
PO
4
, 5 mM ATP, 5 mM MgCl
2
, 1 mM DTT, and
52
10% glycerol, at pH 7.4. Proteasome activity of 100 μg of the cell lysate was
measured using 20S proteasome activity assay kit (Chemicon) following
manufacturer’s instructions. Proteasome activity was quantified by the fluorescence
given out by the cleavage of proteasome substrate (LLVY-AMC) using a 380/460 nm
filter set and presented as percentage inhibition of the control sample.
2.5 Nitrite determination with 2,3-diaminonaphthalene (DAN)
.
NO production was represented by the total nitrite presence in the cell medium
detected by DAN. Briefly, PC12 cells differentiation medium was replaced with
HEPES buffer containing NGF and cAMP in order to prevent interference caused by
high level of nitrite presented in the RPMI. The cells were exposed to MG132 (0.5
μM) at 37 °C for 24 hr; 10 μl freshly prepared DAN (0.5 mg/ml in 0.62 M HCl) was
added to 1 ml of the samples or positive control and stirred immediately in the reaction
buffer containing 40 μM NADPH with or with the presence of nitrate reductase (10
units/ml) . After 30 min incubation at 37 °C in the dark, the reaction was terminated
by 50 μl of 2.8N NaOH. Formation of 1-H-naphthotriazole was measured with a
fluorescence spectrophotometer (Molecular devices, SpectraMax, USA) with
excitation wavelength 365 nm and emission wavelength 450 nm.
2.6 Caspase 9 and 3 activation measurement
Activation of Caspase 9 was observed by western blot using anti-Caspase 9 antibody
to detect the appearance of active caspase 9 (cleaved form) at different MG132 or
53
MG132 + L-NAME (a general NOS inhibitor) treatment doses. Caspase 3 activity was
measured by a colorimetric assay kit (Promega) following manufacturer’s instruction.
Briefly, untreated cells or cells treated with MG132 (0.5 μM) or pretreated with L-
NAME (3 mM) for 20 min then added with MG132 (0.5 μM). 100 μg of protein was
incubated with a colorimetric substrate, Ac-DEVD-pNA for 24hr at 22
o
C. The release
of chromophore p-nitroaniline was quantified by a spectrophotometer at 405nm. Z-
VAD-FMK treated cells were included as negative control.
2.7 Statistical analysis
Data are reported as means ± S.D. of at least three independent experiments.
Significant differences between mean values were determined by student t-test. Means
were considered to be statistically distinct if P < 0.05.
3. Results
3.1 MG132 treatment leads to elevated nNOS content in PC12 cells
Differentiated PC12 were treated with different concentration of MG132 for 24 and 48
hours. Total proteins were harvested and analyzed by western blot using antibody
against nNOS. As showed in Fig. 9, nNOS protein level increased by MG132 in a
dose- and time-dependent manner. More than one nNOS bands were observed at high
concentrations (>0.5 μM) and prolonged (48 hr) MG132 treatment which could be
54
representing partially degrade nNOS proteins or special nNOS spicing variants existed
in PC12 cells. In addition, iNOS and eNOS proteins were not detected in all treatment
conditions (data not shown).
3.2 Increase in nNOS protein content leads to increase in cytotoxicity and decrease in
neuronal viability, which correlated with proteasome inhibition
In order to verify the hypothesis that increased nNOS content leads to increased
nitrative stress and causing progressive neuronal cell death by enhancing overall
apoptotic and/or necrotic events in cells, MTT assay and LDH release assay were
utilized to assess neuron viability and cytotoxicity after MG132 treatment, for an
increase in nNOS content is associated with increased nitrative stress leads to neuronal
cell death.
Differentiated PC12 were treated with different concentrations of MG132 for 24 hours
Fig. 9 Inhibition of proteasome-ubitquitin degradation system leads to increase in nNOS
content.
Differentiated PC12 cells were treated with different concentration of MG132 (0 to
2.5 μM) in the presence of differentiation medium for 24 or 48 hr. Total proteins were
separated on 10 % SDS PAGE gel. nNOS protein content was detected by western blot
using α-nNOS antibody. Dose- and time-dependent increase in nNOS protein level was
observed with increase of MG132 concentration.
55
and their viability was assessed by MTT assay. As showed in Fig. 10A, a dose-
dependent decline of PC12 viability was observed. In order to verify that NO
production contributed to the decreased viability, PC12 cells were pretreated with L-
NAME, which is a general NOS inhibitor, before treating with MG132. Interestingly,
PC12 viability was partially rescued by pre-treating with L-NAME suggesting that
NOS played a role in the decreased viability. Effect of NOS accumulation on
cytotoxicity was investigated by LDH release assay (Fig. 10B). A dose-dependent
increase of cytotoxicity was observed with the increase of MG132 concentration and
again the increase in LDH release can be partially rescued by pre-treating with L-
NAME. The inhibition of proteasome by the different doses of MG132 used in the
MTT assay was also studied. A positive correlation between proteasome inhibition
and decline of cell viability was observed (Fig. 10C). Thus, the data suggests that
MG132 inhibition of the proteasome system caused an elevation of nNOS, which
leads to a decline neuronal viability and increased cytotoxicity.
Fig. 10
A)
56
Fig. 10 Increase in nNOS content by inhibiting proteasome is correlated to increase
in cytotoxicity and decrease in neuronal viability, which correlated to the inhibition of
proteasome.
PC 12 cells were treated with different concentration of MG132 (0 to 10 μM)
for 24 hr in the presence of differentiation medium. A) Cell viability was measured by
MTT assay. Does-dependent decrease of viability caused by MG132 was observed
(solid bar). MTT activity was partially rescued by pretreating the cells with 3mM L-
NAME for 30min prior to the addition of MG132 (open bar).B) Cytotoxicity was
detected by LDH release assay. Cytotoxicity was increased in a dose-dependent
manner with the increase of MG132 concentration. C) Proteasome activity was
detected by 20S proteasome assay kit. The inhibition of proteasome (- ●-) was
positively correlated to the decrease in cell viability (- ■-).
B)
C)
Fig.10 Continued
57
3.3 Accumulation of nNOS protein by MG132 treatment leaded to enhanced
.
NO
production
Production of
.
NO was reflected by the level of nitrite in the medium as measured by
DAN assay with or without the presence of nitrate reductase. Nitrate reductase
converts nitrate, which is decomposed from ONOO
-
, to nitrite. PC12 were treated with
0.5 μM of MG132 in HEPES buffer for 24 hours. Total nitrite level in the medium
was measured by DAN assay as mentioned in the method section. Without the
presence of nitrate reductase, no significant change in total level of nitrite was
observed in PC12 cells treated with MG132 as compare to the control (Fig. 11A).
However, with the presence of nitrate reductase in the DAN assay, total nitrite level
was increased by 55% (Fig. 11B). This data suggesting that there was an increase in
ONOO
-
production in PC12 cells after treating with MG132, i.e. increased nitrative
stress.
58
Fig. 11 Enhanced nNOS causes increase in
.
NO and thus enhances ONOO-
formation.
Differentiated PC12 cells were treated with 0.5 μM MG132 in
HEPES buffer containing NGF and cAMP. Level of NO produced by PC12
cells was reflect by the level of total nitrite level in the culture media by DAN
assay with or without the presence of nitrate reductase as mentioned in the
method section. A) Total nitrite level remained unchanged after MG132
treatment. B) Nitrate level increased after MG132 treatment as reflected by the
increase in total nitrite level with the presence of nitrate reductase in the assay.
7-NI pre-incubation inhibited the production of
.
NO.
A)
B
59
3.4 Increased ONOO
-
production activated JNK signaling pathway and decreased
neuron viability.
Since ONOO
-
has been reported to activate JNK and an increase in ONOO
-
production
was indicated by an overall increase in nitrite level by DAN assay with the presence of
nitrate reductase in the current study, the effect of enhanced ONOO
-
production was
assessed by MTT assay. By inhibiting nNOS with 7-NI (nNOS specific inhibitor), cell
viability was significantly rescued. Interestingly, SP600125, (JNK inhibitor) also
shown similiar effect on rescuing PC12 viability as 7-NI (Fig. 12A). This data
suggests that increased nitrative stress rendered PC12 cells more susceptible to cell
death and it is JNK mediated.
Level of phosphorylated JNK was measured by immubloting utilizing antibody
against phosphorylation JNK (p-JNK). The level of p-JNK1, which is predominant
form of JNK in brain, was increased after MG132 (0.5 μM) treatment (Fig. 12B). 7-NI
was able to repress the phosphorylaytion of JNK further supporting the fact that nNOS
played a role in the activation of JNK1 in MG132 treated PC12 cells. It has been
suggested that upon activation, JNK1 translocates to the mitochondria and
phosphorylates (inhibits) mitochondrial outer member proteins such as Bcl
XL
which
trigger downstream apoptotic cascades. By immubloting the same sample with p-
Bcl
XL
antibody. We ascertained the levels of BCL
XL
phosphorylation was increased
after MG132 treatment which correlated with JNK1 activation (Fig. 12B) and the level
of BCL
XL
phosphorylation was attenuated by either 7-NI or SP600125.
60
Fig. 12 Reduction of cell viability by MG132 is
.
NO and JNK mediate.
PC12 cells were treated with 0.5 μM MG132 for 24hr in the presence of
differentiation medium. A) Cell viability was measured by MTT assay. MG132
treatment reduced cell viability which can be significantly rescued by
preincubation of 7-NI or SP600125. B) Phosphorylation of JNK and Bcl
XL
were
detected by immunoblotting using antibodies against p-JNK and p-Bcl
XL
,
respectively. Increase in p-JNK and p-Bcl
XL
levels after MG132 treatment were
attenuated by 7-NI or SP600125.
A)
B)
61
3.5 Activate JNK signaling pathway leads to neurodegeneration in a mixed necrotic
and mitochondrial mediated apoptotic mechanism with caspases activation
7-NI rescued neuron viability and repressed JNK and Bcl
XL
phosphorylation after
MG132 treatment which implies that increased nitrative and nitrosative stress as
showed in chapter I through upregulation of nNOS may activate apoptosis through
JNK phosphorylation. To determine if activation of JNK leads to mitochondrion-
driven apoptosis under current experimental condition, the level of the activation of
caspases 9 and 3 using western blot and a colorimetric assay system, respectively, was
assessed. A dose-dependent cleavage of pro-caspase 9 to active caspase 9 was
observed and interestingly the activation of caspase 9 was inhibited by L-NAME (Fig.
13A, B). The activity of caspase 3 was also increased by MG132 treatment and
inhibited by pre-incubation with either the general NOS inhibitor (L-NAME) or
nNOS specific inhibitor (7-NI) (Fig. 13C). Both L-NAME and 7-NI equally
attenuated on caspase 3 activation suggesting that apoptosis happened in the current
experimental model is mitochondrial dependent and nNOS mediated.
62
Fig. 13
A)
B)
63
Fig. 13 MG132 induced
.
NO production causes caspase 9 and 3 activation which can
be reversed by NOS inhibitor (L-NAME and 7-NI).
PC12 cells were treated with increasing doses of MG132 (0 to 10 μM) for 24
hr. A) 100 μg total protein was harvested and western blotted with anti-caspase 9
antibody. An enhanced cleavage of caspase 9 (active form) was observed with the
increase of MG132 concentration. B) Cleavage of pro-caspase 9 was inhibited by
treating cells with L-NAME (3 mM). C) Caspase 3 activity was measured by a
colorimetric assay system mentioned in the method section. MG132 (0.5 μM)
significantly increased Caspase 3 activation and pre-incubation of general NOS
inhibitor (3 mM L-NAME) or nNOS specific inhibitor (10 μM 7-NI) inhibited the
activation. Z-VAD-FMK, which inhibits caspase 3, was included as negative control for
MG132 or control cells. *, # p<0.05
Fig. 13: Continued
C)
64
4. Discussion
PD is characterized by the selective loss of dopaminergic neurons accompanied by a
compromised proteasome degradation system which leads to formation of Lewy
bodies. Various studies have also demonstrated that proteasome activity declines with
age. It is well established that degradation of nNOS is proteasome dependent. Basze
of the above information, we utilized the well known dopaminergic cell line PC12 to
study if the elevation of nNOS content could be related to the regulation of cell death.
PC12 is a model for dopaminergic cells due to its ability to produce dopamine. Keller
et al., 2000 reported that dopamine was able to inhibit proteasome activity and its
inhibitory ability is dependent in part on reactive oxygen species (Keller, Huang et al.
2000). It has been showed that mitochondria damage by
.
NO is potentiated by
dopamine (Antunes, Han et al. 2002), in which may lead to increase reactive oxygen
(ROS) and nitrogen species (RNS) generation. Taken together, it may be surmised that
the increased ROS and RNS production can further inhibit the proteasome through a
positive feedback mechanism. Increased level of ROS and RNS has also been shown
to mediate downstream apoptotic events that followed proteasome inhibition.
In this study, differentiated PC12 were treated with MG132 to mimic the proteasomal
impairment that occurs in PD. The proteasome system was not completely shut down
by the highest concentration of MG132 treatment (10 μM), thus the experimental
system was similar to PD in which the proteasome system was compromised but not
65
completely disrupted. Data from the DAN assay suggested an increase in ONOO
-
production since only with presence of nitrate reductase, an increase in total nitrite
level can be observed. Nitrate reductase is responsible for converting nitrate, which is
decomposed from ONOO
-
, to nitrite. It has been reported that uncoupled nNOS
produces reactive oxygen species (ROS). ROS rapidly react with
.
NO to form ONOO
-
(Pou, Keaton et al. 1999; Delgado-Esteban, Almeida et al. 2002). Thus it may be
surmised that in the current experimental model, inhibition of proteasome causing
accumulation of nNOS and part of the undegraded nNOS could be uncoupled that
leaded to the formation of ROS such as O
2
.-
in lieu of NO thus caused the formation of
ONOO
-
. Furthermore, the presence of Cyclic AMP (cAMP) during differentiation of
PC12 cells might also favor ONOO
-
formation. The presence of cAMP in the
differentiation medium enhances the terminal and irreversible differentiation of PC12
into dopaminergic neurons (Michel, Vyas et al. 1995) by phosphorylates and activates
protein kinase A (PKA), which in turn phosphorylates and activates cAMP-response
element binding protein (CREB); CREB bindings to promoters and activate the
transcription of several important neuronal survival factors. However, it has been
reported that cAMP can decrease the level of bioactive
.
NO by upregulated uncoupled
nNOS, which significantly enhancing reactive oxygen species generation and
ultimately enhanced the formation of ONOO
-
from
.
NO and O
2
.-
(Boissel, Bros et al.
2004). These uncoupled nNOS were accumulated due to proteasome inhibition thus
generate high level of ONOO
-
. The formation ROS through uncoupled nNOS could
further inhibits the proteasome.
66
It is unlikely that iNOS plays a role in the increased ONOO
-
formation in proteasomal
impairment induced neuronal cell death because induction of iNOS was not detected
in our experimental model. This finding is supported by Griscavage et al, 1996 and
Conner et al, 1997, that proteasome inhibition blocks induction of iNOS by preventing
activation of nuclear factor-kB (NF-kB). eNOS was also not detected after MG132
treatment. Taken together, it may be surmised that compromised proteasome leaded to
accumulation of nNOS, which may be uncoupled.
Levels of 3-nitrotyrosine, a biomarker of nitrative stress cause by ONOO
-
and other
reactive nitrogen species attack, have been reported to be elevated in several
neurodegenerative diseases (Beal, Ferrante et al. 1997; Halliwell 1997; Greenacre and
Ischiropoulos 2001). Thus, it is reasonable to interpret that the enhanced formation of
ONOO
-
from
.
NO which is produced by nNOS in our experimental model is
contributes to neuronal loss by activating apoptosis. It has been reported that ONOO-
can induce the activation of multiple apoptotic caspases (Zhuang and Simon 2000),
(Heales, Bolanos et al. 1999).
.
NO or ONOO
-
induced apoptotic events can be
classified as guanyl cyclase dependent or independent pathways. The c-Jun N-terminal
Kinase (JNK) pathway is considered as guanyl cyclase independent. In this study,
JNK was found to be activated after nNOS upregulation and the repressed by nNOS
inhibitor suggesting that reactive nitrogen species such as
.
NO or ONOO
-
played a role
in JNK activation. Phosphorylated JNK (active) causes phosphorylation (inhibition) of
67
anti-apoptotic proteins such as Bcl
XL
, which eventually leads to release of cytochrome
c that activates downstream caspases.
The significance of the current study demonstrates the impairment of proteasome leads
to increased nitrative stress in dopaminergic model. nNOS accumulation, which is due
to proteasome inhibition, was probably uncoupled, which produced ROS and
enhanced ONOO
-
formation. Furthermore, we have shown that the
.
NO or ONOO
-
produced by nNOS rendered dopaminergic neurons more susceptible to cell death
through activating JNK dependent mitochondrial apoptotic cascades. This provides a
potential mechanism for age dependent development of PD pathology.
68
CHAPTER III
Neuronal nitric oxide synthase: implication for
environmental toxin induced apoptosis in dopaminergic
neurons
1. Introduction
The first clear environmental toxin that could induce Parkinsonism came from MPTP
(1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine), which was a contaminating compound
found in a meperidine analog called MPPP (1-methyl 4-phenyl 4-
propionoxypiperidine) (Ziering, Malatestinic et al. 1970). MPTP can induce all the
typical Parkinsonian symptoms in primates and humans. It also causes preferential
loss of dopaminergic neurons in substantia nigra (Seniuk, Tatton et al. 1990; Muthane,
Ramsay et al. 1994). MPTP is a lipophilic compound that can freely cross the blood-
brain-barrier (BBB) (Markey, Johannessen et al. 1984). When in the brain, MPTP can
be metabolized by monoamine oxidase B (MAO-B) to form 1-methyl-4-phenyl-2,3-
dihydropyridinium (MPDP
+
) in non-dopaminergic cells (Chiba, Trevor et al. 1984).
Then MPDP
+
will undergo spontaneous oxidation to 1-methyl-4-phenylpyridinium ion
(MPP
+
), which is exocytosed into the extra-cellular space, and taken up primarily by
dopaminergic neurons through dopamine transporters (Javitch, D'Amato et al. 1985;
69
Bezard, Gross et al. 1999). Once inside the dopaminergic neurons, MPP
+
rapidly
accumulates in the mitochondrial matrix via a passive mechanism strictly dependent
on the transmembrane potential (Hoppel, Grinblatt et al. 1987; Davey, Tipton et al.
1992). In the matrix, MPP
+
binds and inhibits electron transfer in NADH-ubiquinone
oxidoreductase, ie. Complex I (Ramsay, Kowal et al. 1987). MPTP induces a vast
array of deleterious effects in the neurons including inhibition of mitochondria
complex I activity, decrease of ATP synthesis (Chan, DeLanney et al. 1991), increase
of O
2
.-
generation (Rossetti, Sotgiu et al. 1988), upregulation of pro-apoptotic proteins
(Vila, Jackson-Lewis et al. 2001), and increase of NO production (Smith, Swerdlow et
al. 1994; Przedborski, Jackson-Lewis et al. 1996; Matthews, Beal et al. 1997; Du, Ma
et al. 2001) that eventally lead to the activation of apoptotic pathway. Mitochondira
seem to be pivotal in these series of events.
Paraquat (N,N´-dimethyl-4-4´-bipyridium), which is a
potent herbicide, is strikingly similar in chemical
structure to Parkinsonism-inducing neurotoxin MPP
+
(Fig. 14). There have been reports documenting lethal
poisoning by dermal exposure to paraquat (Smith 1988). Lung, liver, and kidney are
the prime damage sites after acute paraquat poisoning, however, damage to the brain
has also been observed (Grant, Lantos et al. 1980; Hughes 1988). Paraquat was shown
to be transported across the blood-brain-barrier via amino acid transporters and then
be taken up by dopaminergic neurons via dopamine transporter (Shimizu, Ohtaki et al.
Fig. 14 Paraquat has a similar
chemical structure to MPP
+
.
70
2001; Shimizu, Matsubara et al. 2003), albeit Richardson et al. suggested alternative
transport system other than dopamine transporter (Richardson, Quan et al. 2005).
In fact, epidemiologic studies have shown that there was increased risk for PD after
chronic exposure to paraquat (Lewin 1985; Liou, Tsai et al. 1997), and so it is
suggested to be a potential etiologic factor in PD (Andersen 2003). Several reports
have shown that paraquat treatment in mice resulted in decreased motor activity, death
of substantia nigra dopaminergic neurons (Brooks, Chadwick et al. 1999), and up-
regulation and aggregation of α-synuclein (Manning-Bog, McCormack et al. 2002),
all of which are cardinal features of PD. To date, paraquat is still the most commonly
used herbicide worldwide. Therefore, it is critical that we understand the mechanism
by which paraquat contributes to the development of PD in order to formulate a
preventive/therapeutic strategy.
Oxidative stress has been implicated in the initiation and progression of PD (Jenner
and Olanow 1996). Oxidation of dopamine and concurrent generation of reactive
oxygen species are responsible for the high basal level of oxidative stress in SNpc.
Accumulation of environmental toxins, like paraquat, in SNpc dopaminergic neurons
can further tip the balance of oxidant-antioxidant and subsequently lead to oxidative
damage and apoptotic cell death. It has been established that paraquat can generate
O
2
.-
by redox cycling with cytosolic diaphorases. It can also induce up-regulation of
nitric oxide synthases and ·NO production. ·NO and O
2
·
-
can react at diffusion-
71
controlled rate to form ONOO
-
, which can chemically modify protein by tyrosine
nitration or trigger apoptosis. A serious of morphological evidences also point out
mitochondria as the early target of paraquat. Administration of paraquat in cultured
cells resulted in a decrease of mitochondrial matrix electron density, matrix swelling,
disruption of cristae structure, and breakage of mitochondria (Hirai, Witschi et al.
1985; Ueda, Hirai et al. 1985; Wang, Hirai et al. 1992), all of which closely resemble
mitochondria permeability transition (MPT), which inducing apoptosis.
Apoptotic cell death has been suggested to be the common pathway for neuronal cell
loss in neurodegenerative diseases (Cotman and Anderson 1995; Thompson 1995).
Apoptotic cells have also been identified in the substantia nigra region of PD patients
(Mochizuki, Goto et al. 1996). Interestingly, paraquat treatment in various animal and
cell culture models showed that apoptosis, at least in part, accounted for the loss of
neuronal cells, and the process is mediated by the generation of reactive oxygen
species, since addition of antioxidants (e.g. coenzyme Q
10
, vitamin E) can efficiently
reduce apoptotic cell death (Melchiorri, Del Duca et al. 1998; Li and Sun 1999;
McCarthy, Somayajulu et al. 2004). In cerebellar granule cell, exposure to paraquat
caused decrease of cytochrome c content in mitochondria, caspase 3 activation, and
apoptosis (Gonzalez-Polo, Rodriguez-Martin et al. 2004). Paraquat has also been
shown to induce calcium-dependent mitochondria membrane permeability transition
in isolated rat liver mitochondria (Costantini, Petronilli et al. 1995). Taken together,
72
paraquat neurotoxicity may involve mitochondria permeability transition-mediated
apoptosis.
Paraquat has been known to produce O
2
.-
by redox cycling with cellular diaphorase
(Day, Patel et al. 1999; Margolis, Porasuphatana et al. 2000), and significantly
decreases intracellular GSH in cultured dopaminergic neurons (Yang and Tiffany-
Castiglioni 2005). In the same system, paraquat can decrease mitochondrial trans-
membrane potential (Yang and Tiffany-Castiglioni 2005), possibly by inhibiting
complex I activity (Fukushima, Yamada et al. 1994; Tawara, Fukushima et al. 1996).
In rat liver mitochondria, an outer membrane-associated enzyme with NADH-quinone
oxidoreductase activity was shown to produce O
2
.-
in the presence of paraquat and
NADH (Shimada, Hirai et al. 1998). In addition to O
2
.-
,
.
NO production is also
involved in paraquat cytotoxicity. In guinea pig lungs exposed to paraquat,
.
NO
synthesis was found to be significantly increased (Berisha, Pakbaz et al. 1994). In rats,
paraquat exposure caused wet dog shakes (WES) that can be suppressed by non-
selective NOS inhibitor N-nitro-L-arginine (Hara, Mukai et al. 2000). Administration
of paraquat by microdialysis probe to rat brain results in exocytosis of glutamate into
the extracellular space that activates NMDA receptor and calcium influx. Increase of
intracellular calcium activates nNOS, and increases production of
.
NO (Shimizu,
Matsubara et al. 2003). Since nNOS utilizes electron from NADPH to generate
.
NO,
the electron maybe snapped by paraquat during the process. Thus, nNOS can
potentially act as a diaphorase protein in providing the electron that is necessary for
73
paraquat radical formation. In this chapter, the inherent role of nNOS in mediating
paraquat-toxicity through acting as a paraquat diaphorase and its implication in the
development of PD will be discussed.
2. Materials and Methods
2.1 Chemicals
All chemicals, except stated otherwise, were purchased from Sigma (St Louis, MO).
2.2 Animal
Fisher 334 rat at different ages (6, 14 and 24 months) were purchased from the
National Institute of Aging (NIA). Each rat was individually housed in the animal
facility under standard conditions (12/12 light-dark cycle, humidity at 50±15%,
temperature 22±2
o
C and 12 air changes/hr). All procedures were approved by the local
Animal and Care and Use Committee.
2.3 Cell viability and cytotoxicitiy measurement
PC cells was differentiated in differentiation medium [RPMI medium + 1% donor
horse serum + 100ng/ml nerve growth factor with or without 50ng/ml cyclic AMP].
Cell viability was assessed by incubating cells with MTT reagent (5 mg/ml) (Sigma)
for 30min at 37
o
C in a humidified 5% CO
2
incubator. The reduced intracellular
74
formazan product was dissolved by replacing 2 ml of differentiation medium with the
same volume of DMSO. The absorbance at 490nm was measured with a microplate
reader. Data are expressed as the percentages of cell viability calculated by the
following equation:
Cell viability=(OD
treatment group
/OD
control group
)×100%
For Cytotoxicity measurement, Lactate dehydrogenase (LDH) release assay kit
(Promega) was used following manufacturer’s instructions. The release of LDH from
cells represents an index of cytotoxicity or necrosis.
2.3 CN-PAGE and sample preparation
CN-PAGE was first described in (Schagger, Cramer et al. 1994) and ingredients of the
gels was described in the paper. Briefly, linear polyacrylamide gradient gels (5-25%)
were created using gradient former (Bio-rad). The separating gel contained
PC12 total lysate was solubilized in 10% Dodecyl maltoside (detergent to protein ratio
(mg/mg) is 3:1). Solubilized sample was put on ice for 20 min and then spun down at
12,000g for 5min. 0.001% ponceau S was added to samples before electro- ionic
detergent dodecyl maltoside and loaded onto the gel to follow the migration front.
Supernatant (50 μg) was loaded onto the CN-PAGE gel and electrophoresis for 2 hr at
150 V or until the dye front reach the bottom of the gel.
75
2.4 In-gel paraquat formation assay
CN-PAGE with PC12 total lysate (50 μg) separated on it was soaked in reaction buffer
containing 50 mM Tris-HCl, pH 7.8, 4 mM PQ, 0.1 mM NADPH and 0.1 mM
Nitroblue tetrazolium (NBT). Formation of paraquat radical was observed by the
formation of blue color band due to the electron from paraquat radial was donated to
NBT which turned NBT into blue color.
2.5 Paraquat radical formation assay
0.5 mg PC12 total lysate was added into 1 ml of PBS in a spectrophotometer curvette
and then deoxygenated by purging with argon gas for 3 min. Then 5 mM of NADPH
and 5 mM of paraquat was added to the purged sample and the formation of paraquat
radical was measured at 600 nm.
2.6 Western blotting
Total brain homogenate were separated on the CN-PAGE and electro-transferred to
PVDF membrane at 40 V, overnight. The membrane was stained with a-nNOS
(Sigma) to detect for the presence of nNOS.
2.7 NADPH consumption assay
500 μg of total brain homogenate from rat at different age (6, 14 and 26 month) was
added to reaction buffer containing 50 mM Tris-HCl, pH 7.8, 4 mM PQ and 0.1 mM
76
NADPH. Consumption of NADPH was measured at absorbance at 340 nm using
kinetic mode for 20 min, interval between each reading is 60 sec.
3. Results
3.1 Paraquat dose-dependently decreases cell viability and increase cytotoxicity
Differentiated PC12 was treated with different dosages of paraquat. The effects of
paraquat on cell viability and cytotoxicity were measured by MTT and LDH release
assays, respectively. Paraquat dose-dependently decreases differentiated PC12 cell
viability and increases LDH release (Fig. 15, 16).
Fig. 15 Paraquat dose-dependently decreases cell viability.
PC12 cells have been differentiated for 5 days with NGF+cAMP.
Cells are treated with paraquat at indicated concentrations for 24h, and MTT
reduction by cells was measured at 540 nm. EC
50
of paraquat was calculated
as 155μM.
77
Fig. 16 Paraquat dose-dependently increases LDH release.
PC12 cells have been differentiated for 5 days with NGF + cAMP. Cells are
treated with paraquat at indicated concentrations for 24h, and paraquat toxicity was
measured by the degree of LDH release. LC
50
of paraquat was calculated as 196 μM.
78
3.2 DPI reduces paraquat toxicity
It has been hypothesized that the paraquat toxicity is attributed to its potential to
generate O
2
.-
due to redox cycling ability by snapping the electron from a protein
diaphorase, which refers to enzymes that utilize electron from either NADPH or
NADH for normal physiological functions, and donating it to oxygen to form O
2
.-
. To
determine if paraquat toxicity is affected by inhibiting protein diaphorases, DPI, which
is a flavin- containing protein inhibitor and paraquat were co-incubated with PC12
cells and paraquat toxicity was measured. Strikingly that DPI is able to completely
inhibit paraquat toxicity (Fig. 17).
Fig. 17 DPI has complete protecting effect against paraquat-induced LDH
release.
PC12 cells have been differentiated for 5 days with NGF + cAMP.
Cells are treated with 200 μM paraquat for 16h with or without the presence of
1μM DPI. Paraquat toxicity was measured by the degree of LDH release.
79
3.3 DPI inhibites the formation of paraquat radical
The above data suggests that flavin- containing protein plays a signficant role in
paraquat toxicity, by perhaps donating an electron to paraquat for paraquat radical
formation. Since nNOS is also a flavin- containing protein which utilizes NADPH for
generating
.
NO. Thus, it is hypothesized that nNOS could potentially be a paraquat
diaphorase. The formation of paraquat radical was measured after DPI and L-NAME
treatment. Interestingly, DPI can completely inhibit paraquat radical formation (Fig.
18). In addition, L-NAME can largely inhibit the paraquat radical formation.
Theoretically, by supplementing L-Arg, it may serve to ensure coupling of the NOS
since L-Arg is the substrate for NOS during
.
NO production. This will lead to
reduction in the electron leakage and hence decrease paraquat radical formation.
However, according to Fig. 18, L-Arg supplementation shows little effect in reducing
paraquat toxicity. Taken together, these results support the idea that NOS plays a very
significant role in the formation of paraquat radical in PC12 cells and uncoupling of
nNOS is not essential for paraquat radical formation.
80
Fig. 18 PQ radical generation is NOS dependent and does not require uncoupling
of NOS.
Differentiated PC12 was incubated with DPI, L-NAME, L-Arg or L-Arg
+ L-NAME at the indicated concentrations. Formation of paraquat radical was
measured by spectrophotometer at λ = 600 nm
81
3.4 L-NAME dose-dependently relieves paraquat toxicity
Since L-NAME which is a NOS inhibitor, is able to significantly reduce paraquat
radical formation, the next question is whether L-NAME would be able to relieve
paraquat toxicity. As shown in Fig. 19, L-NAME dose dependently rescues cell
viability as indicated by an increase in MTT reduction.
Fig. 19 L-NAME dose-dependently relieves paraquat toxicity.
Differentiated PC12 was treated with paraquat 150 μM was co-incubated with
various concentration of L-NAME as indicated. The effect of L-NAME co-incubation
on cell viability was assessed by MTT assay.
82
3.5 nNOS induces paraquat radical formation in rat brain homogenate
To further strengthen the evidence that NOS, particularly nNOS, is the protein
diaphorase responsible for paraquat radical formation and toxicity in brain. Rat brain
homogenate was separated using a 5-25% Clear Native –PAGE electrophoresis (CN-
PAGE). The gel was then soaked in reaction buffer containing nitrobluetetrasodium
(NBT), NADPH and with / without paraquat. Under normal condition, the NADPH
reductase protein will utilize the electron from NADPH for its normal physiological
function (e.g. electron transfer chain, NO synthesis, etc). Since paraquat is a
very strong redox cycler, it can easily remove the electron from the NADPH reductase
portion of nNOS and in turn reduces oxygen to form O
2
.-
. NBT then accept the
electron from the O
2
.-
and/or paraquat radical resulting in the formation of a blue color.
Blue-NBT only appeared in the presence of paraquat suggesting the reaction is
paraquat dependent (Fig. 20A). Experiments using L-NAME (a general NOS
inhibitor) confirmed the importance of NOS in paraquat toxicity; as the inhibitor was
able to prevent blue-NBT formatiom, i.e. the reaction is NOS dependent (Fig. 20A).
The blue-NRT band was confirmed to be nNOS using western analysis. The same
protein sample was separated on a different lane of the same clear native gel,
transferred to a PVDF membrane, and immunoblotted for nNOS using α-nNOS
antibody. The nNOS signal corresponded to the blue-NBT band indicating that band
consists of nNOS.
83
Fig. 20 nNOS induces paraquat formation.
A) Differentiated PC12 lysate (50 μg) was incubated with or without L-NAME for
30 min at RT and then separated on a 5-25% clear-native gel. The gel was then incubated
with reaction buffer containing nitrotetrazolium blue, NADPH (0.1 mM) with or without the
presence of paraquat (0.1 mM). Formation of oxidized nitrobluetetrazolium (blue band) was
observed after 1 hr incubation at RT. B) Same lysate from A) was separated on another lane
of the clear native gel and transferred to a PVDF membrane then immunoblotted against
nNOS.
A)
B)
84
3.6 Paraquat radical formation increased with respect to age
It has been shown earlier that nNOS level increases with age (Fig. 3). It would be
significant to study whether the elevated nNOS level enhances paraquat radical
formation. Rat brain homogenate from different ages (6, 14 and 24 months) was
incubated with paraquat in the presence of NADPH. Generation of paraquat radical
was measured by the paraquat dependent consumption of NADPH since the process
utilizes NADPH as the electron donor. Fig. 21 shows that consumption of NADPH
increased with age which implicates an increased paraquat radical formation during
aging.
Fig. 21 Paraquat dependent NADPH consumption increases with age implicating
enhanced paraquat radical formation.
500 μg of total brain homogenate from rat at different age (6, 14 and 26
month) was added to reaction buffer containing PQ (4 mM) and NADPH (0.1
mM). Consumption rate of NADPH was measured at absorbance at 340 nm using
kinetic mode for 20 min, interval between each reading is 60 sec using a
spectrophotometer.
85
4. Discussion
Paraquat is very similar in chemical structure to a well known Parkinsonism-inducing
agent, MPP
+
. Administration of paraquat in mice resulted in an array of Parkinsonism
symptoms (Brooks, Chadwick et al. 1999; Manning-Bog, McCormack et al. 2002). It
has been shown that paraquat is transported through the blood-brain-barrier by an
amino-acid transport system in an animal model, and taken up by dopaminergic
neurons via a carrier-mediated process in a cell culture system (Shimizu, Ohtaki et al.
2001; Shimizu, Matsubara et al. 2003; Richardson, Quan et al. 2005). Once taken up
by dopaminergic neurons, paraquat causes extensive neuronal death, in part through
apoptosis, which can be rescued by antioxidants (e.g. vitamin E and coenzyme Q
10
)
(Melchiorri, Del Duca et al. 1998; Li and Sun 1999; McCarthy, Somayajulu et al.
2004). In cerebellar granule cells, paraquat caused a decrease of mitochondria
cytochrome c content, and activation of caspase 3, indicating a mitochondrion-driven
apoptosis (Gonzalez-Polo, Rodriguez-Martin et al. 2004).
Paraquat is known to produce O
2
·-
by redox cycling with cellular diaphorases, e.g.
NOS (Day, Patel et al. 1999; Margolis, Porasuphatana et al. 2000). By treating PC12
lysate with DPI, which is a diaphorases inhibitor, completely blocked the formation of
paraquat radical. Treatment of DPI also relieved paraquat induced cytotoxicity. These
data further confirmed the critical role of diaphorases in paraquat induced toxicity.
NOS have been hypothesized as a potential diaphorase that plays a role in paraquat
86
radical formation since it utilize NADPH as an electron donor for the generation of
.
NO and that electron can be given to paraquat by forming paraquat radical. In the
present study, data obtained from clear native in-gel assay provided interesting
information about the functional role of NOS in paraquat radical formation. By
treating with PC12 lysate with L-NAME (NOS inhibitor) before incubating with
paraquat and NADPH, L-NAME abolished the formation of blue color NRT band
which indicated that NOS is the essential protein in the formation of paraquat radical.
Immunoblotting of the clear native gel against α-nNOS antibody revealed the identity
of the blue color NRT band as nNOS. Increased paraquat radical formation could be
due to uncoupling of nNOS, which caused electron leakage during the production of
.
NO. Prevent uncoupling nNOS with substrate L-Arg did not reduce paraquat radical
formation significantly suggesting that uncoupling of nNOS may not be an essential
criteria for the radical formation process. The exact mechanism underlying the radical
formation process requires further investigation. Nevertheless, it can be surmised that
paraquat snaps the electron from nNOS by forming paraquat radical which then
donates the electron to O
2
that forms O
2
.-
.
In addition to O
2
·-
, ·NO is also involved in paraquat cytotoxicity. Up-regulation of
iNOS and eNOS genes expression has been shown following paraquat treatment
(Drummond, Cai et al. 2000). Administration of paraquat in rat brain induces
activation of NMDA receptor and Ca
2+
influx that could activate NOS (Shimizu,
Matsubara et al. 2003). Most importantly, paraquat-induced Parkinsonism symptoms
87
in rats can be alleviated by NOS inhibitors (Hara, Mukai et al. 2000). Together with
the data from the earlier chapters that showing elevated nNOS level and activity are
correlated to increased cell death through JNK-mediated mitochondrion-driven
apoptosis and in this chapter we have shown that nNOS activates paraquat by acting
acts paraquat diaphorase. One can conclude that increase incidence of environmental
toxin induced PD in aging is tightly related to the increase in level of nNOS. We have
also shown that nNOS level and NADPH consumption elevated with age suggesting
that aged animal is more susceptible to paraquat toxicity. Data from this study
provided important information in developing preventive/therapeutic strategies against
environmental toxin-related PD.
88
SUMMARY
Aging is considered as a risk factor for many degenerative diseases; particularly PD.
Multiple cellular functions are affected by aging process, which includes, but not
limited to, proteasome and mitochondrial functions. Proteasome dysfunction can lead
to accumulation of proteins, such as NOS that has been highlighted in this thesis.
Lewy body formation is a pathological hallmark of PD, which implicates a
compromised proteasome in PD. Proteins found in Lewy body are often nitrated
suggesting enhanced production of
.
NO by NOS. Interestingly,
.
NO has also been
shown to inhibit mitochondrial functions through inhibition of various components of
mitochondria such as complex I nitration which has been implicated in PD. Thus, the
hypothesis being tested in this dissertation is that nNOS accumulates in brain during
aging because of proteasome dysfunction, causing increased nitrative stress by
elevating
.
NO production, which compromises mitochondria function through protein
post-translational modification and triggers mitochondrial caspases induced
apoptosis.
The present work utilized aging rat and PC12 dopaminergic cell models to decipher
the inter-relationships between aging,
.
NO, proteasome and mitochondria functions.
The data presented in this dissertation showed that proteasome activity declines with
aging leads to accumulation of nNOS that causing an increase in nitrative stress in the
brain. Two important mitochondrial biogenergetics enzymes: 1) SCOT and 2) F
1
-
89
ATPase have been found to be nitrated during aging. Nitration of these two enzymes
both resulted in decreases in their respective enzymatic activity. A compromised
energy production by aged mitochondria s supported by a decrease in RCR ratio and
enhanced mitochondria cytochrome c release.
By using proteasome inhibitor (MG132), proteasome dysfunction during aging / PD
was mimicked in PC12 dopaminergic cell model. We found that accumulation of
nNOS due to compromised proteasome system and enhanced formation of ONOO
-
,
which is a strong nitrating agent, leading to protein nitration. This data coincided with
the findings that nitration was enhanced in aged rat brain. We also found that
enhanced ONOO
-
activated JNK signaling pathway that triggered mitochondrial
dependent apoptosis cascade by activating caspases 9 and 3.
It has been demonstrated in our laboratory that when JNK is activated, it translocates
to mitochondria and inhibits pyruvate dehydrogenase (PDH) activity. PDH is an
enzyme that converts pyruvate into acetyl-CoA that feeds into the TCA cycle. When
PDH is inhibited, utilization of glucose by mitochondria to generate ATP is impaired.
The only alternative energy source for the brain becomes ketone bodies. However, as
our data indicated, that SCOT (the limited enzyme in ketolysis) is also inhibited by
nitration during aging. Thus, aged brain will be facing a critical situation that both
source of fuels: glucose and ketone bodies are not being utilized. Our data indicates
that accumulation of nNOS produces
.
NO at a level that brings multiple insults to the
90
bioenergetics process by decreasing the generation of reducing equivalents (SCOT)
and impairment of oxidative phosphorylation (F
1
-ATPase). Brain mitochondria are
fragile and turnover slowly thus impairment of brain mitochondria functions renders
the brain extremely susceptible to an energy crisis when the animal is aged and very
likely enhancing cell death (Fig. 22).
Fig. 22 Schematic diagram shows the proposed relationships between aging
induced neurodegeneration due to upregulation of nNOS caused by proteasomal
dysfunction.
Arrows and dashed lines indicate activation and repression of the
downstream components, respectively.
91
Another critical aspect covered in this dissertation is the potential inherent role of
nNOS in environmental toxin induced PD. We have shown that nNOS functions as a
paraquat diaphorase in dopaminergic PC12 cells and brain homogenate. Together with
the data from the earlier chapters that showing nNOS level elevated during aging, we
showed that nNOS dependent generation of paraquat radical increased with respect to
aging. Thus, one can conclude that environmental toxin induced PD is tightly related
to aging due to elevated nNOS level and it plays important roles in development of the
disease include but not limited to: 1) compromising energy production, 2) enhance
neuronal apoptosis by increasing nitrative stress and 3) function as a paraquat
diaphorase which converts paraquat to paraquat radical that damages neurons in case
of environmental toxin induced Parkinsonism.
92
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Abstract (if available)
Abstract
Nitric oxide synthase (NOS) is a flavin- and heme- containing enzyme that catalyzes the metabolism of L-arginine to L-citrulline and nitric oxide (.NO) in the presence of O2 and NADPH. Neuronal NOS (nNOS) is a Ca2+-calmodulin-dependent isoform of NOS that is constitutively expressed in neuronal cells. The cellular level of nNOS is regulatory by its turnover through degradation by the proteasome. Various studies have demonstrated that proteasome activity declines with age. Furthermore, dysfunction proteasome is implicated in Parkinson's disease (PD) in which the formation of Lewy bodies and a progressive degeneration of dopaminergic neurons are observed. However, to date, there has been no extensive study undertaken to investigate the role of proteasome function, aging, and nNOS level with respect to viability of dopaminergic neuron. Here, we propose that aging-induced dysfunction of proteasome leads to accumulation of nNOS protein, thereby increasing the production of reactive nitrogen species (RNS), such as .NO and peroxynitrite (ONOO-), and thus results in neuronal death due to increased nitrative / nitrosative stress. In this dissertation, by using brains from rats of different age and a PC12 dopaminergic cell model, it is demonstrated that nNOS protein levels increased with age and this correlated with an increase in both nitrosative and nitrative stress. Under conditions of elevated nNOS expression, two important mitochondrial enzymes involved in mitochondria bioenergetics, succinyl-CoA:3-oxoacid CoA-transferase (SCOT) and F1-ATPase, were found to be nitrated. Nitration of SCOT and F1-ATPase lead to a decrease in their activities, thus suggesting a compromised energy production at the level of reducing-equivalent generation and oxidative phosphorylation, respectively.
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Asset Metadata
Creator
Lam, Yeung
(author)
Core Title
Role of neuronal nitric oxide synthase in aging and neurodegeneration
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Molecular Pharmacology
Publication Date
11/05/2009
Defense Date
10/23/2007
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
mitochondria,neurodegeneration,nitric oxide,nitric oxide synthase,OAI-PMH Harvest
Language
English
Advisor
Cadenas, Enrique (
committee chair
), Alkana, Ronald (
committee member
), Hodis, Howard Neil (
committee member
)
Creator Email
ylam@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-m905
Unique identifier
UC1458951
Identifier
etd-Lam-20071105 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-586789 (legacy record id),usctheses-m905 (legacy record id)
Legacy Identifier
etd-Lam-20071105.pdf
Dmrecord
586789
Document Type
Dissertation
Rights
Lam, Yeung
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Repository Name
Libraries, University of Southern California
Repository Location
Los Angeles, California
Repository Email
cisadmin@lib.usc.edu
Tags
mitochondria
neurodegeneration
nitric oxide
nitric oxide synthase