University of Southern California Dissertations and Theses

C-jun N-terminal Kinase (JNK) mediated inhibition of Pyruvate Dehydrogenase (PDH) activity and its effect on mitochondrial metabolism during brain aging

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C-jun N-terminal Kinase (JNK) mediated inhibition of Pyruvate Dehydrogenase (PDH) activity and its effect on mitochondrial metabolism during brain aging

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Content C-JUN N-TERMINAL KINASE (JNK) MEDIATED INHIBITION OF
PYRUV ATE DEHYDROGENASE (PDH) ACTIVITY AND ITS EFFECT ON
MITOCHONDRIAL METABOLISM DURING BRAIN AGING

by

Qiongqiong Zhou
______________________________________________________________

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 2006

Copyright 2006 Qiongqiong Zhou

i
DEDICATION
To my beloved parents…

Guoyi Zhou and Yafei Wang
I hope one day that I can come back home and stay with you just like in those
old days…

To my dearest sister…
Bili Zhou
I wish you happy in every single day and night!

… …
To my darling husband…
Cheng Zhang
Love you forever!

ii

ACKNOWLEDGMENTS

Millions of thanks to my dearest professor Dr. Enrique Cadenas, he has given
me tremendous guidance and great encouragement during my graduate study.
Without his support, this thesis would have been impossible. Besides, his
kindness and generousness have set an excellent example for me, which will be
incredibly valuable in my future life and career. By his guidance and
encouragement, I grow up here today in research and science.

I would also like to express my sincere thanks to my dissertation committee: Dr.
Roberta Brinton and Dr. Tzung Hsiai. Their suggestions and enormous patience
are deeply appreciated. I am also indebted to Dr. Derick Han, Dr. Jon Nilson
and Dr. Shuhua Chen for their assistance in my experiment and research plan. I
also want to thank Dr. Robin Mockett for critical reading of the manuscript.

Finally, I deeply thank my parents, my sister and my dear husband for their love
and continuous support in my Ph.D. pursuit.
iii
TABLE OF CONTENTS
DEDICATION
ACKNOWLEDGMENTS
LIST OF FIGURES
ABSTRACT
INTRODUCTION TO THE DISSERTATION
1. Brain Aging
2. Increased oxidative stress may serve as a stimulus of JNK
activation during brain aging
3. Mitochondrial function and aging
4. Mitochondrial JNK signaling during brain aging and
age-related neurodegenerative disease

CHAPTER I.
JNK DIRECTLY TARGETS MITOCHONDRIA AND
REGULATES MITOCHONDRIAL PYRUV ATE
DEHYDROGENASE (PDH) ACTIVITY IN PRIMARY
CORTICAL NEURONS
Introduction
Materials and Methods
Results
Discussion

CHAPTER II.
JNK MEDIATES DECLINE OF PDH ACTIVITY DURING BRAIN
AGING
Introduction
Materials and Methods
Results
Discussion
Table 1: LC-MS/MS analysis of protein spot A, B and C from
Sypro-ruby stained 2D gel of mitochondrial proteins
SUMMARY

BIBLIOGRAPHY
ii
iii
v
vii
1
1
2
3
6
8
8
13
21
36

42

42
47
52
66

62

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77
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LIST OF FIGURES

FIGURE 1: Mitochondria pyruvate dehydrogenase, TCA cycle and ATP
production

FIGURE 2: Marker proteins and their activities in isolated rat brain
mitochondria

FIGURE 3: Anisomycin stimulates JNK activation in primary cortical
neurons

FIGURE 4: Active JNK associates with mitochondria in primary cortical
neurons

FIGURE 5: H
2
O
2
induces the rapid activation of JNK and its association with
mitochondria in cultured primary cortical neurons

FIGURE 6: Active JNK associates with the outer membrane of mitochondria

FIGURE 7: JNK-mediated phosphorylation of mitochondrial PDH

FIGURE 8: PDH was specifically phosphorylated and inhibited by incubation
with JNK

FIGURE 9: Regulation of PDH activity in primary cortical neurons by JNK

FIGURE 10: PDH inhibition mediated increase of lactic acid production and
decrease of ATP

FIGURE 11: JNK protein levels and activity in rat brain during aging

FIGURE 12: Mitochondrial association of JNK and its activity in rat brain
during aging

FIGURE 13: PDH activity declines with age in rat brain

FIGURE 14: Identification and localization of phosphorylated- or
non-phosphorylated-forms of PDH E1I subunit on the mitochondrial proteome
2D gel

4
16

21

23

25

28

30

33

34

35

53

56

58

61
v
FIGURE 15: Percentages of phosphorylated- and non-phosphorylated-forms of
PDH E1I subunit in rat brain during aging

FIGURE 16: Protein expression levels of PDH E1I subunit in brains from rats
of different ages

FIGURE 17: Age affects the levels of ATP, GSH and lactic acid in rat brain.

FIGURE 18: A summary of the JNK signaling-mediated inhibition of PDH
during brain aging
63

64

65

76

vi

ABSTRACT

Mitochondrial dysfunction has often been found to occur in brains with
advancing age or neurodegerative diseases. Recent work demonstrated that
upon activation, JNK translocates to mitochondria and regulates mitochondrial
functions in cultured cells. Using experimental models of cultured primary
neurons and isolated brain mitochondria, we found that: (i) both anisomycin, a
potent JNK activator, and hydrogen peroxide can induce the rapid activation of
JNK and its translocation to mitochondria in cultured primary cortical neurons,
(ii) mitochondria-associated active JNK can be degraded by Proteinase K,
which indicates that active JNK associates with the outer membrane of
mitochondria, and (iii) the association of active JNK with mitochondria causes
an increase of pyruvate dehydrogenase (PDH) phosphorylation and inhibition
of its activity, which results in an increase of lactic acid concentration and
decrease of ATP level in the cells. These results indicate that active JNK
might mediate communication between the cytosol and mitochondria,
regulating mitochondrial metabolism according to the cytosolic environment.
Using an aged rat model, we found that: (i) JNK activity and its translocation to
mitochondria were increased in the brain during aging, whereas PDH activity
was diminished, with increased phosphorylation of the PDH E1I subunit with
age in rat brains, and (ii) both ATP levels and GSH/GSSG ratios tended to
vii
decrease during aging, whereas concentrations of lactic acid were significantly
increased. These results indicate that PDH inhibition mediated insufficiency
of energy production and the availability of reducing molecules (GSH), that
acidosis may play an important role in brain aging, and that the inhibition of
PDH may be mediated by increased translocation of JNK to mitochondria
during aging. The studies acquire further significance, because mitochondrial
metabolic activity is critical to nervous system function. An abnormally high
level of JNK activity in the brain may cause severe inhibition of PDH, which
may contribute to the development of the pathological stages of many
neurodegenerative diseases.

1
INTRODUCTION OF THE DISSERTATION

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. The brain
is particularly susceptible to the effects of aging, because of its high metabolic
rate and relatively reduced capacity for cellular regeneration compared with
other organs (Andersen, 2004). During aging, there is a significant loss of
brain weight and volume, which may be caused by increasing neuronal
apoptosis triggered by mitochondrial damage and dysfunction (Pollack et al.,
2002), as well as by a reduced regeneration capacity of neurons, which are
highly differentiated post-mitotic cells. Because of its high metabolic rate, the
brain is particularly sensitive to mitochondrial dysfunction, as symptoms in
the brain usually appeared first when the mitochondrial respiratory chain is
inhibited by toxic compounds, such as rotenone, although those compounds
have universal effects on the whole body (Sherer et al., 2003). Many of the
most common and important neurodegerative diseases (Parkinson’s disease,
Alzheimer’s disease and Huntington’s disease) have been found to be
2
associated with deficiencies in mitochondrial metabolic enzymes, such as
pyruvate dehydrogenase (PDH) and some respiration complexes (Murphy,
2000).

2. Increased oxidative stress may serve as a stimulus of JNK activation
during brain aging

The decrease in brain neurological activities of normal aging is also closely
related to brain oxidative stress, which is increased during aging, as indicated
by increased amounts of oxidative stress markers, e.g.TBARS (thiobarbituric
acid reactive substances) and protein carbonyl content (Blass et al., 2000).
These changes in the cell redox status may serve as stimuli, thus activating
some stress-sensitive signaling cascades, such as the activation of MAPK(s),
specifically the c-Jun N-terminal kinase (JNK).

JNK, also known as stress activated protein kinase (SAPK), is a member of
the mitogen-activated protein kinase (MAPK) subfamily. JNK is activated in
response of a variety of cellular stress that include free radicals, oxidative
stress, heat shock, osmotic shock, protein synthesis inhibitors and
inflammatory cytokines (Bendinelli et al., 1996; Li and Jackson, 2002;
Stadheim and Kucera, 2002; Tournier et al., 2000); activation entails the
3
phosphorylation of its threonine and tyrosine residues at specific positions by
the upstream JNK kinases (JNKK, also called MAP kinase kinases, MKK),
Three major isoforms of JNK have been identified: JNK1 and JNK2 are
expressed ubiquitously, whereas the expression of JNK3 appears to be limited
to the brain, heart and testis (Gupta et al., 1996; Mohit et al., 1995). These
three JNK isoforms exhibit differences in specificity toward substrates and
binding proteins and in their regulation by upstream kinases and scaffold
proteins (Davis, 1999; Gupta et al., 1996).

JNK has been found to be activated by oxidative stress induced by a variety of
metabolites or pharmaceutical drugs in the mammalian brain or cultured
neurons. Because oxidative stress, a stimulus of JNK, increases in the brain
during aging, it is reasonable to surmise that the activity of JNK may also
increase during brain aging.

3. Mitochondrial function and aging

As powerhouses of the cell, mitochondria produce most of the energy for
cells. For oxidation of each glucose molecule, there are about 36 molecules of
ATP produced in mitochondria at variance with the modest energy (2ATP)
generated by the cytosolic pathway. Mitochondria use pyruvate
4
dehydrogenase (PDH) for oxidative decarboxylation of pyruvate to
acetyl-CoA, which is then completely oxidized to CO
2
through the
tricarboxylic acid cycle (TCA cycle). The reaction catalyzed by PDH is the
entry point of glucose metabolism into mitochondria. The NADH produced by
pyruvate decarboxylation and TCA cycle acts as electron donor to the electron
transport chain in the mitochondria: the electrons from NADH are
transported to oxygen by the proton-pumping electron transport chain, and the
backflow of the pumped protons results in ATP formation through the
mitochondrial ATP synthase (Nicholls, 2002) (see Fig. 1).

Figure 1 Mitochondria pyruvate dehydrogenase, TCA cycle and ATP production
Figure 1
5
Most biochemical studies of mammalian mitochondrial activity indicate a
decline in electron transport activity, and a decreased bioenergetic capacity
with age (Cortopassi and Wong, 1999). The mitochondrial alterations
observed during aging include an increased content of oxidation products and
a diminished function. The activities of mitochondrial nitric oxide synthase
(mtNOS), NADH dehydrogenase and cytochrome c oxidase all decline with
aging in the rodent brain.

PDH is an important multi-subunit enzyme complex located in the
mitochondria matrix, but its activity in relation to aging has not been
thoroughly explored. PDH

links glycolysis to the TCA cycle, and thereby
plays a central role in energy metabolism, especially when the TCA cycle in
the other hand is the major metabolic pathways for fuel molecule catabolism.
As a corollary, adequate electron flux through PDH is particularly important
in tissues with a high

ATP requirement, such as brain, when considering that
glucose is the sole fuel for the brain. During starvation and diabetes, PDH is
inactivated in many tissues to conserve three-carbon compounds (pyruvate,
lactate) for gluconeogenesis (Huang et al., 2003), which serves the purpose of
providing brain with its unique fuel. A decrease in PDH activity limits the
production of acetyl-CoA, thus resulting a decreased turnover of the TCA
cycle (Sugden and Holness, 2003). Alternative metabolic pathways, such as
6
those involving fatty acid and amino acid metabolism, are stimulated in an
attempt to produce acetyl-CoA; however, an energy deficit remains, especially
in the central nervous system, because the brain is more dependent on glucose
metabolism and there is no alternative source of acetyl-CoA from fat
metabolism in the brain. Genetic defects in PDH cause abnormal brain
development during the fetal stage (Mine et al., 2003), and patients with
neuromuscular diseases show a decreased rate of pyruvate oxidation (Ngo and
Barbeau, 1978). Furthermore, PDH activity is decreased by 41% in
Alzheimer’s disease and this PDH deficiency appears to contribute to the
progression of the disease rather than as a consequence(Butterworth and
Besnard, 1990; Perry et al., 1980; Sheu et al., 1985). However, very little
work has been contributed to the study of the age-related changes of PDH
activity.

4. Impact of mitochondrial JNK signal on brain aging and age-related
neurodegenerative diseases

JNK is considered to be a central signal transducer in neuronal death in the
mammalian brain (Herdegen and Waetzig, 2001b): activation of JNK
pathways enhances neuronal cell death in cultured primary neurons, in
knockout mice mole, JNK knockout protects against excitotoxicity, MPTP and
7
hypoxia (Hunot et al., 2004; Kuan et al., 2003; Mielke and Herdegen, 2000;
Yang et al., 1997), and the activity of JNK is significantly increased in the
brains of patients with Parkinson’s or Alzheimer’s diseases patients (Peng and
Andersen, 2003; Zhu et al., 2001). Neurodegeneration is also associated with
deficiencies in mitochondrial metabolic enzymes. For instance, deficiency of
PDH activity was found in many Alzheimer’s disease (AD) patients (Sheu et
al., 1985), and dysfunction of Complex I is also associated with Parkinson’s
disease (Shults, 2004). These observations suggest that an important
mechanism in neurodegeneration may be JNK regulation of mitochondrial
metabolic enzymes. Neurodegeneration may thus involve abnormal
phosphorylation of mitochondrial proteins, causing severe problems in energy
production in biological system, especially in the brain, an organ with
high-energy demands for its proper function.

Therefore, we hypothesized that age-dependent translocation of active JNK to
mitochondria regulates mitochondrial functions, which may contribute to the
development of mitochondrial damage inherent in the brain aging and
age-related neurodegenerative disorders. This hypothesis was addressed with
experimental models that assessed the effect of JNK on isolated rat brain
mitochondria, cultured primary cortical neurons, and in brains of rats of
different ages.
8
CHAPTER I

JNK DIRECTLY TARGETS MITOCHONDRIA
AND REGULATES MITOCHONDRIAL
PYRUVATE DEHYDROGENASE (PDH)
ACTIVITY IN PRIMARY CORTICAL
NEURONS

Introduction
c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein
kinase (MAPK) subfamily, is phosphorylated and activated in response to a
variety of cellular stresses that include free radicals, oxidative stress, heat
shock, osmotic shock, protein synthesis inhibitors, and inflammatory
cytokines (Bendinelli et al., 1996; Li and Jackson, 2002; Stadheim and Kucera,
2002; Tournier et al., 2000). Three major isoforms of JNK have been
identified. JNK1 and JNK2 are expressed ubiquitously, whereas the
expression of JNK3 appears to be limited to the brain, heart, and testis (Gupta
et al., 1996; Mohit et al., 1995). JNK was first identified by its ability to
phosphorylate the transcription factor c-Jun on two serine residues in the
9
NH
2
-terminal activation domain; this increases the transcription activity of the
AP-1 complex (Fos/c-Jun) (Gupta et al., 1996). Besides c-Jun, JNK can also
phosphorylate and activate other transcription factors, such as ATF2 and Elk-1
(Cavigelli et al., 1995; Gupta et al., 1995). In addition, a wide variety of
proteins, such as p53, Bcl-2, and the glucocorticoid receptor, are also
substrates of JNK (Fuchs et al., 1998; Giasson and Mushynski, 1997;
Maundrell et al., 1997; Rogatsky et al., 1998).

Recent studies have also demonstrated that active JNK may directly affect
mitochondrial functions through the phosphorylation of undefined substrates
in mitochondria. The direct association of JNK with mitochondria has been
observed in multiple myeloma cells and human U-937 cells (Ito et al., 2001;
Kharbanda et al., 2000). Kharbanda et al. observed that ionizing radiation
exposure induces translocation of JNK to mitochondria and association of
JNK with the anti-apoptotic Bcl-x
L
protein (Kharbanda et al., 2000).
Previous work from this lab showed that recombinant active JNK caused
phosphorylation of Bcl-2, Bcl-x
L
and several other unidentified mitochondrial
proteins in isolated rat brain mitochondria, but along with the release of
cytochrome c and a partial collapse of the mitochondrial membrane potential
(Schroeter et al., 2003). The fact that JNK causes release of cytochrome c in
isolated brain mitochondria indicates that active JNK can directly regulate
10
mitochondrial function without the synthesis of new proteins or through the
regulation of gene transcription. Studies with nerve growth factor
(NGF)-deprived neurons also suggest that JNK may also participate in the
regulation of metabolism in mitochondria. Inhibition of the JNK signaling
pathway prevented the decline in protein synthesis, mitochondrial
dehydrogenase activities, and glucose uptake caused by NGF-deprivation and
JNK inhibition supported neuronal growth in the absence of trophic support
(Harris et al., 2002). However, the mechanism by which JNK modulates
mitochondrial function has not yet been elucidated.

Emerging evidence indicates that reversible phosphorylation, the most
prevalent form of cellular post-translational modification, is an important
mechanism in the regulation of mitochondrial functions (Pagliarini and Dixon,
2006). Mitochondria contain enzymes important in metabolism and energy
production, some of which are regulated by phosphorylation or
dephosphorylation. For instance, phosphorylation of NDUFS4, a subunit of
complex I, increases complex I activity (Papa et al., 2002), while the activity
of pyruvate dehydrogenase (PDH) is inhibited by the phosphorylation of its
E1I subunit. Based on the facts that (i) JNK directly targets mitochondria, (ii)
JNK regulates mitochondrial pyruvate dehydrogenase and (iii) some
mitochondrial metabolic enzymes can be regulated by phosphorylation, it may
11
be surmised that JNK, as a kinase, may potentially regulate mitochondrial
metabolism through the phosphorylation of some metabolic enzymes in
mitochondria. The identification of mitochondrial proteins regulated by JNK
may contribute to understanding the mechanism by which JNK modulates
mitochondrial function.

JNK plays an important role in neuronal death in many neurodegenerative
diseases. The activity of JNK is significantly increased in the brain of patients
with Parkinson’s or Alzheimer’s diseases (Peng and Andersen, 2003; Zhu et
al., 2001). Neurodegeneration is also associated with deficiencies in
mitochondrial metabolic enzymes. For instance, deficiency of PDH activity
was found in Alzheimer’s disease (AD) patients (Sheu et al., 1985) and
dysfunction of Complex I is associated with Parkinson’s disease (Shults,
2004). These observations suggest that an important mechanism in
neurodegeneration may be JNK regulation of mitochondrial metabolic
enzymes. Neurodegeneration may thus involve abnormal phosphorylation of
mitochondrial proteins, causing severe problems in energy production in
biological systems, especially in the brain, an organ that demands high energy
levels for its proper function.

12
The purpose of this study was to clarify the potential biological role of JNK
signaling in the regulation of mitochondrial functions, which may be an
important factor in neurodegenerative diseases. To address this question, we
studied the effect of active JNK on isolated brain mitochondria and in cultured
primary cortical neurons. In this study, we examined whether (i) JNK directly
targets mitochondria in neurons, (ii) there are other target proteins for JNK,
besides Bcl-2 and Bcl-x(L), within mitochondria, and (iii) mitochondrial
metabolism is regulated by JNK in neurons.
13
Materials and Methods

Materials
Recombinant active and inactive JNK1 were purchased from Upstate
Biotechnology (Waltham, MA). Active PKA was bought from Novagen
(Madison, WI) and active PKC was purchased from Calbiochem (La Jolla,
CA). [r-
32
P] ATP was bought from MP Biomedicals (Irvine, CA).
SYPRO
TM
Ruby protein gel stain and Mitotracker Red were obtained from
Molecular Probes (Eugene, OR). Antibodies to JNK1, JNK2 and pJNK
(phosphorylated JNK, the active form) were bought from Santa Cruz Biotech
(Santa Cruz, CA). All other chemicals or reagents were obtained from
Sigma-Aldrich (St Louis, MO).

Primary culture and isolation of mitochondria from cells
Primary neuron cultures were prepared from the cortex of Fisher 344 E18 rat
fetuses using a previously published procedure (Pongrac and Rylett, 1998).
After 10 days in culture, neurons were treated with or without anisomycin for
15 min or H
2
O
2
for 30 min to activate JNK. In order to obtain mitochondrial
protein, a mitochondria isolation kit (Pierce, Rockford, IL) was used to isolate
mitochondria from primary cultured neurons. Proteins extracted from neurons
14
or mitochondria were then subjected to immunoblotting with anti-JNK1,
anti-JNK2 or anti-pJNK antibodies.

Immunocytochemistry
Neurons were cultured on poly-d-lysine-coated two-well chamber slides for
7–10 days prior to experimental treatments. Neurons were first incubated
with MitoTracker Red (100 nM) for 10 min. Then neurons were exposed to
anisomycin (25 µg/ml) for 15 min or H
2
O
2
(100 µM for 30 min). Neurons
were then washed with PBS and fixed with 95% methanol for 5 min at 4°C.
Nonspecific antibody binding was blocked by incubation with 1% normal
horse serum (Vector; Burlingame, CA). The neurons were then incubated with
monoclonal anti-phosphor-JNK antibody (Santa Cruz, CA) overnight at 4°C.
After washing in PBS, the neurons were incubated in fluorescein-conjugated
horse-anti-mouse secondary antibody (Vector; Burlingame, CA) for 30 min at
RT. Slides were examined using a Zeiss LSM-510 laser scanning confocal
microscope (Carl Zeiss Inc.; Thormwood, NY) in USC Doheny Eye Institute.
Fitc images were captured using a 488 nm Argon laser with a 505-530 nm
barrier filter. Rhodamine images were captured using a 543 nm
Helium-Neon laser with a 560 nm barrier. A plan-neofluar x40 (N.A. 0.75) oil
immersion objective lens was used for imaging of the fluorescently labeled
cells.
15
Isolation of rat brain mitochondria
Whole brain mitochondria were isolated from adult male Wistar rats. The
brains were excised, rinsed in ice-cold isolation buffer, pH 7.4, containing
sucrose (250 mM), Hepes (20 mM), EDTA (1 mM), EGTA (1 mM),
dithiothreitol (DTT; 1 mM), protease inhibitor cocktail (sigma-Aldrich; St
Louis, MO) 100 Sl per brain, using a Dounce homogenizer to give a 5% (w/v)
homogenate. Non-synaptosomal mitochondria were isolated by differential
centrifugation followed by discontinuous Percoll density-gradient
centrifugation, according to procedures described previously (Anderson and
Sims, 2000). Briefly, the homogenate was centrifuged at 1330 g for 5 min to
remove nuclei and cell debris. The supernatant was centrifuged at 21,200 g
for 10 min, and the resultant pellet was resuspended in 15% Percoll and
centrifuged at 21,200 g for 10 min to remove the fat. Then, the loose pellet
was layered onto a preformed discontinuous gradient of 23%/40% Percoll.
The gradient was centrifuged at 31,000 g for 5 min. Mitochondrial fractions
were collected and washed with isolation buffer 2 times. The resulting
mitochondrial pellet was resuspended in isolation buffer containing 0.5% BSA
and then washed twice in BSA-free isolation buffer.

The purity of brain mitochondria was assessed by monitoring the presence of
the marker proteins and measuring the amounts of enzymatic activity (Fig. 2).
16
Cytosolic contamination was assessed by monitoring for the cytosol marker
protein -actin, which was absent in mitochondrial fractions when assessed by
immunoblot. Microsomal contamination was assessed by measuring the
activity of microsomal marker protein NADPH-Cytochrome P450 reductase,
using the method of Swanson (Swanson, 1950). The viability and integrity
of mitochondria from brain tissue was determined based on the respiratory
control ratio, i.e. R.C.R, the ratio of ADP-stimulated (state 3) respiration to
resting (state 4) respiration. An R.C.R value of approximately 5 was
obtained routinely, when it was measured using either succinate with rotenone,
or glutamate as substrate.

Fig. 2
A B
0
2
4
6
8
10
Crude
Homogenate
Mitochondria
Figure 2. Marker proteins and their activities in isolated rat brain mitochondria
(A) Equal amount of protein (30 µg) from crude homogenate (CH) and mitochondrial
fraction (MF) were subjected to SDS gel electrophoresis, followed by immunoblot with
an anti--actin (cytoskeletal marker). (B) The activities of NADPH-cytochrome P450
reductase (microsomal marker) were determined by measuring the reduction speed of
cytohrome c in 0.3 M phosphate buffer (pH 7.8), containing 75 µM cytochrome c and 0.1
mM NADPH.
-actin
CH
MF
17
In vitro kinase assay
Intact mitochondria (100 µg) were apportioned into mitochondrial kinase
buffer (MKB), containing isolation buffer supplemented with ATP (0.6 mM)
or [r-
32
P] ATP (10 uCi/vial; 6000 Ci/mmol), beta-glycerophosphate (25 mM)
and MgCl (10 mM), and energized with succinate (5 mM). Mitochondria were
treated with 0.3 µg of recombinant active JNK1 or 0.3 µg of recombinant
inactive JNK1 incubated for 30 min at 37°C under gentle agitation. Following
the incubation and washing steps, mitochondria were collected by
centrifugation (6000 g, 3 min), and prepared for two-dimensional isoelectric
focusing (2D IEF)/SDS-PAGE, proteinase K treatment or PDH enzyme
activity assay.

Proteinase K treatment of mitochondria
Mitochondria from in vitro kinase assay or isolated from stimulated neurons
were resuspended in isolation buffer at 1 mg/ml and treated with 50 µg/ml of
proteinase K at 4ºC for 20 min. PMSF (2 mM) was added to terminate the
reaction and mitochondria were collected by centrifugation.

2D gel and mass spectrometric analysis
Mitochondrial proteins were separated based on their isoelectric points on
precast gel

strips (17 cm) with a linear gradient of pH 3–10 from Bio-rad
18
(Hercules, CA), using the Bio-rad Protean IEF System. Mitochondrial

sample
(100 µg) was solubilized in the rehydration buffer

(6 M urea, 2 M thiourea,
2% Nonidet P-40, 2% IPG buffer (pH

3–10), and 0.1 M dithiothreitol) for 30
min

at room temperature before the rehydration of the strips. The immobilized
pH gradient

(IPG) strips were rehydrated overnight in rehydration buffer with
dissolved mitochondrial protein. The program utilized was the following:
250 V rapid voltage ramping for 30 min, 10,000 V slow voltage ramping for
60 min, 10,000 V rapid voltage ramping for 50KVhrs. The strips were
incubated first in Equilibration buffer I with 6

M urea, 20% glycerol, 2% SDS,
2% DTT, and 0.375 M Tris (pH 8.8) for 10 min at room temperature, then in
Equilibration buffer II with 6

M urea, 20% glycerol, 2% SDS, 2%
iodoacetamide, and 0.375 M Tris (pH 8.8). They were then

loaded onto 10%
SDS-PAGE gel and run at 50 V overnight. The gels were stained with
SYPRO
TM
Ruby protein gel stain first, then exposed to film for 2 days.

Finally, the [r-
32
P] labeled protein was excised from the gel (according to the
spot on the film), and sent for LC-MS/MS (liquid chromatographic mass
spectrometric) sequence analysis at the USC Proteomics Core Facility.

SDS-PAGE gel and immunoblot
Mitochondria was lysed in RIPA buffur (Tris-HCl: 50 mM, NP-40: 1%,
Na-deoxycholate: 0.25%, NaCl: 150 mM, EDTA: 1 mM, pH 7.4), then
19
loading buffer was added and the protein was heated for 5 min at 95°C.
After cool down, protein was loaded 50 µg/well and resolved in 12%
SDS-PAGE gels. For autoradiography, the gels were fixed, dried and
exposed to film over night. For immunoblot, the proteins on the gels were
transferred to nitrocellulose membrane, blocked and detected by antibodies at
concentrations indicated by the manufacturers.

PDH enzyme activity assay
PDH activity was assayed at 37 °C by measuring the reduction of NAD
+
at
340 nm upon the addition of 0.5 mM NAD
+
, 200 µM TPP (thiamine
phosphate), 40 µM CoASH, and 4.0 mM pyruvate to 50 µg/mL mitochondrial
proteins. The assay was done in the presence of 2.5 µM rotenone to prevent
NADH consumption by complex I. Mitochondria was sonicated (30 s, setting
of 3.0, 100% pulse rate, VWR Scientific) in a buffer containing 35 mM
KH
2
PO
4
, 5.0 mM MgCl
2
, 2.0 mM NaCN, 0.5 mM EDTA, 0.25% Triton X100
and 1 X phosphatase inhibitor at pH 7.25 (Humphries and Szweda, 1998).

Lactic acid and ATP concentration measurements
Cells were treated with 25 µg/ml of anisomycin in fresh medium for 15 min.
Medium was collected, acidified by adding an equal volume of perchloric acid
(2 M) and centrifuged for 10 min at 12,000 g. The supernatant was neutralized
20
with KHCO
3
(3 M) and recentrifuged at 12,000 g. of Extract (100 µl) was
added to 500 µl of reaction buffer and the concentration of lactic acid was
measured using a lactic acid assay kit (r-Biopharm, Germany). Cells were
lysed by perchloric acid (2 M), and neutralized as described above. ATP
concentration in the cell extract was determined by using ATP determination
kit (Molecular Probes, OR).

Methyl thiazolyl tetrazolium (MTT) cell viability assay
Neurons were cultured in 6-well plates. After treatment, neurons were
incubated with 0.5 mg/ml of MTT for 90 min at 37°C. Then, cells were
lysed in DMSO and the absorption was measured using a spectrophotometer
(Bio-rad; Hercules, CA) at a wavelength of 490 nm.

Statistical analysis
Data are reported as means ± S.D. of at least three independent experiments.
Significant differences between mean values were determined by the student
t-test. Means were considered to be statistically distinct if P < 0.05.

21
Results

Anisomycin, a potent JNK activator, induces the rapid activation and
translocation of JNK to mitochondria in cultured primary cortical
neurons

Although the translocation of active JNK to mitochondria has been shown in
multiple myeloma cells and human U-937 cells (Ito et al., 2001; Kharbanda et
al., 2000), little work has been done to show this process in primary cultured
cortical neurons. To determine if JNK directly targets mitochondria, primary
cultured neurons were supplemented with anisomycin, a classic JNK activator
p pJ JN NK K
p46
JNK1
-Actin
p46
p54
p46
Anisomycin
Control 10 Ag/ml 25 Ag/ml 50 Ag/ml
Figure 3. Anisomycin stimulates JNK activation in primary cortical neurons
Primary neurons were treated with anisomycin or vector control for 15 min, as described
in the Methods section. Total cell lysate (30 µg per lane) was subjected to
immunoblotting with anti-JNK1, anti-JNK2 or anti-pJNK antibodies. -actin was
included as a loading control.
Fig. 3
JNK2
p54
22
(Stadheim and Kucera, 2002). This treatment led to a rapid activation of JNK
(mostly the p46 form), evident at a dose of 10 µg/ml for 15 min (Fig. 3),
without affecting total JNK protein levels.

To assess the subcellular distribution of active JNK upon anisomycin
treatment, mitochondrial proteins were isolated from neurons and equal
amount of proteins were subjected to immunoblot analysis with anti-pJNK
antibody. Figure 4A shows that anisomycin treatment induced a greater
association of active JNK with mitochondria. Equal loading of the lanes was
confirmed by immunoblot analysis of the mitochondrial cytochrome c oxidase
(COX).
The translocation of active JNK to mitochondria was further confirmed by
immunocytochemistry (Fig. 4B): green fluorescence represents antibody
against pJNK, red fluorescence represents mitochondria labeled using
Mitotracker Red, and yellow fluorescence represents the co-localiztion of
pJNK and mitochondria; the later indicates that following anisomycin
treatment, JNK becomes activated and is translocated to mitochondria.

The cellular distribution of active JNK was also determined by comparing
total cellular proteins and mitochondrial proteins using immunoblots against
23
pJNK. Figure 4C shows that active JNK mainly locates in the mitochondrial
fraction. A Coomassie blue stained protein gel indicates the equal amounts of
total cell lysate and mitochondrial proteins.

Control Anisomycin
Control
Anisomycin
Mitotracker Red
pJNK
Merge
pJNK
COX
B
A
Fig. 4
24
Mitochondira
Anisomycin
pJNK
Total cell lysate
Mitochondira
Total cell lysate
Mitochondira
Control
Figure 4. Active JNK associates with mitochondria in primary cortical neurons
(A) Mitochondria were isolated from neurons that were treated with anisomycin (25 µg/ml)
for 15 min or vector control. Mitochondrial proteins were subjected to immunoblotting
with anti-pJNK antibody. Cytochrome c oxidase (COX) immunoblotting was performed
as a control for equal loading. (B) Neurons were immobilized on slides, fixed, and
incubated with anti-pJNK antibody followed by fluorescein conjugated secondary antibody.
Mitochondria were stained with the mitochondrial-selective permeant dye Mitotracker Red
(100 nM). Green signal, pJNK; red signal, Mitotracker Red; yellow signal, co-localization
of pJNK and mitochondria signals. (C) Equal amounts of mitochondrial protein and total
cell lysate were subjected to immunoblot against pJNK. Equal loading was indicated by
Coomassie staining.

Fig. 4 (continued)
C
25
H
2
O
2
induces the rapid activation of JNK and association with
mitochondria in cultured primary cortical neurons
Primary cortical neurons supplemented with H
2
O
2
, another JNK activator,
showed JNK activation in a dose-dependent manner of H
2
O
2
(Fig. 5A).
Mitochondria isolated from primary cortical neurons that had been treated
with H
2
O
2
showed an increase association of active JNK with mitochondria
(Fig. 5B). To further confirm JNK association with mitochondria following
H
2
O
2
treatment, immunocytochemistry was also employed. Figure 5C shows
that the treatment with 100 SM H
2
O
2
for 30 min caused JNK activation and
the co-localization of active JNK and mitochondria, as seen by the yellow
fluorescence.

pJNK
-Actin
H
2
O
2
0AM 25AM 50AM 100AM
Total cell lysis
pJNK
H
2
O
2
0AM 100 AM
COX
Isolated mitochondria
Fig. 5
B
A
26
Figure 5. H
2
O
2
induces the rapid activation of JNK and its association with
mitochondria in cultured primary cortical neurons
(A) Primary cultured neurons were incubated with different doses of H
2
O
2
for 30 min. Total
cell lysisates were collected and pJNK was detected by immunoblot. -actin: loading
control. (B) Mitochondria were isolated from neurons treated with or without H
2
O
2
(30
min), and then subjected to immunoblot using antibodies against pJNK or COX (loading
control). (C) Neurons were immobilized on slides, fixed, and incubated with anti-pJNK
antibody followed by fluorescein conjugated secondary antibody. Mitochondria were
stained with the mitochondrial-selective permeant dye Mitotracker Red (100nM). Green
signal, pJNK; red signal, Mitotracker Red; yellow signal, co-localization of pJNK and
mitochondria signals.
C
Control
pJNK
Mitotracker
Red
100AM H
2
O
2
Merge
Fig. 5 (continued)
27
Active JNK associates with the outer membrane of mitochondria
Because active JNK was translocated to mitochondria in primary cortical
neurons, the localization of JNK within mitochondria was explored.
Establishing the localization of JNK within mitochondria may provide a
mechanistic understanding of the regulatory role of JNK in mitochondria.
Mitochondria, isolated from anisomycin-treated neurons, were incubated with
proteinase K, which digests proteins on the outer surface of the outer
mitochondrial membrane. Proteins within the outer mitochondrial membrane
will be protected and not subject to proteinase K digestion. Following
proteinase K treatment, mitochondrial proteins were subjected to immunoblot
analysis with antibodies against pJNK. Figure 6A shows that active JNK,
which was associated with mitochondria, was digested by proteinase K
treatment, indicating that the active JNK was localized on the outer surface of
the outer mitochondrial membrane. Similarly, recombinant active JNK,
which associates with isolated brain mitochondria, was found to be digested
by proteinase K treatment (Fig. 6B). The equal amounts of cytochrome c
indicate in the proteinase K-treated and untreated samples that the integrity of
mitochondria was not affected by the proteinase K treatment. Taken together,
these results demonstrate that JNK only associates with mitochondria and
does not cross the outer mitochondrial membrane.

28
B
Cytochrome c
Cytochrome c
A
Fig. 6
pJNK
Proteinase K +
Proteinase K +
Figure 6. Active JNK associates with the outer membrane of
mitochondria
(A) Mitochondria isolated from primary cortical neurons supplemented with
anisomycin (25 µg/ml, 15min), were treated with proteinase K (50 µg/ml)
for 20 min on ice. Mitochondria were then subjected to immunoblotting
with anti-pJNK antibody. (B) Rat brain mitochondria were incubated with
active JNK1 followed by proteinase K treatment (50 µg/ml) for 20 min on
ice. Mitochondrial association of JNK1 was established by immunoblot
analysis with anti-JNK1 antibody. Cytochrome c oxidase levels were also
determined as a control, to ensure that proteinase K did not digest proteins
inside the mitochondria.
29
JNK-mediated phosphorylation of mitochondrial PDH
It has been reported that active JNK can directly regulate mitochondrial
function by phosphorylation of Bcl-2 family proteins (Kharbanda et al., 2000;
Schroeter et al., 2003). In our previous study, we found that in addition to
phosphorylation of Bcl-2 family proteins, active JNK treatment of isolated
mitochondria caused phosphorylation of many unidentified proteins
(Schroeter et al., 2003). Further identification of these unknown proteins
that are phosphorylated by JNK signaling in mitochondria could provide us
with a better understanding of JNK modulation of mitochondrial functions. To
identify the phosphorylated proteins, isolated brain mitochondria were
incubated with active JNK1 in the presence of [X-
32
P] ATP. The
mitochondrial proteins were then resolved in 2D IEF/SDS gels (Fig. 7A).
Protein spots A and B, which showed increased phosphorylation upon JNK
treatment (Fig. 7B), were identified as the PDH E1I subunit by MS/MS.
Multiple phosphorylation of the PDH E1I subunit resulted in two protein
spots on the IEF/SDS-PAGE gel, due to a change in the isoelectric point (pI)
of PDH E1I subunit caused by phosphorylation.

To exclude the possibility that mitochondria were broken during JNK
incubation, broken mitochondria were also incubated with active JNK. 2D
gel results showed that JNK did not cause PDH phosphorylation in broken
30
mitochondria (data not shown), confirming that the phoshorylation of PDH
was not caused by direct phosphorylation of PDH by JNK in broken
mitochondria. Moreover, the data suggested that intact and functional
mitochondria are required for JNK signaling in mitochondria.

kDa
PI [IEF]
131
78
39
30
18
3 10
A B
A
Fig. 7
31
PI[IEF]
A
B
3 10
kDa
131
39
30
18
PI[IEF]
kDa
131
39
30
18
78
78
A B
Control
Fig. 7 (continued)
Active JNK
Figure 7. JNK-mediated phosphorylation of mitochondrial PDH
Rat brain mitochondria (100 µg in 100 µl) were incubated with active JNK1 (0.3 µg) in the
presence of [X-
32
P] ATP, subjected to 2D IEF/SDS/PAGE and analyzed by autoradiography.
(A) The protein gel was stained by SYPRO
TM
Ruby gel stain. Protein spots A and B with
increased phosphorylation by active JNK1 (see Fig. 7B) were both identified as the PDH
E1I subunit by MS/MS. (B) Representative autoradiogram obtained using inactive JNK1
as control and active JNK1 as treatment.
B
32
Mitochondrial PDH is specifically phosphorylated and inhibited by active
JNK
In order to confirm that PDH phosphorylation of PDH was specific to JNK,
the ability of other active serine-threonine kinases to cause PDH
phosphorylation was tested on isolated brain mitochondria. Figure 8A shows
that only JNK can significantly increase the phosphorylation of PDH E1I
subunit.

PDH consists of three enzymes organized into a high-molecular-mass
complex: pyruvate E1I subunit dehydrogenase (E1), dihydrolipoamide
transacetylase (E2) and dihydrolipoamide dehydrogenase (E3). It has been
well documented that PDH E1I subunit can be phosphorylated at three serine
sites. Phosphorylation of each of these three sites can inhibit PDH activity
(Holness and Sugden, 2003). To further study the consequence of PDH
phosphorylation mediated by JNK, the enzyme activity of PDH was measured
following incubation with active JNK1 and other kinases. PDH activity was
significantly inhibited (by ~20%) after the treatment with active JNK1 (Fig.
8B).

JNK3, another isoform of JNK, is selectively expressed in the nervous system,
which has been suggested to have a preferential role in neuronal apoptosis and
33
neurodegeneration. In the kinase assay with isolated brain mitochondria,
recombinant active JNK3 was also tested and found to have the same effect as
JNK1 on PDH phosphorylation and activity inhibition (data not shown).

*
*
0
20
40
60
80
100
120
Control Active JNK PKA PKC
Average PDH activity
(nmol/mg/min)
*
Fig. 8
B
A
Figure 8. PDH was specifically phosphorylated and inhibited by incubation with JNK
(A) Rat brain mitochodria were incubated with different active kinases (JNK1, PKA, PKC),
with equal amounts of kinase activities, in the presence of [X-
32
P] ATP. Proteins were
washed and subjected to SDS-PAGE and analyzed by autoradiography. (B) Mitochondrial
proteins were collected after treatment with different kinases, and PDH activity was
measured under the conditions described in the Methods section. (“*”: P<0.05; error bars:
Standard Deviation (S.D.), n ] 4)
0
0.5
1
1.5
2
2.5
3
Control Active JNK1 PKA PK C
PDH phosphorylation
34
Regulation of PDH activity in primary cortical neurons by JNK
PDH activity was measured in mitochondria isolated from neurons treated with
anisomycin to activate JNK. Figure 9 shows that after treating the neurons
with anisomycin, the PDH activity was almost completely inhibited. However,
pretreatment of neurons with the JNK inhibitor, SP600125, essentially
abolished this inhibition of PDH activity (Fig. 9). The fact that SP600125
prevented a decrease in PDH activity established a strong link between JNK
and regulation of PDH activity. MTT assay of the neurons shows that cells
were not undergoing apoptosis with anisomycin treatment, indicating that the
inhibition of PDH activity was not a consequence of apoptosis (data not
shown).

0
20
40
60
80
100
120
Control Aniso Aniso+SP600125
Average PDH activity
(nmol/mg/min)
Fig. 9
Figure 9. Regulation of PDH activity in primary cortical neurons by JNK
Primary neurons were pretreated with or without SP600125 (2 µM, 30 min) and then
incubated with anisomycin (25 µg/ml, 15 min). Mitochondria were isolated and PDH
activity was measured by the method as described in the Methods section. (“*”: P<0.05;
error bars: Standard Deviation (S.D.), n ] 4)
35
Impact of PDH inhibition on cellular metabolism
The inhibition of PDH activity blocks the entry of pyruvate into the TCA
cycle, but leads to pyruvate convertion to lactic acid by lactate dehydrogenase,
which results in accumulation of lactic acid and decrease of energy production.
In order to test this effect of PDH inhibition in the present study, lactic acid
concentration and ATP levels were measured after the cells were treated with
anisomycin. There was an increase (~25%, n=6) of lactic acid in the medium
and decrease (~15%, n=10) of ATP in the cells after 15 min treatment of
anisomycin, but these changes were not observed in the cells pre-treated with
SP600125 (Fig. 10).

0
0.25
0.5
0.75
1
1.25
1.5
ATP L a ctic Acid
Control
Anis o
Anis o+SP
Relative Levels of ATP\lactic acid
*
*
*
Figure 10. PDH inhibition mediated increase of lactic acid production and decrease of
ATP
Primary neurons were pretreated with or without SP600125 (2 µM, 30 min) and then
incubated with anisomycin (25 µg/ml, 15 min). Medium was collected for the measurement
of lactic acid and cells were lysed for the assay of ATP concentration, as described in the
Methods section.
Fig. 10
36
Discussion

In this work, it was shown that treatment of neurons with anisomycin or H
2
O
2
activated JNK and caused JNK translocation to mitochondria. The activation
of JNK by anisomycin requires the participation of MLK7 (Wang et al., 2005),
a JNK upstream kinase, while the activation of JNK by H
2
O
2
is most likely
mediated through the ASK-1/MKK4/6 pathway (Nemoto et al., 2000).
Although JNK was likely activated by different mechanisms, once activated,
JNK was found to translocate to mitochondria following either anisomycin or
H
2
O
2
treatments. This is in agreement with previous studies that showed
translocation of JNK to mitochondria following UV exposure or phorbol ester
treatment (Ito et al., 2001; Kharbanda et al., 2000). In addition, we observed
that active JNK was able to associate with isolated mitochondria following a
brief incubation in vitro. These results suggest that once activated, JNK
associates with mitochondria, regardless of the initial stimulus. It is reasonable
to predict that activation of JNK probably causes a conformational change in
the protein, which allows JNK to interact with various proteins on the outer
mitochondrial membrane. Mitochondrial proteins, such as Sab and Bcl-x
L
,
have been reported to interact with JNK (Kharbanda et al., 2000; Wiltshire et
al., 2002).

37
Proteinase K treatment revealed that active JNK only associated with the outer
membrane of the mitochondria and did not cross into the intermembrane space.
This association of JNK with the outer membrane of mitochondria has several
advantages as a mechanism for the mitochondrion to adjust its functions to
changes in the cytosolic environment: a) transporting a protein from the
cytosol into mitochondria requires unfolding, transportation, and refolding
processes, which may delay the reaction time and result in loss of kinase
activity, b) most of JNK upstream kinases are located in the cytosol, and upon
activation by its upstream kinases in the cytosol, JNK can easily and quickly
associate with the outer membrane of mitochondria, and c) although there
are phosphatases in mitochondria, there is little evidence suggesting that they
can dephosphorylate JNK. Upon association with the outer membrane of
mitochondria, JNK is still available to the phosphatase in the cytosol.
Overall, the localization of JNK at the outer membrane of the mitochondria
allows active JNK to function as a bridge between the cytosol and
mitochondria and provides mitochondria with information about the cytosolic
environment in real time.

The in vitro kinase assay followed by 2D gel electrophoresis, revealed that
several mitochondrial proteins were phosphorylated, directly or indirectly, by
active JNK. The protein with the strongest signal was the PDH E1I subunit, a
38
mitochondrial matrix protein. However, because active JNK only seems to
associate with the outer membrane of mitochondria, this suggests that JNK
must initiate a signal cascade that can cross the mitochondrial membranes and
affect matrix proteins. How JNK modulates signal pathways across the outer
and highly impermeable inner membrane to the matrix remains unknown.
Second messengers may play a key role in signal transduction across
mitochondrial membranes by JNK. One possible signaling pathway modulated
by JNK to alter PDH activity could be mediated through a change of calcium
level in mitochondria. Calcium has been shown to upregulate PDH activity by
inhibiting pyruvate dehydrogenase kinase (PDK) and activating PDH
phosphatase (Holness and Sugden, 2003). Our previous work showed that the
association of active JNK with mitochondria causes a decrease of mitochondrial
membrane potential, a driving force for calcium transport across the inner
mitochondrial membrane (Cortassa et al., 2003; Dougherty et al., 2004). The
decrease of membrane potential may lead to a lower calcium level in the
mitochondrial matrix, resulting in increased phosphorylation of PDH by
activation of PDK and inhibition of PDH phosphatase. Another possible
mechanism is that JNK may be directly regulating calcium channels on the
mitochondrial membrane by phosphorylating calcium channel subunits and
causing the efflux of calcium from mitochondria. A recent study (Margineantu
et al., 2002) reveals a discrete, heterogeneous distribution of PDH complexes in
39
the matrix of mitochondria, which suggests that other mitochondrial matrix
enzymes may also have a discrete distribution, and together with PDH, form a
metabolic compartmentalized unit in the matrix. If other metabolic enzymes are
closely related, JNK signaling may also regulate other metabolic enzymes in
mitochondria. The exact mechanism by which JNK transduces signals in
mitochondria needs be further explored.

Previous studies have shown that three serine sites on PDH E1I subunit can be
phosphorylated and inhibit PDH activity (Holness and Sugden, 2003). Our
results demonstrated that PDH phosphorylation by JNK corresponded with an
inhibition of PDH activity in isolated mitochondria and in cells. The inhibition
of PDH following JNK activation in cells was more effective than JNK induced
inhibition of PDH in isolated brain mitochondria. This suggests that association
of JNK with mitochondria and regulation of mitochondrial function by JNK
requires other cofactors that were not present in the in vitro system.

Taken together, our findings imply that an abnormally high level of JNK
activity in the brain may cause severe inhibition of PDH, leading to decreased
energy production. The inhibition of PDH by JNK and consequent decrease in
energy production may play an important role in the pathogenesis of many
neurodegenerative diseases. PDH

links glycolysis to the TCA cycle, the
40
biochemical pathway responsible for most NADH and FADH
2
generation in
the cells. As a consequence, PDH activity is particularly important in tissues
with a high

ATP requirement, such as brain. A decrease in PDH activity limits
the production of acetyl-CoA, thus resulting in a decreased turnover of the
TCA cycle (Sugden and Holness, 2003). Alternate metabolic pathways, such
as those involving fatty acid and amino acid metabolism, are stimulated in an
attempt to produce acetyl-CoA; however, an energy deficit generally remains,
especially in the central nervous system, because the brain is more dependent
on glucose metabolism, and there are no alternative sources of acetyl-CoA
from fat metabolism in the brain. Genetic defects in PDH causes abnormal
brain development during the fetal stage (Mine et al., 2003), and patients with
neuromuscular diseases show a decreased rate of pyruvate oxidation (Ngo and
Barbeau, 1978).

PDH was reported to be deficient in the Alzheimer’s brain (Perry et al., 1980),
which has subsequently been confirmed in at least three other laboratories
(Butterworth and Besnard, 1990; Sheu et al., 1985; Yates et al., 1990), with no
conflicting reports. Deficiency of PDH activity not only occurs in regions of
the brain that are neuropathologically damaged in AD, but also in regions that
are histopathologically normal, suggesting that the abnormalities in PDH
activity are more likely part of the disease process rather than consequences of
41
tissue damage (Butterworth and Besnard, 1990; Perry et al., 1980; Sheu et al.,
1985; Yates et al., 1990). However, no evidence has been reported for
abnormalities in the genes encoding the components of PDH in AD. These
observations support the notion that post-translational modifications, such as
phosphorylation, of PDH proteins may be the major cause of its activity
inhibition in the progress of AD. Because JNK activity has been shown to be
increased in AD, our work suggests that increased JNK activity may be
responsible for decreases in PDH activity observed in AD.

In summary, the data obtained in this study with isolated rat brain
mitochondria, using active recombinant JNK, and cultured primary cortical
neurons, supports the hypothesis that JNK directly targets mitochondria and
regulates mitochondrial metabolism by inhibiting PDH. An abnormally high
level of JNK activity in the brain may cause severe inhibition of PDH, which
may contribute to the development of the pathological stages of many
neurodegenerative diseases.
42
CHAPTER II

JNK MEDIATES THE DECLINE OF PDH
ACTIVITY DURING BRAIN AGING

Introduction

Aging is a process marked by a general decline of physiological functions,
including a pronounced effect on brain activities, such as neuromuscular
coordination, cognitive performance and environmental awareness. The
decrease in these neurological activities during normal aging has been found
to be directly related to brain oxidative stress, which is increased, as indicated
by elevated levels of oxidative stress markers, such as TBARS (thiobarbituric
acid reactive substances) and protein carbonyl content (Blass et al., 2000).

The c-Jun N-terminal kinase (JNK), a subfamily of MAP kinases, is activated
by phosphorylation of its threonine and tyrosine residues in specific positions.
This cytoplasmic reaction requires the preceding activation of the upstream
kinases which constitute the family of JNK kinases (JNKK, also called MAP
kinase kinases, MKK) (Herdegen and Waetzig, 2001a). The JNK family
43
consists of three isoforms: JNK1, JNK2 and JNK3 (Kyriakis et al., 1994),
which exhibit differences in specificity toward substrates and binding proteins,
and in their regulation by upstream kinases and scaffold proteins (Davis, 1999;
Gupta et al., 1996). JNK is considered to be a central signal transducer in
neuronal death in the mammalian brains (Herdegen and Waetzig, 2001b),
which can be activated under oxidative stress. Several studies have
demonstrated that activation of JNK pathways enhances neuronal cell death in
cultured primary neurons or in models of JNK knockout mice (Mielke and
Herdegen, 2000). Increased JNK activity has been found frequently in the
brains of patients with Parkinson’s disease (PD) or Alzheimer’s disease (AD)
(Peng and Andersen, 2003; Zhu et al., 2001). As oxidative stress increases in
the brain during aging, it is very likely that the activity of JNK will also
increase in the brain. Although the expression and activity of JNK have been
widely studied in aging associated neurodegenerative diseases, the effect of
JNK on brain aging has barely been explored.

Recent studies showed that active JNK might directly affect mitochondrial
functions through the phosphorylation of undefined substrates in mitochondria
(Schroeter et al., 2003). Mitochondria play a key role in brain aging, as these
organelles are a major source of oxidants, a target for radical damaging effects,
and a source of pro-apoptotic factors (Cadenas and Davies, 2000). The
44
mitochondrial alterations observed upon aging include an increased content of
oxidation products and a diminished functional activity. The activities of
mitochondrial nitric oxide synthase (mtNOS), NADH dehydrogenase, and
cytochrome c oxidase also decline with aging in rodent brain.

Pyruvate dehydrogenase (PDH) is an important a multi-subunit enzyme
complex located in the mitochondrial matrix, the activity of which has not
been thoroughly explored as a function of aging. PDH catalyzes the oxidative
decarboxylation of pyruvate to form acetyl CoA, NADH, and CO
2
. This
reaction bridges the anaerobic and aerobic brain energy metabolisms, and it is
the entry point of carbohydrates into the tricarboxylic acid cycle. PDH is
composed of three major subunits: E1, E2, and E3. The E1 subunit is a
tetramer that contains two I and two _ subunits, with molecular weights of 41
and 36 kDa, respectively. E2 is covalent bound to a lipoyl moiety, which acts
as a swing arm and allows the E2 to catalyzed the acyl transfer to CoA. E3
catalyzes the reoxidation of the dihydrolipoyl moiety with NAD
+
as the
ultimate electron acceptor. Furthermore, a variety of substrates and cofactors
pyruvate, NAD
+
, thiamine pyrophosphate (TPP), and coenzyme A (Co-A) are
all required for PDH activity. The activity of this enzyme can be regulated by
its substrates and products, cofactors, nucleotides, [Ca
2+
], [Mg
2+
] and
reversible phosphorylation. The target of phosphorylation/dephosphorylation
45
is the E1I subunit (E1I-PDH), which has three phosphorylation sites: Ser
264
,
Ser
271
and Ser
203
, and phoshorylation of each of these three sites can inhibit
PDH activity. Taken together, the complexity of PDH, including its multiple
subunits, strict cofactor requirements, and stringent regulation, make it a
vulnerable target for deregulation and subsequent inactivation during aging or
pathological conditions, including neurodegenerative disorders. Decreased
PDH activity was found in human brain homogenates from patient with AD,
which may be at least partially responsible for mitochondrial dysfunction and
impaired brain energy metabolism associated with AD. However, very little
work has been done to study the age-related changes in PDH activity.

Previous work described in Chapter I showed that JNK is activated by H
2
O
2
and anisomycin, and that it translocates to mitochondria after activation. The
mitochondrion-associated active JNK induces phosphorylation of the
E1I-PDH in brain mitochondria, which causes inhibition of PDH activity. The
work presented here employed this inhibition effect of JNK on PDH activity
and hypothesized that elevated JNK activity associated with mitochondria
during brain aging may mediate the inhibition of PDH activity by increasing
the phosphorylation on E1I-PDH. The current study showed that: 1) the
activity of JNK in mitochondria increases during aging in the rat brain, 2) and
PDH phosphorylation was increased as a function of the increased JNK
46
activity in mitochondria, and 3) that PDH activity decreased as a function of
age.
47
Materials and Methods

Materials
Antibodies against JNK1, JNK2 and were purchased from Santa Cruz Biotech
(Santa Cruz, CA). Antibodies against JNK3 and pJNK were bought from
Upstate Biotechnology (Waltham, MA). Antibody against the E1I-PDH was
obtained from Mitoscience (Eugene, OR). All other chemicals or reagents
were obtained from Sigma-Aldrich (St Louis, MO).

Animals
Male Fisher 344 rats of different ages (6 months, 14 months and 24 months)
were purchased from the National Institute on Aging (Baltimore, MD). Each
rat was maintained in one cage in the animal facility under standard conditions
(12-h light/12-h dark cycle, humidity at 50 ± 15%, temperature 22 ± 2°C, and
12 air changes/h) for 3 days to recover from the shipment stress.

Isolation of Mitochondria
Whole brain mitochondria were isolated from Fisher 344 rats of different ages
in a parallel process. The brains were excised, rinsed in ice-cold isolation
buffer, pH 7.4, containing sucrose (250 mM), Hepes (20 mM), EDTA (1 mM),
EGTA (1 mM), dithiothreitol (DTT; 1 mM), protease inhibitor cocktail
48
(sigma-Aldrich; St Louis, MO) 100 Sl per brain, using a Dounce homogenizer
to give a 5% (w/v). Non-synaptosomal mitochondria were isolated by
differential centrifugation followed by discontinuous Percoll density-gradient
centrifugation, according to procedures described previously (Anderson and
Sims, 2000). Briefly, the homogenate was centrifuged at 1330 g for 5 min to
remove nuclei and cell debris. The supernatant was centrifuged at 21,200 g
for 10 min, and the resultant pellet was resuspended in 15% Percoll and was
centrifuged 21,200 g for 10 min to remove the fat. Then the loose pellet was
layered onto a preformed discontinuous gradient of 23%/40% Percoll. The
gradient was centrifuged at 31,000 g for 5 min. Mitochondrial fractions were
collected and washed with isolation buffer 2 times. The resulting
mitochondrial pellet was resuspended in isolation buffer and then the protein
concentration was determined using Bio-rad Protein Assay for further
analysis.

SDS-PAGE Gel and Immunoblot Analysis
Mitochondria or total brain homogenates were lysed in RIPA buffer containing
Tris-HCl (50 mM), NP-40 (1%), Na-deoxycholate (0.25%), NaCl (150 mM),
EDTA (1 mM), pH 7.4. Then, loading buffer was added and the protein was
heated for 5 min at 95°C. After cool down, proteins were loaded at 50
µg/well and resolved in 12% SDS-PAGE gels. The proteins on the gel were
49
then transferred to PVDF membrane, blocked and detected by antibodies at
concentrations indicated by the manufactures. Immunoblot bands of targeted
proteins were quantified by Scion Image beta 4.0.2.

PDH enzyme activity assay
PDH activity was determined at 37°C by measuring the reduction of NAD
+
at
340 nm, upon the addition of 40 µM CoASH to 50 µg/mL mitochondrial
proteins, in a buffer containing 35 mM KH
2
PO
4
, 5.0 mM MgCl
2
, 2.0 mM
NaCN, 0.5 mM EDTA, 0.5 mM NAD
+
, 200 µM TPP, 4.0 mM pyruvate, 1 X
protein phosphatase inhibitor cocktail and 2.5 µM rotenone. The assay was
done in the presence of rotenone to prevent NADH consumption by
NADH:ubiquinone oxidoreductase (complex I). Before the assay,
mitochondria were disrupted by 2% Chaps in the isolation buffer.

2D gel and MS sequence
Mitochondrial protein was separated by isoelectric point (pI) on precast gel

strips (17 cm) with a linear gradient of pH 3–10 from Bio-rad (Hercules, CA)
by using the Bio-rad Protean IEF System. Mitochondrial

samples (300 µg)
were solubilized in the rehydration buffer

[6 M urea, 2 M thiourea, 2%
Nonidet P-40, 2% IPG buffer (pH

3–10), and 0.1 M dithiothreitol] for 30 min

at room temperature before the rehydration of the strips. The immobilized pH
50
gradient

(IPG) strips were rehydrated overnight in rehydration buffer with
dissolved mitochondrial protein. The program utilized was the following:
250 V rapid voltage ramping for 30 min, 10,000 V slow voltage ramping for
60 min, 10,000 V rapid voltage ramping for 50KVhrs. The strips were
incubated first in Equilibration buffer I with 6

M urea, 20% glycerol, 2% SDS,
2% DTT, and 0.375 M Tris (pH 8.8) for 10 min at room temperature, then in
Equilibration buffer II with 6

M urea, 20% glycerol, 2% SDS, 2%
iodoacetamide, and 0.375 M Tris (pH 8.8). They were then

loaded onto 10%
SDS-PAGE gels and run at 50 V overnight. The gels were fixed overnight and
first stained with Pro-Q
®
Diamond phosphoprotein gel stain from Molecular
Probes (Eugene, OR). After imaging, gels were then stained with SYPRO
TM

Ruby protein gel stain. Images of the protein gels were taken with a
VersaDoc
TM
imaging system (Bio-rad (Hercules, CA). Densities of protein
spots were quantified by Scion Image beta 4.0.2. Protein spots of interested
(candidate spots of E1I-PDH: based on the molecular weight, pI value and
phosphorylation signals) were excised from the 2D gel and sent for
LC-MS/MS sequence at USC Proteomics Core Facility.

Lactic acid and GSH concentration measurements
Total brain homogenates were lysed in an equal volume of perchloric acid (2M)
and centrifuged for 10 min at 12,000 g. Supernatants were neutralized with
51
KHCO
3
(3 M) and recentrifuged at 12,000 g. Extracts (50 µl) were added to 500
µl of reaction buffer and the concentration of lactic acid was measured using a
lactic acid assay kit (r-Biopharm, Germany). Samples for ATP measurement
were prepared in the same way as for lactic acid measurment, and ATP levels
were determined using an ATP determination kit (Molecular Probes). Equal
volumes of 5% metaphosphoric acid were added to total brain homogenates
right after the homogenization to stabilize the GSH and GSSG. GSH and GSSG
were detected by HPLC as described (Han et al., 2003).
Statistical analysis
Data are reported as means ± S.D. of at least three independent experiments.
Significant differences between mean values were determined by the student
t-test. Means were considered to be statistically distinct if P < 0.05.

52
Results

To determine whether expression and activities of JNK change with age in
vivo, we compared the basal levels and activities of JNK in the brains of male
Fischer 344 rats at different ages (6 months, 14 months and 24 months). Total
brain homogenates were used to measure expression and activities of JNK.
As shown in Figure 11A, the expression level of JNK1 in the brains increased
about 1.2-fold (n ] 3) between the ages of 6 and 14 months, and it remained
the same between the ages of 14 and 24 months. The basal levels of JNK2/3
exhibited no significant change from ages of 6 to 24 months (Fig. 11B, C).
JNK activity was determined using antibodies against pJNK, as dual
phosphorylation of JNK is essential for kinase activity. Figure 11D showed
that the level of pJNK was significantly increased during aging in the rat brain
(n ] 3), which is consistent with previous finding (Suh, 2001). that JNK
activity was constitutively high and significantly increased in older rats.

53
0
0. 5
1
1. 5
6M 14M 24M
JNK1
Actin
p46
0
0. 5
1
1. 5
6M 14M 24M
JNK2
Actin
p54
p46
Fig.11
B
A
54
JNK3 p46
pJNK
p54
p46
Actin
Actin
0
0.2
0.4
0.6
0.8
1
1.2
6M 14M 24M
0
1
2
3
4
5
6M 14M 24M
Fig.11 (continued)
D
C
Figure 11. JNK protein levels and activity in rat brain during aging
Total brain homogenates were taken from rats of different ages (6 months, 14 months and
24 months). Levels of JNK1 (A), JNK2 (B), JNK3 (C) and pJNK (D) were detected by
immunoblot using different antibodies (n ] 3). Actin levels were monitored as a control for
equal loading.
55
It has been reported that JNK translocates to mitochondria upon its activation.
Therefore, during aging, the increase of total JNK activity may result in an
increase of JNK translocation to mitochondria. To assess whether the protein
levels and activity of JNK increase with age, brain mitochondria were isolated
from rats of different ages and immunoblot analysis was employed to
determine the levels of JNK1, JNK2, JNK3 and pJNK. Figure 12A shows that
the association of JNK1 with mitochondria was significantly increased during
aging, whereas mitochondrial association of JNK2/3 remained unaltered.
Although JNK2 has isoforms of p46 and p54, only p54 was found to be
associated with mitochondria; p46 was not detected in mitochondria. (Fig.
12B, C) Mitochondrial association of pJNK was significantly increased with
age in the rat brains (P < 0.05, n ] 3, Fig. 12D).

56
0
0. 5
1
1. 5
6M 14M 24M
0
0. 5
1
1. 5
6M 14M 24M
JNK1
COX
B
Fig. 12
A
p46
JNK2
p54
p46
COX
57
0
0.2
0.4
0.6
0.8
1
1.2
6M 14M 24M
0
1
2
3
4
5
6M 14M 24M
Figure 12. Mitochondrial association of JNK and its activity in rat brain during aging
Brain mitochondria were isolated from rats of different ages (6 months, 14 months and 24
months). Levels of JNK1 (A), JNK2 (B), JNK3 (C) and pJNK (D) were detected in
mitochondrial proteins from rats of different ages by immunoblot using different antibodies
(n ] 3). COX levels are shown as a control for equal loading.
Fig. 12 (continued)
D
*
*
C
pJNK
p54
p46
JNK3
p46
COX
COX
58
The work described in described in Chapter I in this dissertation, revealed that
mitochondrial association of active JNK causes inhibition of mitochondrial
PDH activity. Therefore, an enhancement of mitochondrial JNK activity with
age may also lead to a decrease of PDH activity, which may contribute to the
development of the pathological stages of many neurodegenerative diseases. In
order to assess whether PDH activity decreases with age, this enzyme activity
was measured in brain mitochondria from rats of different ages. Figure 13A
shows that there was a significant decrease of PDH activity (~25% in
14-month-old rat, ~45% in 24-month-old rat, P < 0.05, n ] 3) during aging in
the rat brain. Figure 13B shows the actual rate of PDH activity, indicating that
PDH activity from the younger rat not only has a faster initial rate, but that the
activity also persists longer than in the mitochondria from the older rat.

0
50
100
150
200
250
6M 14M 24M
*
*
Fig. 13
A
59
PDH activity is, in part, controlled by phosphorylation/dephosphorylation,
where a specific pyruvate dehydrogenase kinase (PDK) phosphorylates three
serine residues of E1I-PDH, thereby inactivating pyruvate oxidation. Previous
work from this laboratory, as described in Chapter I, showed that JNK could
inhibit PDH activity by increasing the phosphorylation level of E1I-PDH.
During aging, more active JNK was translocated to mitochondria and decreased
B
0
0.1
0.2
0.3
0.4
0 40 80 120 160
Time (seconds)
Absorption 340nm
6 Mon
14 Mon
24 Mon
Fig. 13 (continued)
Figure 13. PDH activity declines with age in rat brain
Brain mitochondria (50 µg) from rats of different ages (6 months, 14 months and 24
months) were lysed in 20 µl of Miotchondria isolation buffer containing 2% Chaps. PDH
activity was determined at 37°C, by measuring the reduction of NAD
+
at 340 nm upon the
addition of 40 µM CoASH to 50 µg/mL mitochondrial proteins, in a buffer containing 35
mM KH
2
PO
4
, 5.0 mM MgCl
2
, 2.0 mM NaCN, 0.5 mM EDTA, 0.5 mM NAD
+
, 200 µM
TPP, 4.0 mM pyruvate, 1 X protein phosphatase inhibitor and 2.5 µM rotenone (n = 4).
Figure 13A shows the initial rates of PDH activity normalized as nmol of NADH
production per minute per mg of mitochondrial protein. Figure 13B shows the absorption
change at a wavelength of 340 nm over the time course.
60
PDH activity was detected, which may also be due to the JNK-mediated
phosphorylation of E1I-PDH. To validate this notion, the extend of E1I-PDH
phosphorylation was determined. First, the localization of phosphorylated or
non-phosphorylated-forms of E1I-PDH was identified on 2D gels by staining
mitochondrial proteins with Pro-Q
®
Diamond stain, followed by LC-MS/MS
(liquid chromatographic mass spectrometric) analysis. Fig. 14A shows that
phosphorylated protein spots B and C were the two major
phosphorylated-proteins in the mitochondrial proteome, and both were
identified by LC-MS/MS as the PDH E1I subunit (Table1). This result was
consistent with our previous work using radioactive [-
32
P] ATP labeling of
mitochondrial proteins, which showed that protein spots B and C (same
localization on 2D gel) were two major sites of mitochondrias phosphorylation.
Both spots were identified as the PDH E1I subunit. According to the molecular
weight and pI value, spot A was suspected to be non-phosphorylated E1I-PDH
(Table1). After sequencing, it was confirmed that spot A was the
non-phosphorylated form of E1I-PDH.

61
C
A
C
B
B
B
A
Figure 14. Identification and localization of phosphorylated- or
non-phosphorylated-forms of PDH E1K subunit on the mitochondrial proteome 2D gel
Figure 14A shows the Pro-Q Diamond stain of mitochondrial proteins in a 2D gel. Protein
spots B and C were the proteins with the highest signals of phosphorylation. Figure 14B is
the Sypro-ruby stain of mitochondrial proteins in a 2D gel and protein spot A was the PDH
E1I subunit, which was identified by LC-MS/MS. Protein spots B and C in Figure B
correspond to protein spots B and C in Figure A, respectively, and they were both also
identified by LC-MS/MS as the PDH E1Isubunit.
Fig. 14
62
Table 1
Protein
Spot
Total Score Sequence Coverage Numbers of
peptides
Matched
Protein ID number
in NCBI
A 309 18% 13 gi|34879653
B 242 18% 5 gi|34879653
C 488 35% 11 gi|34879653
Table 1 LC-MS/MS analysis of protein spot A, B and C from Sypro-ruby stained 2D gel
of mitochondrial proteins

Because the phosphorylated and non-phosphorylated forms of PDH had been
mapped on the 2D gel of the mitochondrial proteome, the percentage of
phosphorylated E1 I-PDH could be quantified based on the protein amount on
2D gels. Figure 15A shows the protein spots (SYPRO
TM
Ruby protein gel
stained) of phosphorylated or non-phosphorylated forms of E1I-PDH from rats
of different ages (left: 6 months, middle: 14 months, right: 24 months).
Protein spots B and C represent the phosphorylated forms of E1I-PDH; protein
spot A is the non-phosphorylated form. The percentages of phophorylated and
non-phosphorylated forms of E1I-PDH protein were calculated based on the
signals of SYPRO
TM
Ruby stained protein spots (Fig. 15B). The percentage of
phosphorylated-form2 of E1I-PDH was increased significantly during aging
from 14 months old to 24 months old, and the percentage of the
non-phosphorylated-form, the actual functional form, decreased during aging (n
] 5, P < 0.05). The immunoblot of total mitochondrial proteins with antibody
anti-E1I-PDH antibody revealed no significant changes of the total protein
63
amount of E1I-PDH in rats of different ages (Fig. 16). Therefore, it can be
concluded that the amount of the non-phosphorylated form of E1I-PDH (the
active form) decreases during aging, which may contribute to the age related
decrease of PDH activity.

A C
A
C A
C
B B
B
6Mon 14Mon 24Mon
A: non-phosphorylated-form of PDH
B: phosphorylated-form 1 of PDH
C: phosphorylated-form 2 of PDH
0
20
40
60
80
100
phosphorylation-
form 1
phosphorylation-
form 2
non-
phosphorylation-
form
Relative Protein Amoun
6M
14M
24M *
*
Figure 15. Percentages of phosphorylated- and non-phosphorylated-forms of PDH
E1K subunit in rat brain during aging
Proteins from brain mitochondria from rats of different ages (6 months, 14 months and 24
months) were separated in 2D gels and stained with Sypro-ruby. Figure 15A shows the
phosphorylated- or non-phosphorylated-forms of the PDH E1I subunit. The protein
amounts of phosphorylated- or non-phosphorylated-forms of PDH E1I subunit were
quantified based on the intensity of Sypro-ruby staining (Figure 15B).
B
A
Fig. 15
64
PDH links glycolysis to the TCA cycle that is a major biochemical process
response for most reduced NADH and FADH
2
generation in the cells. As a
consequence, adequate flux through PDH is particularly important for tissues to
maintain a reducing environment and high ATP production. Inhibition of PDH
may also cause an increase in abundance of lactic acid, because of the increased
availability of pyruvate to lactate dehydrogenase. Higher levels of lactic acid
were detected in the spinal fluid of patients with defects in the PDH enzyme. To
assess these related metabolic effects of PDH inhibition, levels of ATP, GSH
and lactic acid were measured in brain homogenates from rats of different ages.
0
0.3
0.6
0.9
1.2
6M 14M 24M
E1K-PDH
COX
Fig. 16
Figure 16. Protein expression levels of PDH E1K subunit in brains from rats of
different ages
Brain mitochondria were isolated from rats of different ages (6 months, 14 months and 24
months) and PDH E1I subunit was detected by immunoblot using an anti-E1I-PDH
antibody (n = 4). COX levels are showed as a control for equal loading.
65
Trends of decrease in ATP levels and GSH/GSSG ratios were observed, and a
significant increase of lactic acid concentraion was detected as a function of age
in rat brains (Fig. 17). These metabolic changes implicate PDH in the inhibition
of brain functions during aging.

Altogether, it can be concluded that the decrease of PDH activity is at least
partially due to the increase of phosphorylation of E1I-PDH, which may be
caused by the increased mitochondrial association of active JNK during aging.
0
2
4
6
8
10
12
6M 14M 24M
Lactic acidµg/mg
0
0.2
0.4
0.6
0.8
1
1.2
6 Mon 14 Mon 24 Mon
0
5
10
15
20
25
30
6 Mon 14 Mon 24 Mon Relative ATP level
GSH/GSSG
Fig. 17
Figure 17. Age affects the levels of ATP, GSH and lactic acid in rat brain.
Total brain homogenates were obtained from rats of different ages. Levels of ATP (A),
GSH, GSSG (B) and lactic acid (C) were measured as described in the Methods section.
66
Discussion

Mammalian brains barely use metabolic intermediates from fatty acids, but
they are mostly dependent on glucose metabolism, which provides most of the
ATP consumed during neuronal excitation. Since PDH plays a central role in
controlling the use of glucose-linked substrates as sources of oxidative energy,
the regulation of this large complex is critical for normal brain function.
Previous work from this laboratory, as described in the Chapter I, revealed
that mitochondrion-associated active JNK initiates an unknown signal cascade
and leads to the phosphorylation of E1I-PDH, and therefore inhibition of
PDH activity. The present study investigated the inhibitory effect of JNK on
PDH in the aging rat brain. JNK activity is increased in mitochondria during
brain aging, whereas PDH activity declined, with an increased
phosphorylation on E1I-PDH. These results, together with the previous
findings, suggest that increased mitochondrion-associated active JNK is
responsible for the increased phosphorylation on E1I-PDH during the aging
process in the brain. And the increased phosphoyrlation levels of E1I-PDH
contributes to the decline of PDH activity during aging in the brain. The
data presented here provide new evidence that high levels of JNK activity
during brain aging may cause appreciable inhibition of PDH, and thus
diminish the energy supply, which may contribute to the development of the
67
pathological stages of many neurodegenerative diseases.

PDH was reported to be deficient in the AD brain in 1980 (Perry et al., 1980).
That finding has subsequently been confirmed in at least three other
laboratories, with no contravening reports (Butterworth and Besnard, 1990;
Perry et al., 1980; Sheu et al., 1985; Yates et al., 1990). The deficiency of
PDH activity occurs not only in regions of brain that are neuropathologically
damaged in AD, but also in regions that are histopathologically normal, which
indicates that the decreased PDH activity happens in an early stage of the
disease and it can be a cause of AD, rather than a consequence of AD
(Butterworth and Besnard, 1990; Perry et al., 1980; Sheu et al., 1985; Yates et
al., 1990). Therefore, rescue of PDH activity in an early stage of AD can be a
new therapeutic strategy for AD. Lipoic acid has been proposed as an
excellent agent for such a treatment. Lipoic acid is a naturally occurring
disulfide compound; once reduced to dihydrolipoic acid, this is recognized as
an essential cofactor for PDH enzyme (Holmquist et al., 2006). Supplement
with lipoic acid to PDH may rescue the decreased PDH activity caused by the
phosphorylation of E1I-PDH. Another ideal agent for the treatment of early
stage of AD would be acetyl-L-carnitine (ALCAR) (Calabrese et al., 2002;
Calabrese et al., 2003; Martin et al., 2005). The presence of
acetylcarnitine-CoA transferase in the brain allows the entry of ALCAR acetyl
68
units into the TCA cycle in mitochondria (Bresolin et al., 1982). By providing
a source of acetyl-CoA alternative to pyruvate decarboxylation, ALCAR may
stimulate aerobic energy metabolism bypass the PDH. ALCAR also acts
indirectly as an antioxidant, which can be explained by increased NADH
production from the TCA cycle after the ALCAR administration. Because the
NADH in the mitochondria can act as an reducing equivalent to reduce
NADP
+
back to NADPH by the NAD(P)H transhydrogenase.

Increased oxidative stress is presented during brain aging. Oxidative stress is
thought to be an early event in AD, and it is also considered to be as an
inducer of the JNK signaling pathway in the brain. Endogenous JNK
activation, especially at a low level, may reflect a chronic and cumulative
stress process that contributes to mitochondrial dysfunction during brain aging.
Our data showed that JNK activity and its association with mitochondria were
significantly enhanced during aging, which suggests that during aging the
regulation of mitochondria by JNK is more potent in the aged brain. In
another study, mitochondria, as the major cellular source of free radicals and
oxidants, were demonstrated to be able to regulate JNK activation by releasing
of H
2
O
2
(Dougherty et al., 2004; Nemoto et al., 2000). In our study, PDH
activity was declined in the brain during aging, which leads to a decreased
NADH level in the mitochondria. The decreased NADH level may
69
consequently effect NADPH reducing power, thus to weaken the defense
mechanism against oxidative stress (H
2
O
2
) in the mitochondria. Therefore, it
may be surmised that increased oxidant production by mitochondria at older
ages results in increased JNK activity, which may in turn regulate
mitochondria and lead to further increases in production of oxidants (H
2
O
2
).
This crosstalk between mitochondria and the JNK signaling pathway forms a
feedback loop, which may amplify the regulatory effects of JNK on
mitochondria during the aging process. The regulatory effects of JNK on
mitochondria may include: 1) release of cytochrome c inducing of neuronal
death, 2) phosphorylation of components of the respiratory chain, causing
insufficient ATP generation and more free radical release, and 3) mediating
phosphorylation of E1I-PDH, resulting in inadequate reducing power
(NADH).

With the implications of JNK in many neurodegenerative diseases, there have
been intensified efforts (Scapin et al., 2003) to use JNK inhibitors as novel
neuroprotective strategy (Hunot et al., 2004), however, it demands for the
careful differentiation of physiological and pathological functions of JNK
isoforms. A major challenge to understanding the function of JNK in vivo is
imposed by the complexity of the JNK gene family. Ten JNK isoforms
resulting from alternative splicing of three genes have been identified, which
70
exhibit differences in specificity toward substrates and binding proteins and in
the regulation by upstream kinases and scaffold proteins. These observations
suggest the importance in further delineation of the mechanisms underlying
JNK isoform-specific functions and that, in many cases, JNK inhibitors for
therapeutic use should aim to target individual JNK isoforms. In the rodent
brain, the understanding of the different functions of individual JNK isoforms
has evolved only recently. Our data showed indicates that increased
mitochondrion-associated active JNK is mostly contributed by increased
association of JNK1 with mitochondria. Although JNK3 was considered to be
more directly related to neurodegeneration, its association with mitochondria
does not change during aging in the rat brains. These observations suggest that
JNK1 is the major isoform that leads to the phosphorylation of E1I-PDH and
consequent activity inhibition of PDH in the brain during aging. This finding
is consistent with previous report that obese Jnk1
-/-
mice, had significantly
lower blood glucose concentrations than obese Jnk1
+/+
or Jnk2
-/-
mice
(Hirosumi et al., 2002).

The work presented here also showed several innovate points in terms of
methodology development, including PDH activity measurement and
quantification of phosphorylated and non-phosphorylated proteins.
Age-related trends in PDH activity have been measured in several studies,
71
with inconclusive results. PDH activity was reported to decline in
synaptosomes from rat cerebral cortex (Curti and Benzi, 1989), but an earlier
study suggested there was no change in PDH activity during brain
aging(Leong et al., 1981). In those studies, strong detergent (triton X-100),
sonication or freeze-thaw cycles were used to break mitochondria, which may
cause incomplete breaking or introduce damage to the enzymes. If the
mitochondria are not completely broken, the rate of the PDH reation will
probably be limited by the rate of entry of NADH into the matrix; therefore,
the actual level of PDH activity could be concealed during the measurements.
In our work, we used CHAPS, a new gentle detergent, to thoroughly break
mitochondria without damaging the enzymes. We found that after substrates
were added into the reaction solution, PDH instantly responded to the
substrates and showed NADH production (a marker of PDH activity), whereas
previous work reported that to measure PDH activity, the enzyme needed to
be pre-incubated with substrate for at least 15 min. This delayed response may
be due to the incomplete breakage of the mitochondria, with the
pre-incubation allowing some substrate, e.g. NAD
+
, to get into the
mitochondrial matrix. In the present study, the phosphorylated and
non-phosphorylated E1I-PDH were mapped on the 2D gel by using Pro-Q
Diamond stain followed by LC-MS/MS, then the percentage of
phosphorylated protein was calculated based on the SYPRO-Ruby stained
72
protein signal. By the integration of 2D gel protein separation, protein gel
stain and LC-MS/MS results, we were able to determine for the first time the
percentages of phosphorylated and non-phosphorylated protein at the same
time. Comparing with the conventional method of only determining the
phosphorylated protein, our method gives new concept and special importance
when the non-phosphorylated protein is the actual functional form.

Phosphorylation/dephosphorylation is a major mechanism of regulation PDH
activity under physiological conditions. Our data showed that phosphorylation
of E1I-PDH increased significantly in 24-month-old rat brain, and there was
only a slight increase in 14-month-old rat compared with 6-month-old rat.
However, PDH activity declined significantly in the brains both of 14-month-
and 24-month-old rats when they were compared with the 6-month-old rats.
This inconsistency of phosphorylation and inhibition of PDH activity in the
14-month-old rat indicates that there might be other factors influencing PDH
activity. Oxidative stress has been considered as one possible mechanism
responsible for damage to the PDH complex (Martin et al., 2005).

In conclusion, the increased JNK activity associated with mitochondria during
brain aging is closely related to the decrease of PDH activity. Our findings
certainly provide a new perspective in the study of pathological development
73
of age-related neurodegenerative diseases, as well as several innovate
therapeutic interventions for the treatment of those diseases.
74
SUMMARY

Mitochondrial dysfunction has often been found to increase in the brain as a
function of age or in neurodegenerative diseases. Recent work demonstrated
that upon activation, JNK translocates to mitochondria and regulates
mitochondrial functions in cultured cells. The activity of JNK in the brain is
increased with age and is significantly eveluated in the brains of the patients
with Parkinson’s or Alzheimer’s diseases. Therefore, we hypothesized that
age-dependent translocation of active JNK to mitochondria regulates
mitochondrial functions, which may contribute to the development of
mitochondrial damage inherent in the aging process of the brain.

In the presented work, we found that total JNK activity and the translocation
of JNK to mitochondria were increased as a function of aging in rat brains.
The co-localization of active JNK with mitochondria was also observed in
primary cultured neurons under the confocal microscope. By using a
mitochondrial proteomic approach (2D electrophoresis followed by
LC-MS/MS), the mitochondrial protein pyruvate dehydrogenase E1I subunit
was identified, exhibiting increased phosphorylation both in aged rat brain and
or in isolated brain mitochondria incubated with active JNK in vitro.
Pyruvate dehydrogenase E1I subunit (E1I-PDH) is one component of
75
pyruvate dehydrogenase complex (PDH), and the phosphorylation of
E1I-PDH causes inhibition of PDH activity. Therefore, PDH activity and
other related metabolites were measured. An inhibition of PDH activity,
increase of pyruvate and lactate concentration, and decrease of ATP
production were observed after up-regulation of JNK activity in cultured
primary neurons and in isolated brain mitochondria. In the brains of aging rats,
we found that PDH activity was diminished, with an increase of E1I-PDH
phosphorylation, which might be explained, at least in part, by enhanced JNK
translocation to mitochondria during aging. Increased concentration of lactic
acid and pyruvate were also observed in aged rat brain, which may have
resulted from decreased PDH activity (see Figure 18).

In summary, we found that JNK directly targets mitochondria and regulates
mitochondrial metabolism through inhibition of PDH activity. The increased
JNK activity associated with mitochondria is closely related to the decrease of
PDH activity during brain aging. Our findings elucidated the processes
involved in the regulatory mechanisms that coordinate mitochondrial
functions with the rest of the cell, the metabolic network that control cellular
energy levels and its impairment during brain aging.

76
Fig. 18
Figure. 18. A summary of the JNK signaling-mediated inhibition of PDH during brain
aging
77
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Abstract (if available)

Abstract Mitochondrial dysfunction has often been found to occur in brains with advancing age or neurodegerative diseases. Recent work demonstrated that upon activation, JNK translocates to mitochondria and regulates mitochondrial functions in cultured cells. Using experimental models of cultured primary neurons and isolated brain mitochondria, we found that: (i) both anisomycin, a potent JNK activator, and hydrogen peroxide can induce the rapid activation of JNK and its translocation to mitochondria in cultured primary cortical neurons, (ii) mitochondria-associated active JNK can be degraded by Proteinase K, which indicates that active JNK associates with the outer membrane of mitochondria, and (iii) the association of active JNK with mitochondria causes an increase of pyruvate dehydrogenase (PDH) phosphorylation and inhibition of its activity, which results in an increase of lactic acid concentration and decrease of ATP level in the cells. These results indicate that active JNK might mediate communication between the cytosol and mitochondria, regulating mitochondrial metabolism according to the cytosolic environment. Using an aged rat model, we found that: (i) JNK activity and its translocation to mitochondria were increased in the brain during aging, whereas PDH activity was diminished, with increased phosphorylation of the PDH E1[alpha]

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Asset Metadata

Creator Zhou, Qiongqiong (author)

Core Title C-jun N-terminal Kinase (JNK) mediated inhibition of Pyruvate Dehydrogenase (PDH) activity and its effect on mitochondrial metabolism during brain aging

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology

Publication Date 11/14/2006

Defense Date 10/05/2006

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag neurodegeneration JNK mitochondria aging,OAI-PMH Harvest

Language English

Advisor Cadenas, Enrique (committee chair), Brinton, Roberta Diaz (committee member), Hsiai, Tzung K. (committee member)

Creator Email qiongqiz@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m133

Unique identifier UC1145815

Identifier etd-Zhou-20061114 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-21408 (legacy record id),usctheses-m133 (legacy record id)

Legacy Identifier etd-Zhou-20061114.pdf

Dmrecord 21408

Document Type Dissertation

Rights Zhou, Qiongqiong

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