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Energy metabolism and inflammation in brain aging: significance of age-dependent astrocyte metabolic-redox profile
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Energy metabolism and inflammation in brain aging: significance of age-dependent astrocyte metabolic-redox profile
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i
ENERGY METABOLISM AND INFLAMMATION IN BRAIN AGING
SIGNIFICANCE OF
AGE-DEPENDENT ASTROCYTE METABOLIC-REDOX PROFILE
by
TIANYI JIANG
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)
June 2014
Copyright 2014 Tianyi Jiang
ii
DEDICATION
To my parents-Jianming Jiang and Fangmin Ren, my fiancé e Mian Zhou,
and my grandparents
iii
ACKNOWLEDGEMENTS
First I would like to thank my mentor, Dr. Enrique Cadenas, for his patient guidance
every step of the way throughout my five years’ Ph.D. studies. I benefited a lot from his
wisdom and critical thinking in the pursuit of science and truth. Besides, his humble and
amiable personality will also shape mine in the future. I am also truly grateful for what he
has always been striving to do: keep the projects going and encourage, trust, and support
me no matter what.
I greatly appreciate my dissertation committee members: Dr. Curtis Okamoto and Dr
Wei-Chiang Shen for their continuous support.
I am thankful to all of the former and current laboratory members. Dr. Fei Yin gave
me invaluable suggestions when I was at early stages of my Ph.D. studies. Dr. Amit Ag-
garwal, Dr. Harsh Sancheti, Zhigang Liu, Ishan Patil, and Dr. Ryan Hamilton all helped
me in some way. Besides, my work was greatly benefited from other people outside the
lab. I am thankful to Jia Yao for collaboration and helpful discussions of science. I am
thankful to Jennifer Mao, Shuhua Chen, Dr. Fan Ding, and Yang Li for the technical sup-
port. I am thankful to Dr. Jason Arimoto for experimental procedures.
Last but not least, I would like to express my deep appreciation to my parents, my
grandparents and my fiancé e for their unconditional love and support. I would also like to
thank all my dear friends here and back in China. May our friendship last forever.
iv
ABSTRACT
Aging is risk factor for diseases in both peripheral and central nervous system. Stud-
ies in this dissertation are aimed at investigating brain aging in the context of mitochon-
drial energy-redox axis and inflammation.
The progress of a hypometabolic state inherent in brain aging was studied in an ani-
mal model consisting of Fischer 344 rats of young, middle, and old ages. Dynamic mi-
croPET scanning demonstrated a significant decline in brain glucose uptake at old ages,
which was associated with a decrease in the expression of insulin-sensitive neuronal glu-
cose transporters GLUT3/4 and of microvascular endothelium GLUT1. Brain aging was
associated with an imbalance of the PI3K/Akt pathway of insulin signaling and JNK sig-
naling and a downregulation of the PGC1 mediated transcriptional pathway of mito-
chondrial biogenesis that impinged on multiple aspects of energy homeostasis. R-(+)-
lipoic acid treatment increased glucose uptake, restored the balance of Akt/JNK signaling,
and enhanced mitochondrial bioenergetics and the PGC1 -driven mitochondrial biogene-
sis. It may be surmised that impairment of a mitochondria-cytosol-nucleus communica-
tion is underlying the progression of the age-related hypometabolic state in brain; the ef-
fects of lipoic acid are not organelle-limited but reside on the functional and effective co-
ordination of this communication that results in improved energy metabolism.
This study, however, did not distinguish the effects of aging on specific cell types in
the brain. Astrocytes fulfill important functions in terms of energy supply to neurons, re-
dox homeostasis and inflammation. Age-dependent metabolic-inflammatory axis was ex-
amined in primary astrocytes isolated from brain cortices of 7-, 13-, and 18 month-old
v
Sprague Dawley male rats. Astrocytes showed an age-dependent increase in mitochon-
drial oxidative metabolism respiring on glucose and/or pyruvate substrates; this increase
in mitochondrial oxidative metabolism was accompanied by increases of
COX3/18SrDNA values, thus suggesting an enhanced mitochondrial biogenesis. En-
hanced mitochondrial respiration in astrocytes limits the substrate supply from astrocytes
to neurons; this may be viewed as an adaptive mechanism to altered cellular inflammato-
ry-redox environment with age. These metabolic changes were associated with an age-
dependent increase in hydrogen peroxide generation (largely ascribed to an enhanced ex-
pression of NOX2) and NFκB signaling in the cytosol as well as its translocation to the
nucleus. Astrocytes also displayed augmented responses with age to inflammatory cyto-
kines, IL-1β and TNFα. Activation of NFκB signaling resulted in increased expression of
nitric oxide synthase 2 (inducible nitric oxide synthase), leading to elevated nitric oxide
production. IL-1β and TNFα treatment stimulated mitochondrial oxidative metabolism
and mitochondrial biogenesis in astrocytes. It may be surmised that increased mitochon-
drial aerobic metabolism and inflammatory responses are interconnected and support the
functionality switch of astrocytes, from neurotrophic to neurotoxic with age.
Since lipoic acid showed beneficial effects in terms of restoring brain energy metabo-
lism in old Fischer 344 rat brain cortices, it would be intriguing to investigate its effects
on astrocytes in relevance to astrocyte metabolic-inflammatory axis. R-(+)-Lipoic acid
treatment of primary astrocytes –isolated from 24 month-old Fischer 344 rat brain corti-
ces– induced a shift of metabolic phenotypes in old astrocytes: lipoic acid suppressed ox-
idative metabolism but augmented anaerobic glycolysis. Lipoic acid treatment diminished
H
2
O
2
generation, which was associated with decreased NOX4 levels. Lipoic acid sup-
vi
pressed redox-sensitive NFκB cytosolic signaling and its nuclear translocation, which
were induced by IL-1β stimulation. Reduced NFκB signaling resulted in decreased ex-
pression of iNOS, which –along with decreased level of nNOS– lead to attenuation of
NO generation. The effects of lipoic acid may be interpreted in the context of brain aging,
where reactive astrocytes deprive neurons of metabolic intermediates and release neuro-
toxic inflammatory mediators. Lipoic acid seems to reverse the metabolic and inflamma-
tory phenotypes of old astrocytes to that of young and neurotrophic astrocytes.
In summary, studies in this dissertation investigate mitochondrial energy-redox axis
in brain aging with focus on astrocytic metabolic and inflammatory changes underlying
the transition of astrocytes from neurotrophic to neurotoxic. The balance of redox-
sensitive insulin signaling and JNK signaling is surmised as a determinant of brain aging.
The beneficial effects of lipoic acid on restoring brain energy metabolism and reversing
the phenotypes of old astrocytes can be explained by its thiol-disulfide exchange reac-
tions. Furthermore, studies in this dissertation demonstrated the therapeutic potential of
redox modulators such as lipoic acid to promote healthy aging and ameliorate age-related
diseases by acting in the integrated energy, redox, and inflammatory environment in brain
aging.
vii
TABLE OF CONTENTS
Dedication ………………………………………………………….……………………..ii
Acknowledgements ……………………………………………………………………...iii
Abstract …………………………………………………………………..………………iv
Chapter One: Mitochondrial Energy- Redox Aspects of Brain aging………...……….….1
1.1 Mitochondrial Energy-Redox Axis………………………...............................1
1.1.1 Mitochondrial Energy Metabolism……………...……………….….3
1.1.2 Mitochondrial Redox Homeostasis………………………………….8
1.1.3 Interdependence of Mitochondrial Energy-Redox Axis…………...12
1.2 Metabolic Triad: Mitochondria, Insulin signaling, and JNK signaling…..….14
1.3 Biochemistry and Biology of Lipoic Acid………………….………………..18
References…………………………………………………..……………………21
Chapter Two: Lipoic Acid Restores Age-Associated Impairment of Brain Energy Metab-
olism through the Modulation of Akt/JNK Signaling and PGC1α Transcriptional Path-
way ………………………………………………………………………...…………….30
Summary…………………………………………………………………………30
Introduction…………………………………………..……………….………….31
Experimental Procedures……………………………..………………………….33
Results…………………………………………………..………………………..39
Discussion……………………………………………….………………………51
References………………………………………………..………………………56
Chapter Three: Astrocytic Metabolic and Inflammatory Changes as a Function of Age
viii
……………………………………………………..……………………………………..60
Summary…………………………………………………………………………60
Introduction………………………………..………………………….………….61
Experimental Procedures…………………..…………………………………….63
Results……………………………………..……………………………………..66
Discussion…………………………………..……………………………………78
References………………………………..………………………………………82
Chapter Four: The Metabolic-Inflammatory Phenotype in Old Astrocytes: Effect of Lipo-
ic Acid ……………………………………….…………………………………………..86
Summary…………………………………………………………………………86
Introduction…………………………..……………………………….………….87
Experimental Procedures………………..……………………………………….88
Results…………………………………..………………………………………..91
Discussion………………………………………………………………………100
References………………………………………………………………………103
Chapter Five: Conclusion and Future Directions……………………………………….106
CHAPTER ONE
MITOCHONDRIAL ENERGY-REDOX ASPECTS OF BRAIN AGING
Aging is associated with a general decline of a wide array of physiological functions with
those functions that depend on the central nervous system being more affected. Brain is the cen-
ter of thought, emotion, and memory, processes that are highly energy-dependent. Although the
brain represents only 2% of the body weight, it accounts for 20% of the body oxygen consump-
tion and 25% of total body glucose utilization (Magistretti 2000).
The phenotype of brain aging includes cognitive impairment, memory loss, and increased
risk for neurodegenerative disorders. Because of the heavy reliance of brain on energy to per-
form its multiple functions, impairment of energy metabolism is widely recognized as the hall-
mark of brain aging. The hypometabolic state in brain aging may be due to deficiency in sub-
strate supply and metabolism, the latter can be further divided into cytosolic metabolism and mi-
tochondrial catalysis.
1.1 Mitochondrial energy-redox axis
As the powerhouse of the cells, mitochondria provide most of the energy needed for cellular
functions. The generation of the energy currency ATP starts from the oxidation of acetyl-CoA in
the tricarboxylic acid (TCA) cycle with the concomitant generation of reducing equivalents
(NADH, FADH
2
) that provide electrons for the respiratory chain, generating the proton gradient
that is required for ATP synthase (Mathews 2000). Electron leakage leads to the generation of
2
O
2
.–
, which disproportionates to H
2
O
2
, either catalyzed by the superoxide dismutases (matrix
Mn-SOD and intermembrane space Cu, Zn-SOD) or, secondarily, through spontaneous dismuta-
tion (Melov 2000). H
2
O
2
affects mitochondrial function by directly oxidizing proteins, also by
diffusing out of mitochondria and modulating redox-sensitive signaling pathways that impinge
on mitochondrial function.
Steady-state levels of mitochondrial H
2
O
2
are determined by both en-
ergy metabolism and the redox systems that metabolize this species. Therefore, impaired electron
transport chain and compromised H
2
O
2
clearance both contribute to mitochondrial accumulation
of H
2
O
2
, thus highlighting the importance of mitochondrial energy-redox axis (Fig. 1).
NNT
GSH
Trx
NADH NADP
+
O
2
NAD
+
H
2
O
H
2
O
2
H
2
O
2
O
2
. –
cytosol
mitochondria
TCA
RC
Domain
Specific
Signaling
JNK IIS
NADPH
energy redox
H
2
O
2
Fig 1 Mitochondrial energy–redox axis
Reducing equivalents from the tricarboxylic acid cycle flow through the respiratory chain (RC); elec-
tron leak accounts for 2%–3% of O
2
consumed in the form of O
2
.-
and H
2
O
2
. Reduction of H
2
O
2
is supported
by thiol-based systems, for which the ultimate reductant is NADPH. Sources of mitochondrial NADPH: nic-
otinamide nucleotide transhydrogenase (NNT), isocitrate dehydrogenase-2 (IDH2), and malic enzyme. Do-
main-specific signaling entailing regulation of redox-sensitive JNK- and insulin/IGF1 signaling (IIS) path-
ways
3
O
2
H
2
O H
+
e
–
III
I
IV
H
+
ATP
ADP
e
–
GLUT
glucose
ketone
bodies
glucose
pyruvate
acetyl-CoA
succinyl-CoA
succinate
acetoacetate
pyruvate acetoacetate
MCT
NADH
PDH
SCOT
CO
2
+ NADH
HSCoA + NAD
TCA
lactate
acetoacetyl-CoA
1.1.1 Mitochondrial energy metabolism
Brain glucose uptake
Brain energy metabolism is strictly controlled by substrate availability (Fig. 2). In the brain
glucose is the primary energy source while ketone bodies are the secondary fuel source under
metabolically challenging conditions. Glucose uptake in the brain is mainly mediated by the fa-
cilitated glucose transporter family: GLUT1 (55kD) is the major transporter in endothelial cells
driving the translocation of glucose across the blood brain barrier while the 45kD isoform of
GLUT1 is predominantly located in astrocytes. GLUT3 and GLUT4 are specifically expressed in
neurons and are responsible for the direct uptake of glucose from the extracellular space (Squire
2008).
Fig. 2 Mitochondrial energy
metabolism
Glucose is the primary
energy source for the brain,
which undergoes cytosolic me-
tabolism (glycolysis) and mito-
chondrial bioenergetics (TCA
cycle and respiratory chain).
Ketone bodies serve as second-
ary energy sources under meta-
bolic challenging conditions.
4
Astrocyte-neuron metabolic coupling
Astrocytes have a supportive function for neurons in the central nervous system (Kettenmann
2005). Neurons harbor strong aerobic metabolism, but glycolysis is directed mainly to the pen-
tose phosphate pathway leading to regeneration of glutathione (GSH) (Bolañ os et al. 2010). On
the other hand, astrocytes primarily rely on the ATP derived from glycolysis with lactate extru-
sion as the end point (Hu & Wilson 1997; Dimmer et al. 2000; Dienel & Hertz 2001; Korf 2006;
Schurr 2006). Under resting conditions, 85% of the glucose consumed by astrocytes is released
as the form of lactate (Bolañ os et al. 1994), which diffuses out of astrocytes through the mono-
carboxylate transporters 1 (MCT1) along with H
+
, and is taken up subsequently by neurons
through the neuronal isoform of high-affinity MCT2 (Halestrap & Price 1999). Lactate serves as
a key metabolite for neuronal aerobic metabolism in addition to direct glucose utilization by neu-
rons to meet the high energy demand associated with neuronal activity (Turner & Adamson
2011).
The major energetic cost in brain cortex takes place at glutamatergic synapses (Sibson et al.
1998; Alle et al. 2009). Once released into synaptic cleft, glutamate binds to NMDA receptors on
the postsynaptic membrane and induces the influx of Ca
2+
, which modulates the action potential
on postsynaptic membrane and triggers a serious of signaling events. Excessive amount of glu-
tamate in the extracellular space is believed to be the cause of neuronal toxicity and the removal
of which is especially important (Choi et al. 1987). Astrocyte acts as “cleaners” of extracellular
glutamate through highly efficient glutamate transporters on the plasma membrane including
EAAT1 (rodent ortholog: Glast) and EAAT2 (rodent ortholog: GLT1) (Anderson & Swanson
2000; Shigeri et al. 2004). Once inside astrocytes, glutamate is converted to glutamine at the ex-
5
NAD
+
Astrocyte
LDH
NAD
+
pyruvate
lactate
NADH
LDH
glutamate
TCA
glutamate glutamate
glutamine
NH
4
+
+ ATP
glutamine
synthase
glutaminase
NH
4
+
glutamate
Na
+
Na
+
Na
+
K
+
Na
+
ATP
glucose
anaerobic
glycolysis
pyruvate
NADH
lactate
glutamine
ADP + Pi
K
+
Na
+
,K
+
ATPase
glucose
glucose
glucose
Glutamatergic
synapse
glycolysis
Glutamate receptors
Na
+
MCT2
MCT1/4
EAAT
pense of ATP. Glutamine is then transported to neurons where it is deaminated and replenishes
the neuronal glutamate pool.
In the model of astrocyte-neuron metabolic coupling (Fig. 3), the production of lactate by as-
trocytes is determined by the uptake of glutamate. Glutamate transport is associated with an in-
tracellular Na
+
increase correlated with the mobilization of the plasma membrane Na,K-ATPase
activity, and an ensuing substantial increase in ATP consumption (Magistretti et al. 1999). Gly-
colysis is therefore stimulated in astrocytes and yields more pyruvate that is further reduced to
lactate, in turn transported to neurons to meet the energy demand in neuronal activity. Lactate,
once taken up by neurons, is oxidized to pyruvate by LDH1 and pyruvate serves as the substrate
for neuronal TCA cycle.
6
Fig. 3 Astrocyte-Neuron Metabolic Coupling
Neurons release glutamate when in high activity, the uptake of which by perisynaptic astrocytes
stimulates anaerobic glycolysis and the production of lactate. Lactate, as a metabolic intermediate, is trans-
ported to neurons to be integrated in aerobic metabolism.
Brain substrate metabolism
Brain substrate metabolism occurs in both cytosol and mitochondria. In the cytosol, glycoly-
sis starts with glucose as the fuel source and ends with pyruvate as the product. The outcome of
glycolysis includes ATP and NADH. Most of the enzymes in glycolysis are regulated tightly,
which is reasonable given the critical role of glycolysis in maintaining normal cell function. The
enzymatic machinery of glycolysis is regulated allosterically, by signaling pathways, and by the
cell's redox status. Hexokinase is in important enzyme fulfilling two roles: first, it phosphorylates
glucose to prepare it for later metabolism; second, phosphorylation by hexokinase traps glucose
inside cells and prevents it from moving outside (partly the basis of positron emission tomogra-
phy (PET)). Hexokinase can associate physically to the outer membrane of mitochondria through
specific binding to voltage dependent anion channel (VDAC) and gain direct access to ATP gen-
erated by mitochondria. Mitochondrion-bound hexokinase (especially hexokinase 2) is highly
elevated in rapidly growing cancer cells and has been shown to be the driving force for the ex-
tremely high glycolytic rates (Mathupala et al. 2009). Glyceraldehyde 3-phosphate dehydrogen-
ase (GAPDH) is also essential in that it not only generates reducing equivalents in the form of
NADH but also acts as a reversible metabolic switch under oxidative stress. When exposed to
excessive oxidants, GAPDH is inhibited by glutathionylation (Yap et al. 2010) and this inactiva-
tion re-routes temporally the metabolic flux from glycolysis to the pentose phosphate pathway,
allowing the cell to generate NADPH, which serves as the primary electron donor for the thiore-
doxin- and glutathione systems to counteract oxidative stress (Ralser et al. 2007).
7
Pyruvate generated by glycolysis is the substrate for mitochondrial pyruvate dehydrogenase
complex (PDH), which catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA,
which feeds into the TCA cycle. Inhibition of PDH results in impaired glucose utilization and
ATP production. PDH activity was found to decrease with age (Nakai et al. 1997), and its phos-
phorylation (inhibition) is driven by JNK that translocates to mitochondria during aging (Zhou et
al. 2008; Zhou et al. 2009). Additionally, recent work in our laboratory demonstrated an age-
dependent decrease in succinyl-CoA: 3-oxoacid Co-A transferase (SCOT) activity (Lam et al.
2009), a key mitochondrial matrix enzyme for ketolysis that provides the only alternative energy
for brain during glucose starvation. Acetyl-CoA enters TCA cycle and is oxidized to generate
reducing equivalents NADH and FADH
2
for ATP synthesis. Aconitase and α-ketogluterate de-
hydrogenase of TCA cycle have demonstrated an age-dependent decline in their respective en-
zymatic activities (Yarian et al. 2006). It may therefore be surmised, that alterations in activities
of TCA cycle enzymes and enzymes controlling entry to the TCA cycle such as PDH and SCOT
contribute significantly to the age-dependent decline in mitochondrial bioenergetics.
Mitochondrial oxidative phosphorylation is a process that encompasses electron transfer
through the complexes I-IV of the respiratory chain, vectorial H
+
release into the inter-membrane
space, and H
+
re-entry to the matrix through F
0
of complex V with ATP synthesis by F
1
-ATP
synthase. Electron transfer in mitochondria decreases in aged brain (Beckman & Ames 1998;
Navarro & Boveris 2007). Mitochondria isolated from brain of old rats and mice show decreased
electron transfer activity in complexes I, III, and IV (Navarro & Boveris 2004; Navarro &
Boveris 2007; Navarro & Boveris 2008). The inhibition of complex I activity during aging is ac-
companied by a decrease in NAD
+
concentration (Boveris & Cadenas 2000). The presence of
NAD
+
is necessary for the progression of the TCA cycle and decrease in NAD
+
could lead to im-
8
pairment of the turnover efficiency of the TCA cycle, irrespective of the presence of acetyl-CoA.
Moreover, decreased electron transfer can also lead to decreased mitochondrial inner membrane
potential which is observed in aged rat brain (Sastre et al. 1998; LaFrance et al. 2005). Recent
work in our lab also shows that F
1
-ATPase activity of complex V decreases with age due to ni-
tration on Tyr
269
(in the vicinity of the Mg
++
coordination site) suggesting deficient ADP binding
to the active site (Lam et al. 2009).
Given the central roles that mitochondria play in energy supply, tight regulation of mito-
chondrial dynamic remodeling is vital (Hock & Kralli 2009). Mitochondrion is a dynamic orga-
nelle that undergoes biogenesis, fusion/fission, motility, and autophagy. Mitochondrial biogene-
sis is an adaptive response to environmental stimuli to form new mitochondria. It induces mito-
chondrial DNA replication and increases copies of mitochondrial respiratory complexes leading
to the enhancement of mitochondrial oxidative capacity and therefore mitochondrial function.
Fewer mitochondria are found in skeletal muscle of insulin-resistant, obese or diabetic subjects
(Kelley et al. 2002; Morino et al. 2005; Ritov et al. 2005). Mitochondrial biogenesis is mediated
by PGC1α, a transcriptional coactivator that associates with multiple transcription factors/nuclear
receptors and integrates several pathways driven largely by Sirt1 and AMPK, redox- and energy
sensors, respectively. The downstream targets of PGC1α specifically for mitochondrial biogene-
sis include, among others, NRFs, Tfam and ERRα. PGC1α
-/-
mice have reduced mitochondrial
function and oxidative capacity in skeletal muscle, thus establishing PGC1α as a key contributor
to the regulation of mitochondrial biogenesis, oxidative capacity, and energy metabolism (Lin et
al. 2004).
1.1.2 Mitochondrial redox homeostasis
9
Trx2
ox
Trx2
red
GSSG
GSH
NADP
+
NADPH
GR TR GPx Prx3
H
2
O
2
H
2
O
Mitochondrial redox status is dependent on abundant redox couples NADPH/NADP
+
,
GSH/GSSG, and Trx2
red
/Trx2
ox
(Schafer & Buettner 2001; Patenaude et al. 2004) (Fig. 4). Glu-
tathione and thioredoxin constitute two major redox systems, while NADPH acts as the ultimate
electron donor. Mitochondrial redox homeostasis is important for maintaining thiol status of pro-
teins, which are usually subjected to redox regulation (Han et al. 2003).
Glutathione-based systems
Glutathione (GSH) is synthesized in the cytosol from glycine, glutamate, and cysteine in a
two-step process by -glutamylcysteine synthetase and GSH synthase (Han et al. 2006), and is
then imported into mitochondria. As the concentration of GSH in the mitochondria far exceeds
that of any other redox couple (~100-10,000 greater) (Hurd et al. 2005); the mitochondrial redox
Fig. 4 GSH- and Trx-based systems for H
2
O
2
removal in brain mitochondria
GPx: Glutathione peroxidase; GR: Glutathione reductase; TR: Thioredoxin
reductase; Trx: Thioredoxin; Prx: Peroxiredoxin.
10
status can be assessed with the Nernst equation and considering the concentrations of GSH and
GSSG. GSH plays a central role in the protection of mitochondria from oxidative stress by (a) a
direct interaction with oxidants and (b) as an electron donor for enzymes such as glutathione pe-
roxidases (GPxs), glutathione-S-transferases, and glutaredoxins (Grxs) (Mari et al. 2009). Per-
turbation of GSH/GSSG redox status results in the formation of protein mixed disulfides (gluta-
thionylation), which could affect protein functions. Protein-mixed disulfides are specifically re-
duced by Grxs (Grx2 is the mitochondrial isoform) through a monothiol mechanism (Holmgren
& Aslund 1995). Oxidized Grx2 is reduced by GSH, which is regenerated from GSSG by
NADPH-supported glutathione reductase (GR) (Dalle-Donne et al. 2008). Grx2 is constitutively
expressed in neurons and glia in mouse and human brain (Karunakaran et al. 2007).
Thioredoxin-based systems
Thioredoxin system is essential to maintain a reducing environment within the cell. Thiore-
doxin 1 (Trx1) is present in the cytosol and mitochondrial intermembrane space, whereas thiore-
doxin 2 (Trx2) is mitochondrion specific. Trx2 is a small protein (~12 kDa) that contains two
redox-active cysteine residues in the active center, and highly efficient at reducing disulfides in
proteins and peptides (Miranda-Vizuete et al. 2000; Jones 2008). Trx2 reacts with multiple pro-
teins via the transient formation of a mixed disulfide between the target protein and Trx2 fol-
lowed by thiol-disulfide exchange with reduction of the target protein and oxidation of Trx2. Ox-
idized Trx2 is recovered by Trx2 reductase (TrxR2) using NADPH as an electron donor
(Mustacich & Powis 2000).
Homeostasis of mitochondrial oxidants
11
Mitochondria are essential sources of O
2
.–
and H
2
O
2
. Complex I and complex III of the mito-
chondrial respiratory chain were identified as main sites for O
2
.–
generation (Turrens 2003;
Cadenas 2004; Murphy 2009). O
2
.–
is converted to H
2
O
2
by superoxide dismutase (SOD) in mi-
tochondrial matrix (Mn-SOD) and inner membrane space (Cu, Zn-SOD) Removal of mitochon-
drial H
2
O
2
is achieved by glutathione- or thioredoxin-dependent systems with NADPH as the ul-
timate electron donor. Removal of H
2
O
2
by energized brain mitochondria is mainly contributed
by thioredoxin/peroxiredoxin (Trx/Prx) system, and to a lesser extent by GSH/Glutathione pe-
roxidase (GSH/GPx) system (Drechsel & Patel 2010). Glutathione serves as a cofactor for gluta-
thione peroxidases (GPxs). GPx1 localizes in mitochondrial matrix, whereas GPx4 is mainly as-
sociated with the inner membrane and catalyzes the reduction of phospholipid hydroperoxides
(Schuckelt et al. 1991; Ursini et al. 1997). Mitochondrial Prx3 and Prx5 are both involved in
H
2
O
2
elimination; Prx3 specializes in removing H
2
O
2
whereas Prx5 is more efficient at reducing
ONOO
–
(Dubuisson et al. 2004; Peng et al. 2004). Prx3 is associated with Trx2, and the transfer
of electrons in H
2
O
2
elimination starts from NADPH to TrxR2, Trx2, Prx3, and finally to H
2
O
2
(Zhang et al. 2007).
Nitric oxide (NO) is mainly generated by nitric oxide synthases (NOS), of which there are
three isoforms with tissue- and cell-dependent preferential distribution. In the brain, endothelial
nitric oxide synthase (eNOS; NOS-III) performs some physiological functions including vasodi-
lation; neuronal nitric oxide synthase (nNOS; (NOS-I)) provides constitutive level of NO; and
inducible nitric oxide synthase (iNOS; NOS-II) is usually stimulated in inflammation and is re-
sponsible for continuous and high-level production of NO. NO is involved in the regulation of a
broad spectrum of pathophysiological processes in the brain, including (a) its interaction with the
soluble guanylate cyclase, thus yielding cGMP and resulting in signal transduction through
12
cGMP-dependent protein kinases (Jurado et al. 2005), (b) the reversible inhibition of mitochon-
drial cytochrome oxidase, thus transiently inhibiting mitochondrial respiration (Brown & Cooper
1994) and (c) protein post-translational modifications, mainly S-nitrosylation of cysteine resi-
dues (Derakhshan et al. 2007). The impact of NO on mitochondrial function mainly results from
its diffusion from cytosol to mitochondria, although a 144 kD mitochondrial nitric oxide syn-
thase (mtNOS) was reported to be localized in the inner mitochondrial membrane in rat brain
(Riobo et al. 2002; Valdez et al. 2006). NO-induced protein post-translational modifications are
relevant in mitochondria, as these organelles are major cellular sites of O
2
.-
production; NO can
react with O
2
.–
to yield peroxynitrite (ONOO
–
) (Beckman et al. 1990). ONOO
–
is a strong oxi-
dizing and nitrating species that inhibits mitochondrial NADH-ubiquinone reductase (complex I)
activity (Schopfer et al. 2000) with implications for Parkinson’s disease (Bharath & Andersen
2005); ATP synthase, cytochrome oxidase, aconitase, and creatine kinase are inhibited by nitra-
tion or ONOO
–
-mediated oxidation of cysteinyl residues (Konorev et al. 1998; Murray et al.
2003; Han et al. 2005)
1.1.3 Interdependence of mitochondrial energy-redox axis
Mitochondrial redox status cannot be viewed independent of their energy status, for (a) the
generation of oxidants reflects mitochondrial energy-transducing capacity and (b) mitochondrial
metabolism provides reducing equivalents which are required for the redox system. On the other
hand, mitochondrial energy metabolism cannot be secluded from their redox status, as the redox
modulation on mitochondrial proteins affects their functions and therefore energy-transducing
capacity.
13
Mitochondrial energy status influences oxidants production
Mitochondrial H
2
O
2
generation depends on the mitochondrial energy status: H
2
O
2
generation
is maximal in the resting state (state 4) and negligible in efficient respiration (state 3). H
2
O
2
pro-
duction in state 4 respiration was found to be 4-5 times higher than in state 3 respiration (Chance
et al. 1979; Cadenas et al. 2000). The generation of O
2
.-
by mitochondria is determined by
NADH/NAD
+
ratio, membrane potential, mitochondrial respiration, and ATP production
(Murphy 2009).
NADH-NADPH exchange: the role of nicotinamide nucleotide transhydrogenase (NNT)
As the ultimate electron donor for both the GSH and Trx systems, NADPH supply is critical
to maintain mitochondrial redox homeostasis. Mitochondrial NADPH is formed from mainly
three pathways: (1) NADP
+
-dependent isocitrate dehydrogenase (IDH), (2) malic enzyme, and (3)
nicotinamide nucleotide transhydrogenase (NNT), with the latter providing ~50% of NADPH
from the membrane potential-dependent conversion of NADH (Rydstrom 2006). NNT links mi-
tochondrial energy metabolism and redox status through the conversion of NADH, a reducing
equivalent generated by mitochondrial TCA cycle, and NADPH, the ultimate support for redox
systems. The crucial role of NNT in regulating cellular redox homeostasis, energy metabolism,
and apoptotic pathways was demonstrated in PC12 cells, where knockdown of NNT results in an
altered redox status encompassed by decreased cellular NADPH levels and GSH/GSSG ratios
and increased H
2
O
2
levels. The activation of redox-sensitive signaling (JNK) by H
2
O
2
after NNT
suppression impairs mitochondrial energy-transducing capacity, induces mitochondrion depend-
ent intrinsic apoptosis and results in decreased cell viability (Yin et al. 2012).
14
Protein redox modulation
The sulfhydryl groups in a wide variety of protein cysteine residues are redox-sensitive and
vulnerable to chemical modifications due to redox changes in the cellular environment (Dalle-
Donne et al. 2008). Increased H
2
O
2
leads to oxidation of cysteine residues to sulfonic acid, for-
mation of disulfide bonds, and glutathionylation, all of which alter protein functions.
Protein sulfhydryl groups can form mixed disulfide with GSH due to the changes in cellular
redox environment. The reversible glutathionylation markedly affects protein functions, and was
suggested to act as a protective mechanism to prevent key cysteine residues from irreversible
damages (Klatt & Lamas 2000).
Mitochondrial proteins are also subject to NO modification (S-nitrosylation and nitration),
resulting in collapse of the mitochondrial membrane potential and induction of mitochondrial
permeability transition, leading to mitochondrion-driven apoptosis with the concomitant release
of cytochrome c from the organelles and activation of downstream signaling apoptotic cascades
(Stewart et al. 2000; Cooper et al. 2003; Mannick 2007). Succinyl-CoA-transferase (SCOT) and
F
1
-ATPase were identified to be the targets in the situation of elevated nNOS in brain aging
(Lam et al. 2009).
1.2 Metabolic triad: mitochondria, insulin signaling, and JNK signaling
The mitochondrial energy-redox status determines the release of second messengers such as
H
2
O
2
to the cytosol. H
2
O
2
, diffused from mitochondria, is actively involved in the regulation of
cytosolic redox-sensitive signaling pathways such as insulin signaling and JNK signaling; in turn,
these signaling pathways regulate mitochondrial function, thus establishing a coordinated meta-
15
IRS
PI3K
Akt
PTEN
PIP
2
PIP
3
PDK1
IR
H
2
O
2
Akt
JNK
bolic triad encompassed by an intricate signaling network with close connections to mitochon-
drial energy-redox status (Fig. 5).
IIS and mitochondrial function
In the brain, PI3K/Akt pathway is the major downstream pathway of insulin signaling that
promotes neuronal survival and synaptic plasticity (van der Heide et al. 2006). Glucose trans-
ports into the brain through facilitative transporters. Activation of insulin pathway promotes the
translocation of GLUT4 from an intracellular pool to the plasma membrane (Squire 2008). The
similar effect of insulin was also observed for GLUT3 trafficking (Uemura & Greenlee 2006),
which was induced by increased neuronal activity and was mediated by the NMDAR/Akt de-
pendent nNOS-cGMP-PKG pathway (Ferreira et al. 2011). Chronic insulin administration was
also found to enhance biosynthesis of GLUT3 in rat neurons (Uehara et al. 1997). Insulin signal-
Fig. 5 Metabolic triad in brain aging
Metabolic triad consists of mito-
chondria, insulin signaling, and JNK sig-
naling.
16
ing also impinges on mitochondrial function: (a) insulin signaling supports the functional integri-
ty of the electron transport chain by suppressing the FOXO1-HMOX1 pathway (Cheng et al.
2010); (b) PI3K/AKT pathway regulates mitochondrial biogenesis via ERRα (Li et al. 2013b); (c)
Akt translocates to cardiac muscle mitochondria and activates Complex V (Yang et al. 2009), to
neuroblastoma cells mitochondria where it phosphorylates GSK3β and β subunit of ATP syn-
thase (Bijur & Jope 2003), and to liver mitochondria, where it phosphorylates a number of mito-
chondrial-resident proteins including the subunits α and β of ATP synthase (Li et al. 2013a). Mi-
tochondrial H
2
O
2
is involved in the regulation of insulin signaling, due to the large quantities of
redox-sensitive cysteine residues in IR, IGF-1 receptor and IRS. Oxidation of cysteine residues
to Cys-OH, Cys-SG (S-glutathionylation) or disulfides promotes tyrosine autophosphorylation of
the IR (Storozhevykh et al. 2007), as well as the inhibition of phosphatases, such as PTP1B and
PTEN (Packer & Cadenas 2011), which are both negative regulators of IIS through the
dephosphorylation of IR/IRS and PIP3 (phosphatidylinositol-3,4,5-trisphosphate) respectively.
JNK signaling and mitochondrial function
JNK is a stress-responsive kinase that is involved in the regulation of several cellular pro-
cesses such as transcription, inflammation, and apoptosis (Vallerie & Hotamisligil 2010). JNK is
activated in primary glia cultures in response to TNF / , IL-1, UV light, heat shock, inhibitors
of protein synthesis, and mechanical injury. Incubation of cells with bacterial sphingomyelinase
and a cell-permeable ceramide stimulated JNK activity (Zhang et al. 1996). In primary cortical
neurons, upon stimulation by anisomycin or H
2
O
2
, JNK translocates to mitochondria. Associa-
tion of JNK with outer mitochondrial membrane results in the phosphorylation of multiple pro-
teins: phosphorylation of Bcl-2 and Bcl-x
L
leads to mitochondrion-driven apoptosis (Schroeter et
17
Regulation of
Energy Metabolism
JNK
Translocation to
Mitochondria
JNK
P
P
Regulation of
redox-sensitive
signaling
Regulation
of apoptosis
H
2
O
2
generation
JNK
H
2
O
2
al. 2003); phosphorylation of the E
1α
subunit of pyruvate dehydrogenase (PDH) results in the
inhibition of its activity (Zhou et al. 2008). PDH bridges cytosolic anaerobic and mitochondrial
aerobic energy metabolism and its inhibition results in a bioenergetic deficit expressed as a de-
crease in cellular ATP levels and an increase in lactate formation; the latter suggests a compensa-
tory effect by anaerobic glycolysis. The phosphorylation cascade triggered upon association of
JNK with the outer mitochondrial membrane is likely to be mediated by pyruvate dehydrogenase
kinase-2 (Zhou et al. 2009). JNK bisphophorylation (activation) and its association with the out-
er mitochondrial membrane increased as a function of age in rat brain (Zhou et al. 2009); this
was associated with a decreased in PDH activity and subsequent deficit in energy metabolism.
Thus, JNK is an important negative regulator of mitochondrial metabolic function. Impaired mi-
tochondrial function, on the other hand, is associated with increased release of H
2
O
2
that acti-
vates JNK signaling, which constitute a vicious cycle, leading to mitochondrial failure (Fig. 6).
Fig. 6 Regulation of energy metabolism
by the dynamic interaction be-
tween the redox-sensitive JNK sig-
nalling pathway and mitochondria
Activation (bisphosphorylation) of
redox-sensitive JNK by stress conditions or
mitochondrionally generated H
2
O
2
translo-
cates to the mitochondrion. The association
of JNK with the outer mitochondrial mem-
brane triggers a phosphorylation cascade
(partly mediated by PDH kinase-2) that
results in phosphorylation and inhibition of
PDH, impairment of energy metabolism
and greater generation of H
2
O
2
.
18
IIS-JNK interactions
JNK pathway has been shown to negatively regulate insulin signaling in different cell types
and promotes insulin resistance under conditions including inflammation, diabetes, and aging.
JNK phosphorylates IRS at Ser
307
, counteracting the insulin-mediated tyrosine phosphorylation
(Karpac & Jasper 2009). On the other hand, Akt inhibits JNK activation through the in vitro and
in vivo phosphorylation of MLK3 (mixed lineage kinase 3) (Barthwal et al. 2003). In primary
neurons, Akt regulates JNK signaling by binding to the JIP1 (JNK-interacting protein 1): the
Akt–JIP1 interaction prevents the binding of JIP1 to specific JNK targets, thereby reducing apop-
tosis elicited by excitotoxicity (Kim et al. 2002). In addition, Akt is found to inhibit ASK1 ac-
tivity and its downstream activation of JNK (Kim et al. 2001). The interplay between Akt and
JNK is demonstrated in ischemic brain injury, where Akt phosphorylates the pro-apoptotic Bcl-2
protein Bad and promotes neuronal survival whereas JNK phosphorylates Bad at a distinct site
leading to apoptosis (Wang et al. 2007).
1.3 Biochemistry and biology of lipoic acid
α-Lipoic acid is a naturally occurring dithiol compound synthesized by cells; it exists in both
R- and S-enantiomeric forms. R-Lipoic acid forms a covalent bond with lysine residues, and
therefore serves as an essential cofactor for the mitochondrial E
2
subunit of α-ketoacid dehydro-
genase complexes (Reed & Oliver 1968). Dietary sources of lipoic acid include animal tissues
such as muscle, kidney, liver, heart, and to a lesser extent, plants (Shay et al. 2009a).
Lipoic acid is also used as a dietary supplement in non-covalently bound form. Non-
covalently bound lipoic acid does not exchange with the metabolic cofactor in mitochondria, but
elicits a distinct set of biochemical activities due to its capability of equilibrating between differ-
19
ent subcellular compartments as well as extracellularly. Free Lipoic acid crosses cell membranes
through medium chain fatty acid transporter, Na
+
-dependent vitamin transporter, and a H
+
-linked
monocarboxylate transporter in the intestine (Machlin & Bendich 1987; May et al. 2007;
Takaishi et al. 2007). Lipoic acid distribution is mainly in liver, heart, and skeletal muscle but is
found in other tissues. A few studies showed that lipoic acid can cross blood brain barrier
(Panigrahi et al. 1996; Arivazhagan et al. 2002), but one study debates on its availability in the
brain (Chng et al. 2009).
As a potent redox modulator (Fig. 7), lipoic acid participates in a wide variety of biological
actions based mainly on its thiol-disulfide reaction (Patel 2008; Shay et al. 2009b). Briefly, lipo-
ic acid (a) facilitates GSH synthesis; (b) is involved in the redox control of glucose uptake and
metabolism; (c) was shown to inhibit the JNK pathway and the IRS-1 serine phosphorylation; (d)
increases pyruvate metabolism and PDH activity in hepatocytes (Walgren et al. 2004); (e) induc-
es the Nrf2-dependent transcription of phase II detoxification enzymes by oxidizing Keap1 and
facilitating dissociation of Nrf2 from Keap1 (Fig. 8).
Fig. 7 Lipoic acid: Energy and Redox Modulator
20
Considering the variety of redox-sensitive signaling and transcriptional pathways regulating
brain energy metabolism, lipoic acid has potential of modulating the cellular energy and redox
status. Lipoic acid has been used extensively in Germany for over 60 years as a therapy for dia-
betic neuropathy (Haak et al. 1999). Several clinical trials were also performed to test its thera-
peutic potential: lipoic acid along (Hager et al. 2007), or in combination with ω-3 (Shinto et al.
2014) slowed cognitive and functional decline in patients with Alzheimer’s disease. Moreover,
lipoic acid has been reported as potential therapeutic/nutritional agent in multiple age-related
disease models: lipoic acid has been found to restore the age-dependent impairment of long-term
potentiation (LTP) and glutamate release in rat hippocampus (McGahon et al. 1999); lipoic acid
effectively reverses insulin resistance and increases brain glucose uptake and LTP in a triple
transgenic mouse model of Alzheimer’s disease (Sancheti et al. 2013). Lipoic acid in combina-
tion with L-acetyl-carnitine restores mitochondrial biogenesis in the hippocampus (Aliev et al.
Fig. 8. Biological actions of lipoic acid
21
2009) and protected cortical neurons against -amyloid and H
2
O
2
toxic insults (Zhang et al.
2001).
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30
CHAPTER TWO
LIPOIC ACID RESTORES AGE-ASSOCIATED IMPAIRMENT OF BRAIN ENERGY ME-
TABOLISM THROUGH THE MODULATION OF AKT/JNK SIGNALING AND
PGC1 TRANSCRIPTIONAL PATHWAY
SUMMARY – This study examines the progress of a hypometabolic state inherent in brain aging
with an animal model consisting of Fischer 344 rats of young, middle, and old ages. Dynamic
microPET scanning demonstrated a significant decline in brain glucose uptake at old ages, which
was associated with a decrease in the expression of insulin-sensitive neuronal glucose transport-
ers GLUT3/4 and of microvascular endothelium GLUT1. Brain aging was associated with an
imbalance of the PI3K/Akt pathway of insulin signaling and JNK signaling and a downregula-
tion of the PGC1 mediated transcriptional pathway of mitochondrial biogenesis that im-
pinged on multiple aspects of energy homeostasis. R-(+)-lipoic acid treatment increased glucose
uptake, restored the balance of Akt/JNK signaling, and enhanced mitochondrial bioenergetics
and the PGC1 -driven mitochondrial biogenesis. It may be surmised that impairment of a mito-
chondria-cytosol-nucleus communication is underlying the progression of the age-related hypo-
metabolic state in brain; the effects of lipoic acid are not organelle-limited but reside on the func-
tional and effective coordination of this communication that results in improved energy metabo-
lism.
31
INTRODUCTION
Brain is a highly energy-demanding organ, which represents only 2% of the body weight but
accounts for 25% of the total glucose utilization. Brain aging features pronounced energy deficit
accompanied by neuronal loss, impaired cognition and memory, and increased risk for neuro-
degenerative disorders. This hypometabolic state is a consequence of a decreased energy-
transducing capacity of mitochondria, partly attributed to reduced rates of electron transfer, de-
creased inner membrane potential, and impaired ATPase activity (Navarro & Boveris 2007). The
activity of enzymes or complexes that catalyze the entry of acetyl-CoA into the tricarboxylic acid
cycle, i.e., pyruvate dehydrogenase and succinyl-CoA transferase, decreases as a function of age
in brain (Lam et al. 2009; Zhou et al. 2009), as well as the activity of the tricarboxylic acid regu-
latory enzyme, -ketoglutarate dehydrogenase (Gibson et al. 2004). Mitochondrial biogenesis
could be viewed as an adaptive response to adjust bioenergetic deficits to alterations in the extra-
cellular and intracellular energy–redox status (Onyango et al. 2010).
Mitochondria are effective sources of H
2
O
2
, which is involved in the regulation of redox-
sensitive signaling and transcriptional pathways. Mitochondrial function is also regulated by sig-
naling and transcriptional pathways (Yin et al. 2012; Yin et al. 2013). The PI3K/Akt route of
insulin signaling is implicated in neuronal survival and synaptic plasticity, via –among other ef-
fects– maintenance of the functional integrity of the mitochondrial electron transfer chain and
regulation of mitochondrial biogenesis (Cohen et al. 2004; Cheng et al. 2010); conversely, mito-
chondrially generated H
2
O
2
plays an important role in the insulin receptor (IR) autophosphoryla-
tion in neurons (Storozhevykh et al. 2007). In human neuroblastoma cells, Akt translocates to the
mitochondrion and subunit β of ATPase is a phosphorylation target (Bijur & Jope 2003). Mito-
chondrial oxidants are also involved in the activation of c-Jun N-terminal kinase (JNK) (Nemoto
32
et al. 2000; Zhou et al. 2008), which, in turn, regulates mitochondrial bioenergetics by modulat-
ing the activity of pyruvate dehydrogenase in primary cortical neurons (Zhou et al. 2008). JNK
translocates to the mitochondrion and associates with the outer mitochondrial membrane and
triggers a phosphorylation cascade that results in phosphorylation (inhibition) of the pyruvate
dehydrogenase complex; there is an inverse relationship between the increasing levels of active
JNK associated with the outer mitochondrial membrane and the decreasing pyruvate dehydro-
genase activity in rat brain as a function of age (Zhou et al. 2009). This translated into decreased
cellular ATP levels and increased lactate formation.
R-(+)-lipoic acid (1,2-dithiolane-3-pentanoic acid) acts as a cofactor in energy metabolism
and the non-covalently bound form as a regulator of the cellular redox status. The effects of lipo-
ic acid on the cellular energy and redox metabolism, physiology, and pharmacokinetics have
been extensively reviewed (Patel & Packer 2008; Shay et al. 2009). Lipoic acid modulates dis-
tinct redox circuits because of its ability to equilibrate between different subcellular compart-
ments as well as extracellularly and is an essential cofactor for the mitochondrial E
2
subunit of
-ketoacid dehydrogenase complexes. As a potent redox modulator, lipoic acid participates in a
wide variety of biological actions based mainly on thiol-disulfide exchange reactions with key
redox-sensitive cysteines on target molecules. Considering the variety of redox-sensitive signal-
ing and transcriptional pathways regulating brain energy metabolism, lipoic acid has potential of
modulating the cellular energy and redox status.
This study was aimed at characterizing changes in substrate supply and energy metabolism
and their modulation by signaling pathways, and mitochondrial biogenesis in brain as a function
of age as well as the potential role of lipoic acid in restoring normal brain energy metabolism via
thiol-disulfide exchange reactions.
33
EXPERIMENTAL PROCEDURES
Animals and lipoic acid supplement
Male Fisher 344 rats of different ages (6, 12 and 24 months) were purchased from the Na-
tional Institute of Ageing (NIA). Each rat was individually housed in the animal facility under
standard conditions (12/12 light-dark cycle, humidity at 50 ± 15%, temperature 22 ± 2° C and 12
air changes/h). Rats at different ages (6-, 12- and 24 month old) were fed with 0.23% (wt/vol) R-
(+)-lipoic acid in the drinking water for 3 weeks. Age-matched rats fed with normal water were
used as control groups. All procedures were approved by the local Animal Care and Use Com-
mittee. The examined lipoic acid concentrations (0.08%, 0.14%, and 0.23% (wt/vol) estimated
40.5-, 60.3-, and 99.1 mg/kg per day) in drinking water for 3 weeks revealed that 0.23% (wt/vol)
was more effective in most biochemical assays. Food intake was not affected by lipoic acid sup-
plementation during the three weeks of treatment and there was no statistically significant differ-
ence in body weight between control group and lipoic acid–supplemented group.
Isolation of rat brain mitochondria
Upon completion of LA treatment, both LA-treated and control groups were sacrificed after
euthanasia by CO
2
inhalation for 1-2 min and the brains were rapidly dissected on ice. Cerebel-
lum, brain stem, and hippocampi were removed and the cortices were rapidly minced and ho-
mogenized at 4° C in mitochondrial isolation buffer (MIB) (pH 7.4), containing sucrose (250
mM), HEPES (20 mM), EDTA (1 mM), EGTA (1 mM), plus 0. 5% (w/v) bovine serum albumin
and freshly supplemented with 25 μl/100 ml protease inhibitor cocktail, and 100 μl/100 ml phos-
phatase inhibitors. A portion of the cortex homogenates was collected for the Western Blot anal-
ysis and the rest were then centrifuged at 1500g for 5 min. The post-nuclear supernatants were
34
collected and crude mitochondria were pelleted by centrifugation at 21,000g for 10 min. The re-
sulting mitochondrial pellet was resuspended in 15% Percoll made in MIB, layered over a pre-
formed 23%/40% Percoll discontinuous gradient, and centrifuged at 31,000g for 10 min. The pu-
rified mitochondria were collected at the 23%/40% interface and washed with 10 mL MIB by
centrifugation at 16,700g for 15 min. The loose pellet was collected and transferred to a micro-
centrifuge tube and washed in MIB by centrifugation at 9000g for 8 min. The resulting mito-
chondrial pellet was resuspended in MIB to an approximate concentration of 5 mg/mL. Mito-
chondrial samples were used immediately for respiratory measurements or stored at -80° C for
later protein and enzymatic assays. The purity of the mitochondrial fraction was assessed as pre-
viously described (Zhou et al. 2008).
Membrane preparation
Isolation of membrane-containing fractions was performed as described previously (Piroli et
al. 2007; Grillo et al. 2009). Briefly, rats were decapitated and brain cortices were isolated, fro-
zen on dry ice and stored at −70 °C until use. Brain cortices from each individual rat was ho-
mogenized in ice-cold homogenization buffer (0.32 M sucrose, 2 mM EDTA, 2 mM EGTA, 20
mM HEPES, with 25 μl/100 ml protease inhibitor cocktail, 100 μl/100 ml phosphatase inhibitors)
and centrifuged for 10 min at 500 g at 4° C. The total membrane fraction (supernatant) was saved;
a portion of this fraction was centrifuged at 31,000 g for 30 min at 4° C. The resulting pellet,
which contained the plasma membrane fraction, was resuspended in PBS. Protein concentrations
of the total membrane fraction and the plasma membrane fraction were determined by the meth-
od of Bradford (1976) using bovine serum albumin (BSA) as a standard.
35
DNA isolation and quantification
Total DNA from rat brain was prepared using Wizard Genomic DNA Purification Kit
(Promega Corporation, Madison, WI, USA) and following the manufacturer’s instructions. The
relative copy numbers of mitochondrial and nuclear DNA were determined by real-time PCR
with primers specific to the COX3 (mitochondrial) and 18SrDNA (nuclear) genes, 100 ng DNA,
and SYBRGreen PCR master mix (Bio-Rad, Hercules, CA, USA) on an iCycler real-time PCR
machine (Bio-Rad).
MicroPET imaging
MicroPET imaging was conducted at the Molecular Imaging Center at the Department of
Radiology, University of Southern California, under the guidance of Dr. Peter Conti. Briefly,
both LA treated and control groups were fasted for 6 h on a water only diet and then sedated us-
ing 2% isoflurane by inhalation and administered the radio tracer 2-deoxy-2 [
18
F]fluoro- D-
glucose intravenously. Blood for glucose concentration was measured before the administration
of the tracer to ensure that changes in glucose metabolism during [
18
F]- FDG-PET imaging were
not due to differences in starting blood glucose levels but the intrinsic activity of the brain. Rats
were placed on a scanner bed with a warming bed to maintain animal body temperature and un-
derwent scanning for duration of 10 min using a Siemens MicroPET R4 scanner with a 19 cm
(transaxial) by 7.6 cm (axial) field of view. This system has an absolute sensitivity of 4% with a
spatial resolution of ~1.3 mm at the center of view. This is a non-invasive technique and the rats
were sedated during the entire duration. Additionally, the rats underwent microCT scanning for 5
min (Siemens Inveon) with intravenous contrast material for coregistration with microPET (AM-
IDE, Free Software Foundation, Inc., Boston, MA, USA). This provides high resolution (~1 mm)
36
information of brain structure and enables identification in the extent of brain atrophy. Region of
Interest (ROI) was defined (AMIDE, Free Software Foundation, Inc., Boston, MA), and Stand-
ard Uptake Values (SUV) was calculated based also on dose, time, and body weight.
Polarographic assays and ATP measurements
Oxygen consumption was measured with a Clarktype electrode (Hansatech, Norfolk, UK) as-
sembled to a thermostatic water jacket. The assay buffer consisted of 70 mM sucrose, 220 mM
mannitol, 10 mM KH
2
PO4, 5 mM MgCl
2
, 1 mM EGTA, 2 mM HEPES, and 0.5% (w/v) bovine
serum albumin, pH 7.4. The mitochondrial suspension was maintained under continuous stirring
with a magnetic agitator in the electrode chamber. State 4 respiration was measured with com-
plex I substrates (5 mM glutamate + 5 mM malate) and state 3 respiration in the presence of 0.41
mM ADP. Brain cortex homogenates were lysed in an equal volume of perchloric acid (2 M) and
centrifuged for 10 min at 12000 g. Supernatants were neutralized with KHCO
3
(3 M) and recen-
trifuged at 12000 g. ATP in tissue extracts was quantitatively measured by a bioluminescence
assay that uses recombinant firefly luciferase and D-luciferin (Invitrogen, Carlsbad, CA, USA).
Metabolic flux analysis
Primary cortical neurons from day 18 (E18) embryos of female Sprague-Dawley rats were
cultured on Seahorse XF-24 (Seahorse BioSciences, Billerica, MA, USA) plates at a density of
75,000 cells/well. Neurons were grown in Neurobasal Medium + B27 supplement (Invitrogen,
Carlsbad, CA, USA) for 10 days prior to experiment. Cells were treated with control vehicle, R-
(+) lipoic acid (20 M), LY294002 (50 M), and R-(+) lipoic acid (20 M) + LY294002 (50
M), and the assays were conducted 18 h post-treatment. On the day of metabolic flux analysis,
37
media was changed to unbuffered DMEM (DMEM base medium supplemented with 25-mM
glucose, 1 mM sodium pyruvate, 31 mM NaCl, 2 mM GlutaMax (Invitrogen, Carlsbad, CA,
USA); pH 7.4) and incubated at 37° C in a non-CO
2
incubator for 1 hour. All medium and injec-
tion reagents were adjusted to pH 7.4 on the day of assay. Using the Seahorse XF-24 (Seahorse
BioSciences) metabolic analyzer, 3 baseline measurements of oxygen consumption rate (OCR)
were sampled prior to sequential injection of mitochondrial inhibitors. Three metabolic determi-
nations were sampled following addition of each mitochondrial inhibitor prior to injection of the
subsequent inhibitors. The mitochondrial inhibitors used were oligomycin (4 M), FCCP (car-
bonyl cyanide 4-(trifluoromethoxy)- phenylhydrazone) (1 M), and rotenone (1 M). OCR was
automatically calculated and recorded by the Seahorse XF-24 software. After the assays, protein
level was determined for each well to confirm equal cell density per well.
Enzyme activity assays and H
2
O
2
measurement
ATPase (complex V) activity was measured in purified mitochondria from rat brain cortex:
10 g of broken mitochondria were added to 200 l reaction buffer containing 250 mM sucrose,
50 mM HEPES, pH 8.0, 5 mM MgSO
4
, 2.5 mM sodium phosphoenolpyruvate, 2 g antimycin, 1
l of PK/LDH mixture, and 2.5 mM ATP. Reaction was initiated by addition of 0.35 mM NADH
and initial rates were measured at 340 nm at 25° C (
340
= 6.22 mM
–1
cm
–1
). Complex I activity
was assessed in isolated mitochondria (20 g) using Complex I Enzyme Activity Microplate As-
say Kit (Mitosciences, Eugene, OR, USA) following the manufacturer’s instructions. H
2
O
2
gen-
eration from isolated brain cortical mitochondria was determined by the Amplex Red /Peroxidase
Assay kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.
38
Immunoprecipitation
Immunoprecipitation was used to detect the lysine-acetylation levels of Sirtuin substrates, i.e.,
PGC1α. Brain cortex homogenate was subjected to immunoprecipitation by using Pierce Coated
Plate IP Kit. Immunoprecipitated proteins were boiled in non-reducing sample buffer (Thermo
Scientific, Rockford, IL, USA) and then detected by Western blot.
Western blot analysis
Brain cortex homogenates and mitochondria were solubilized in SDS sample buffer, separat-
ed by SDS/PAGE, and transferred onto PVDF membranes. Using appropriate antibodies, the
immunoreactive bands were visualized with an enhanced chemiluminescence reagent. The blots
were quantified using UN-SCAN-IT gel 6.1 (Silk Scientific, Inc., Orem, UT, USA).
Immunocytochemistry
Primary cortical neurons from day 18 (E18) embryos of female Sprague-Dawley rats were
cultured on pre-coated chamber slides. Neurons were grown in Neurobasal Medium +B27 sup-
plement for 10 days prior to experiment. Cells were treated with either vehicle or R-(+)-lipoic
acid (20 M) for 18 h followed by fixation with 4% paraformaldehyde. For immuno-fluorescent
staining, fixed cells were washed in PBS three times, and then blocked (1hr RT, PBS with 5%
goat Serum and 0.5% triton x-100), immuno-stained using antibodies directed against PDH E
1
(1:200, 4
o
C overnight, Mitosciences, Eugene, OR, USA) and -KGDH (1:200, 4
o
C overnight,
Proteintech Group Inc, Chicago, IL, USA) followed by three times of washing and secondary
antibodies Fluorescein goat anti-mouse and CY3-conjugated goat anti-rabbit (1:500, Chemicon,
Ramona, CA, USA, 1h at RT) respectively. Slides were mounted with anti-fade mounting medi-
39
um with DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were taken
using a fluorescent microscope, normalized and analyzed with the slide book software (Intelli-
gent Imaging Innovations Inc, Santa Monica, CA, USA).
Statistical analysis
Number of animals for statistically significant outcomes in [
18
F]-FDG-PET experiments was
calculated as n = 5 to observe a significance of P < 0.05 for differences between control and
treatment group averages with either 15% or 20% coefficient of variation (CV) (Eckelman et al.,
2007). Data are reported as means ± SEM of at least 5-6 independent experiments. Significant
differences between mean values were determined by Student t-test or one way analysis of vari-
ance (ANOVA) followed by a Newman-Keuls post hoc analysis.
RESULTS
Effects of lipoic acid on brain glucose uptake and glucose transporter expression
Fig. 1A shows the [
18
F]-FDG-PET images (dynamic microPET scanning) of 6- and 24
month-old male rat brains. The standardized glucose uptake value (SUV) that assesses the kinet-
ics of glucose uptake, in the 24 month-old rat brain was significantly lower ( 14%) than that in
the 6 month-old rat brain at the end of the scan (Fig. 1B). There were no significant differences
between 6- and 12 month-old SUV values. Lipoic acid (0.23% wt/vol in the drinking water for 3
weeks) increased SUV by 40% in the 24 month-old rat brains (Fig. 1A,B) but had no effect at
younger ages.
40
Fig. 1 Effect of lipoic acid on brain glu-
cose uptake
(A) [
18
F]-FDG microPET repre-
sentative images of glucose uptake in 6-
month control rats, 24-month control rats,
and 24-month rats fed with lipoic acid as
described in the Methods section. (B)
Glucose Standard Uptake Values (SUV)
as a function of age and effect of lipoic
acid. *p < 0.05, n = 6.
Expression of glucose transporters, which is closely related to glucose supply to the brain, is
shown in Fig. 2. The protein level of neuronal glucose transporter 3 (GLUT3) decreased by 30%
in 24 month-old rat brains compared to the 6 month-old brains, whereas lipoic acid treatment
partly restored GLUT3 in the 24 month-old group (Fig. 2C). Likewise, neuronal GLUT4 expres-
sion decreased sharply with age, and lipoic acid treatment restored its expression slightly (Fig.
2D). GLUT1 (55 kDa), across the blood brain barrier, decreased marginally as a function of age;
lipoic acid, however, had no effect on its expression (Fig. 2A). Interestingly, expression of the
glial glucose transporter, GLUT1 (45 kDa) increased with age, and lipoic acid treatment had no
effect on its expression (Fig. 2B). It is well established that insulin signaling promotes the trans-
location of GLUT4 from a mobilizable pool to the plasma membrane (Grillo et al. 2009). Fig. 2E
41
shows that membrane-bound GLUT4 does not change significantly with age but lipoic acid facil-
itates increased expression of membrane-bound GLUT4 (ratio of GLUT4 in the plasma mem-
brane fraction over that in total membrane fraction) in 6- and 24 month-old rat brains (Fig. 2E).
Fig. 2 Effect of lipoic acid on age-dependent changes in brain glucose transporters expression
Equal amount of homogenate samples from brain cortices of Fischer 344 rats were loaded on the gel.
Expression of (A) endothelial GLUT1 55 kDa; (B) glial GLUT1 45 kDa; (C) neuronal GLUT3; (D) neuronal
GLUT4. (E) Lipoic acid induced the translocation of GLUT4 from the cytosol to plasma membrane. GLUT4
translocation was assessed by the relative expression of GLUT4 on the plasma membrane fraction and total
membrane. Na
+
/K
+
ATPase and -actin were used as loading control for plasma membrane and total membrane,
respectively. Top panel: the purity of plasma membrane fraction was determined by Na
+
/K
+
ATPase and
GAPDH. *p < 0.05, n ≥ 6.
42
Effects of lipoic acid on Akt- and JNK signaling pathways
Phosphorylation of Akt on Ser
473
by upstream signals results in its activation; phosphoryla-
tion on Thr
308
is largely constitutive. Phosphorylation of Akt at Ser
473
in brain cortices from 24
month-old rats is substantially lower than that from 6 month-old rats; treatment with lipoic acid
significantly increased the levels of Akt phosphorylation (Fig. 3A). Phosphorylation of
GSK3at Ser
9
by Akt results in its inhibition: the percentage of GSK3 phosphorylated at Ser
9
decreases with age and lipoic acid significantly increased inhibition of GSK3 (and, thereby its
pro-apoptotic effects) in 12- and 24 month-old rat brains (Fig. 3B). The effects of lipoic acid on
Akt activation (Fig. 3A) tally with those on GSK3 inhibition (Fig. 3B).
JNK activation (phosphorylation) increases with age (Fig. 3C) and dissimilar effects of lipoic
acid were observed on different age groups: lipoic acid increased pJNK expression levels in 6
month-old rat brains, whereas it decreased pJNK levels in 24 month-old rat brains (Fig. 3C).
The overall effect of lipoic acid seems to maintain a similar relative activity of JNK to Akt path-
ways in brain cortices from 6- and 24 month-old rats: this notion is supported by the pJNK/pAkt
ratios depicted in Fig. 3D.
43
Fig. 3 Lipoic acid restored the age-induced imbalance of Akt/JNK signaling
Equal amount of homogenate samples from brain cortices of Fischer 344 rats were loaded on the gel.
(A) Decreased activation (phosphorylation at Ser
473
) of Akt with age. (B) Decreased inactivation (phosphoryla-
tion at Ser
9
) of GSK3 with age. (C) Increased activation of JNK with age; lipoic acid increased JNK phos-
phorylation in the brain cortex homogenate from 6 month-old rats but decreased its phosphorylation in the brain
cortex homogenate from 24 month-old rats. (D) Lipoic acid restored the age-associated imbalance of Akt/JNK
signaling. Relative activity of JNK and Akt signaling was determined by assessing the relative active JNK and
Akt levels on the same membrane. (E) Increased IRS1 Ser
307
phosphorylation with age and reduction by lipoic
acid. (F) Decreased IRS1 Tyr
608
phosphorylation with age and restoration by lipoic acid.*p < 0.05, **p < 0.01,
n ≥ 6.
44
Residing upstream in the insulin pathway, IRS1 bridges insulin receptor and PI3K and is es-
sential for the activation of PI3K/Akt signaling cascade. Phosphorylation of IRS1 at Tyr
608
is re-
quired for the interaction of IRS1 with PI3K and the subsequent activation of PI3K/Akt pathway
(Sun et al. 1993; Rocchi et al. 1995). Conversely, phosphorylation of IRS1 at Ser
307
is inhibitory
and mediated by JNK, placing it as a pivotal node in the crosstalk between the JNK and
PI3K/Akt pathways. The levels of IRS1 phosphorylated at Ser
307
increase in rat brains as a func-
tion of age (Fig. 3E) whereas those phosphorylated at Tyr
608
show a slight decrease (Fig. 3F).
Lipoic acid increased Tyr
608
phosphorylation and decreased Ser
307
phosphorylation of IRS1; the
effects were more pronounced in old animals (24 month-old rat brains) (Fig. 3E,F). The decrease
in Ser
307
phosphorylation of IRS1 elicited by lipoic acid matched its effect on the pJNK/pAkt
ratios (Fig. 3D).
Insulin-like effect of lipoic acid on cellular bioenergetics
Supplementation of primary cortical neurons with lipoic acid resulted in a substantial in-
crease of oxygen consumption rates (OCR) (Fig. 4A): lipoic acid increased basal respiration,
OXPHOS-induced respiration, and maximal respiratory capacity by 27.3-, 33.7-, and 37.5%, re-
spectively. The reserve capacity was augmented by 47.6% by lipoic acid (Table 1). These en-
hancing effects by lipoic acid were suppressed by LY294002, a specific inhibitor of PI3K; this
may be interpreted as lipoic acid exerting its effects upstream of PI3K and in agreement with the
increased levels of IRS1 phosphorylated at Tyr
608
(Fig. 3F). (Similar effects of lipoic acid were
observed in a mixture of hippocampal/cortical neurons from a triplet transgenic mouse model of
Alzheimer's disease). The lipoic acid-mediated increase in the bioenergetic parameters may be
accounted for in terms of an increase in mitochondrial density in primary cortical neurons (pre-
45
treated with 20 µ M lipoic acid for 18 h) as shown by the increased expression of pyruvate dehy-
drogenase E
1
subunit (thus enhancing acetyl-CoA supply to the tricarboxylic acid cycle) and of
-ketoglutarate dehydrogenase (Fig. 4B) and by the DNA
mit
/DNA
nu
values (COX3 and
18SrDNA representing mitochondrial genome and nuclear genome, respectively) (Fig. 4D); the
former data were confirmed by the increased protein expression of pyruvate dehydrogenase E
1
subunit, -ketoglutarate dehydrogenase, and complexes II and V of the mitochondrial respiratory
chain (Fig. 4C). The latter, COX3/18SrDNA ratios, indicate that the increase in mitochondrial
density elicited by lipoic acid supplementation was inhibited by LY294002 and compound C,
inhibitors of PI3K and AMPK, respectively (Fig. 4D). The role of AMPK in mitochondrial bio-
genesis is further examined in Fig. 5.
46
Table 1
Bioenergetic Parameters of Primary Cortical Neurons
Effect of Lipoic Acid
_______________________________________________________________________________________________________
___________________
OCR (pmoles/min)
_________________
Control LA LY294002 LA +
LY294002
_______________________________________________________________________________________________________
Basal respiration 344 ± 27 439 ± 55* 225 ± 18 285 ± 17
OXPHOS-induced respiration 190 ± 29 254 ± 63* 124 ± 20 149 ± 21
H+ leak-induced respiration 154 ± 11 184 ± 31* 100 ± 8 136 ± 12
Maximal respiratory capacity 691 ± 63 950 ± 114* 396 ± 45 467 ± 36
Non-mitochondrial respiration 114 ± 10 142 ± 27* 55 ± 7 66 ± 12
Reserve capacity 347 ± 69 512 ± 127* 171 ± 49 182 ± 40
_______________________________________________________________________________________________________
Assay conditions as described in the Methods section. *p < 0.05 versus control.
p < 0.05
versus lipoid acid treatment group.
Fig. 4 Bioenergetics of primary cortical neurons
(A) Cells were treated with control vehicle, R-(+)-lipoic acid (20 M), LY294002 (50 M), and
R-(+) lipoic acid (20 M) + LY294002 (50 M) for 18 h. Oxygen consumption rate (OCR) was deter-
mined using Seahorse XF-24 as described in the Methods section. (B) Lipoic acid increased expression of
PDH-E
1
and -KGDH. Cells were treated with vehicle or R-(+)-lipoic acid (20 M) for 18 h before they
were subjected to immuno-fluorescent staining. (C) Lipoic acid increased expression of Complex II-
SDHB, CV- , CV- , PDH-E
1
and -KGDH. After treated with vehicle or R-(+)-lipoic acid (20 M) for
18 h, cells were harvested and lysed in Mammalian Protein Extraction Reagent (M-PER). (D) Lipoic acid
increased mitochondrial density and was sensitive to PI3K (LY294002) and AMPK (compound C) inhibi-
tors. *p < 0.05, n = 5 wells per group.
47
Lipoic acid activates AMPK-Sirt1-PGC1 -NRF1 transcriptional pathway and stimulates mito-
chondrial biogenesis
The full activation of PGC1 –the master regulator of mitochondrial biogenesis– requires its
phosphorylation and deacetylation. The phosphorylation of PGC1 by AMPK at Thr
177
and
Ser
538
appears to be a requirement for the induction of the PGC1 promoter (Jager et al. 2007).
AMPK is activated through the phosphorylation at Thr
172
on the (catalytic) subunit; the levels
of AMPK phosphorylated at Thr
172
decreased with age whereas lipoic acid elicited a robust in-
crease of active AMPK in the brain of 12- and 24-month-old rats (Fig. 5A). Also, PGC1 phos-
phorylation by AMPK facilitates the subsequent deacetylation by Sirt1 (Canto et al. 2009). The
expression level of Sirt1, a NAD-dependent deacetylase, remained unchanged during aging but
treatment with lipoic acid significantly increased Sirt1 expression in the brain of 24 month-old
rats (Fig. 5B).
The total PGC1 expression in rat brain cortex decreased as a function of age and lipoic acid
elicited a slight but significant enhancement of the expression levels in the brain cortex of 24
month-old rats (Fig. 5C). The activity of PGC1 is negatively correlated with its relative acetyla-
tion level, which was significantly decreased in the brain of 24 month-old rats upon lipoic acid
treatment (Fig. 5D). It may be surmised that brain aging is associated with an apparent decrease
in PGC1 expression and activity and that the effects of lipoic acid are more evident at old ages.
NRF1 has been identified as a downstream target of PGC1 and an important transcription
factor for mitochondrial biogenesis that not only stimulates the expression of mitochondrial pro-
teins such as OxPhos components but also regulates the expression of Tfam and thereby affects
mtDNA replication and expression (Scarpulla 2008). The activation of NRF1 requires the inter-
action with PGC1 and hence it is not surprising that its expression is regulated by AMPK
48
(Bergeron et al. 2001). NRF1 expression levels decreased as a function of age (Fig. 5E), and li-
poic acid increased its expression in the brains of both 6- and 24 month-old rats.
Taken together, a decreased AMPK-Sirt1-PGC1 -NRF1 transcriptional pathway as a func-
tion of age results in diminished mitochondrial biogenesis; accordingly, DNA
mit
/DNA
nu
values
(COX3 and 18SrDNA representing mitochondrial genome and nuclear genome, respectively)
decreased with age (Fig. 5F). As before, lipoic acid treatment enhanced mitochondrial biogenesis
in brain of old animals (Fig. 5F).
Fig. 5 Effect of lipoic acid on PGC1 transcriptional pathway and mitochondrial biogenesis
Equal amount of homogenate samples from brain cortices of Fischer 344 rats were loaded on the gel.
Percentages (relative to 6-month old rats) of (A) p-AMPK-Thr
172
; (B) Sirt1; (C) PGC1 ; (D) PGC1 acetylation
levels; (E) NRF1. (F) Mitochondrial biogenesis was assessed by the ratio of COX3/18SrDNA, determined by
real-time PCR. *p < 0.05, n ≥ 6.
49
Lipoic acid rescues the decline in mitochondrial bioenergetics associated with age
Data shown on the effects of lipoic acid on the different components of the AMPK-Sirt1-
PGC1 transcriptional pathway and resulting in enhanced mitochondrial biogenesis (Fig. 5) sug-
gest a more robust mitochondrial bioenergetic efficiency. Accordingly, lipoic acid treatment
augmented brain cortex ATP levels (Fig. 6A); ATP content in 24 month-old rats was only 70%
of that in their younger counterparts, while lipoic acid treatment increased it by 15% (Fig. 6A).
The increased ATP levels in the brain cortex of 24 month-old rats was associated with a substan-
tial increase (41%) in ATP synthase activity (Fig. 6B). It was shown previously that the respira-
tory control ratios (RCR) of rat brain mitochondria respiring on complex I substrates (gluta-
mate/malate) decreased as a function of age, and the decline is accounted for largely by an in-
crease in state 4 respiration while state 3 respiration remained somehow constant (Lam et al.
2009). Consistent with this observation, lipoic acid increased the respiratory control ratio of
brain cortical mitochondria, an effect mainly driven by a diminished state 4 respiration (20%);
the latter effect correlated with decreased formation of H
2
O
2
during state 4 respiration (Fig.
6C,D).
Pyruvate dehydrogenase (PDH) catalyzes the oxidative decarboxylation of pyruvate to ace-
tyl-CoA, thus furnishing substrates for the tricarboxylic acid cycle. Inactivation of PDH occurs
upon phosphorylation in the E
1
subunit; hence, an increase in pPDH/PDH values is associated
with limited delivery of activated carbon units to the tricarboxylic acid cycle and diminished
formation of reducing equivalents to support respiratory chain activity. Fig. 6E shows a substan-
tial increase in the pPDH/PDH ratio in the brain of 24 month-old rats as compared with that of 6
month-old animals; these effects are ameliorated by treatment with lipoic acid. It is noteworthy,
that JNK activation (bisphosphorylation) was reported to increase with age in rat brain as well as
50
Fig. 6 Lipoic acid restored age-associated decline in mitochondrial bioenergetics in rat brain cortex
(A) ATP levels in the brain cortex homogenate of 6- and 24 month-old rats. (B) Effect of lipoic acid
on ATPase activity of brain cortex mitochondria of 24 month-old rats. (C) Effect of lipoic acid on respiratory
control ratios and (D) H
2
O
2
production of brain cortex mitochondria of 24 month-old rats. (E) pPDH/PDH
values in brain cortex mitochondria from 6- and 24 month-old rats. (F) Expression of complex II-SDHB,
COX-1, and CV- as a function of age. *p < 0.05, **p < 0.01, ***p < 0.001, n ≥ 6.
it translocation to mitochondria where it triggers a phosphorylation cascade that results in phos-
phorylation (inhibition) of the E
1
subunit of PDH (Zhou et al. 2008). The effect of lipoic acid on
PDH activity is highly likely driven by its inhibition of JNK (see Fig. 3C).
The expression levels of Complex II-SDHB, COX-I, and CV- of the mitochondrial respira-
tory chain decreased with age; in every instance, lipoic acid treatment resulted in an increased
expression of the aforementioned complexes in the brains of 24 month-old rats (Fig. 6F). Lipoic
acid significantly increased complex I activity (30%), whereas there was no significant effect on
complex IV activity (not shown).
51
DISCUSSION
This study characterized the age-associated impairment in brain glucose uptake, mitochon-
drial bioenergetics and biogenesis, and the regulatory signaling and transcriptional pathways that
impinge on the mitochondrial energy-transducing capacity. The beneficial effects of lipoic acid
on energy metabolism in brain cortex reported here are interpreted in terms of lipoic acid-
mediated regulation of redox-sensitive regulatory pathways via thiol-disulfide exchange reac-
tions. A direct interaction of lipoic acid with covalently bound lipoamide in the pyruvate dehy-
drogenase and -ketoglutarate dehydrogenase complexes is ruled out because exogenously ad-
ministered lipoic acid cannot equilibrate with these cofactors.
Insulin signaling affects various aspects of energy metabolism: active Akt promotes glucose
uptake, translocates to mitochondria in human neuroblastoma cells (Bijur & Jope 2003), and is
suggested to maintain mitochondrial electron-transport chain integrity by suppressing
FOXO1/HMOX1 and preventing heme depletion (Cheng et al. 2010). Insulin resistance is a pro-
nounced pathological phenomenon in age-related diseases, as aging is associated with decreases
in the levels of both insulin and its receptor (Frö lich et al. 1998). Although chronic exposure to
high level of oxidative stress could alter mitochondrial function and cause insulin resistance,
modest oxidative conditions are actually required for the activation of insulin signaling (Cho et
al. 2003). Therefore the effect of lipoic acid on insulin signaling most likely lies in its pro-
oxidant feature, oxidizing critical cysteine residues to disulfides. Possible targets of lipoic acid-
mediated oxidation could be the ones with abundant cysteine residues, including insulin recep-
tors (Cho et al. 2003; Storozhevykh et al. 2007), IRS1, and phosphatases (PTEN and PTP1B)
(Barrett et al. 1999; Loh et al. 2009). These thiol/disulfide exchange reactions are likely the basis
for the effects of lipoic acid in increasing phospho-Tyr
608
(Fig. 3F) and decreasing phospho-
52
Ser
307
(Fig. 3E) on IRS1. These effects are supported by the observation that the enhancing effect
of lipoic acid on mitochondrial basal respiration and maximal respiratory capacity was sensitive
to PI3K inhibition (Fig. 4A), thus suggesting that lipoic acid acted upstream of PI3K with IRS1
as one of the most plausible targets. As downstream targets of Akt signaling, the trafficking of
GLUT4 to the plasma membrane was induced by lipoic acid treatment. The effect of lipoic acid
on the biosynthesis of glucose transporters was also insulin-dependent, for chronic insulin ad-
ministration induced biosynthetic elevation of GLUT3 in rat brain neurons and L6 muscle cells
(Bilan et al. 1992; Taha et al. 1995; Uehara et al. 1997). Therefore increased efficiency of glu-
cose uptake into brain by lipoic acid could at least partly be accounted for by its insulin-like ef-
fect.
JNK activation increases in rat brain as a function of age as well as JNK translocation to mi-
tochondria and impairment of energy metabolism upon phosphorylation of the E
1
subunit of the
pyruvate dehydrogenase complex (Zhou et al. 2009). Data in this study indicate that lipoic acid
decreases JNK activation at old ages; this effect might be due to the attenuation of cellular oxida-
tive stress responses; in this context, lipoic acid was shown to replenish the intracellular GSH
pool (Busse et al. 1992; Suh et al. 2004).
Cross-talk between the PI3K/Akt route of insulin signaling and JNK signaling is expressed
partly as the inhibitory phosphorylation at Ser
307
on IRS1 by JNK, thus identifying the JNK
pathway as a negative feedback of insulin signaling by counteracting the insulin-induced phos-
phorylation of IRS1 at Tyr
608
. Likewise, FoxO is negatively regulated by the PI3K/Akt pathway
and activated by the JNK pathway (Karpac & Jasper 2009). Overall, insulin signaling has a posi-
tive impact on energy metabolism and neuronal survival but its aberrant activation could lead to
tumor and obesity (Finocchietto et al. 2011); JNK activation adversely affects mitochondrial en-
53
ergy-transducing capacity and induces neuronal death, but it is also required for brain develop-
ment and memory formation (Mehan et al. 2011). A balance between these survival and death
pathways determines neuronal function; as shown in Fig. 3D, lipoic acid restores this balance
(pJNK/pAkt) that is disrupted in brain aging: in aged animals, lipoic acid sustained energy me-
tabolism by activating the Akt pathway and suppressing the JNK pathway; in young animals,
increased JNK activity by lipoic acid met up with the high insulin activity to overcome insulin
over-activation and was required for the neuronal development.
Given the central role of mitochondria in energy metabolism, mitochondrial biogenesis is
implicated in various diseases. Fewer mitochondria are found in skeletal muscle of insulin-
resistant, obese, or diabetic subjects (Kelley et al. 2002; Morino et al. 2005). Similarly, PGC1α
–
/–
mice have reduced mitochondrial oxidative capacity in skeletal muscle (Lin et al. 2004). Data
from this study showed a reduced mitochondrial density and decreased expression and activity of
PGC1α in brain with age: evidence for the downregulation of the AMPK - Sirt1 pathway and the
PGC1α downstream effector NRF1 is shown in Fig. 5. Lipoic acid significantly enhanced mito-
chondrial biogenesis especially in old rats probably through the activation of AMPK-Sirt1-
PGC1α -NRF1 (Fig. 5). Mitochondrial biogenesis appears to be regulated by both insulin- and
AMPK signaling, as shown by changes in COX3/18SrDNA ratios by inhibitors of PI3K and
AMPK (Fig. 4D).
The increase in bioenergetic efficiency (ATP production) by lipoic acid was associated with
enhanced mitochondrial respiration and increased expression and catalytic activity of respiratory
complexes (Fig. 6). However, this bioenergetic efficiency is dependent on concerted action by
glucose uptake, glycolysis, cytosolic signaling and transcriptional pathways, and mitochondrial
metabolism. The enhancement of mitochondrial bioenergetics by lipoic acid may be driven by its
54
insulin-like effect (evidenced by the insulin-dependent increase in mitochondrial respiration in
primary neurons) and by the activation of the PGC1 transcriptional pathway leading to in-
creased biogenesis (evidenced by increasing expression of key bioenergetics components such as
complex V, PDH, and α-KGDH upon lipoic acid treatment).
The observation that AMPK activity declines with age in brain cortex suggests an impaired
responsiveness of AMPK pathway to the cellular energy status. The activation of AMPK re-
quires Thr
172
phosphorylation by LKB1 and CaMKK with a 100-fold increase in activity, fol-
lowed by a 10-fold allosteric activation by AMP (Hardie et al. 2012). It is highly likely that loss
of AMPK response to AMP allosteric activation is due to the impaired activity of upstream ki-
nases Lipoic acid may act as a mild and temporary stress that activates AMPK, the PGC1 tran-
scriptional pathway, and mitochondrial biogenesis, thereby accounting for increases in basal and
maximal respiratory capacity that enables vulnerable neurons in aged animals to adequately re-
spond to energy deficit, achieving a long-term neuroprotective effect. Hence, activation of
PGC1 by lipoic acid serves as a strategy to ameliorate brain energy deficits in aging.
PGC1 transgenic mice demonstrated enhanced neuronal protection and altered progression of
amyotrophic lateral sclerosis (Liang et al. 2011) and preserved mitochondrial function and mus-
cle integrity during aging (Wenz et al. 2009).
Overall, data in this study unveil an altered metabolic triad in brain aging, entailing a regula-
tory devise encompassed by mitochondrial function (mitochondrial biogenesis and bioenergetics),
signaling cascades, and transcriptional pathways, thus establishing a concerted mitochon-
dria/cytosol/nucleus communication. Specifically, brain aging is associated with the aberrant
signaling and transcriptional pathways that impinge on all aspects of energy metabolism includ-
ing glucose supply and mitochondrial metabolism. Mitochondrial metabolism, in turn, modifies
55
cellular redox- and energy- sensitive regulatory pathways; these constitute a vicious cycle lead-
ing to a hypometabolic state in aging. The prominent effect of lipoic acid in rescuing the meta-
bolic triad in brain aging is accomplished through modulation of regulatory pathways, achieving
an insulin-like effect: augmenting glucose uptake, restoring the Akt/JNK balance, enhancing mi-
tochondrial bioenergetics, and supporting transcriptional pathways that foster mitochondrial bio-
genesis. Moreover, lipoic acid has been reported as potential therapeutic/nutritional agent in mul-
tiple age-related disease models: lipoic acid has been found to restore the age-dependent impair-
ment of long-term potentiation (LTP) and glutamate release in rat hippocampus (McGahon et al.
1999); lipoic acid in combination with L-acetyl-carnitine restores mitochondrial biogenesis in the
hippocampus (Aliev et al. 2009) and protected cortical neurons against -amyloid and H
2
O
2
tox-
ic insults (Zhang et al. 2001).
ACKNOWLEDGEMENTS
Supported by NIH grant RO1AG016718 (to E.C.) and PO1AG026572 (to R.D.B.)
56
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Roles of p21ras and pp70 S6 kinase. J. Biol. Chem. 270, 24678-24681.
Uehara Y, Nipper V, McCall AL (1997) Chronic insulin hypoglycemia induces GLUT-3 protein
in rat brain neurons. Am. J. Physiol. Endocrin. Metab. 272, E716-E719.
Wenz T, Rossi SG, Rotundo RL, Spiegelman BM, Moraes CT (2009) Increased muscle PGC-
1alpha expression protects from sarcopenia and metabolic disease during aging. Proc. Natl.
Acad. Sci. USA. 106, 20405-20410.
Yin F, Boveris A, Cadenas E (2012) Mitochondrial energy metabolism and redox signaling in
brain aging and neurodegeneration. Antioxid. Redox Signal. in press.
Yin F, Jiang T, Cadenas E (2013) Metabolic triad in brain aging: mitochondria, insulin/IGF-1
signalling, and JNK signalling. Biochem. Soc. Trans. 41, 101-105.
Zhang L, Xing GQ, Barker JL, Chang Y, Maric D, Ma W, Li BS, Rubinow DR (2001) Alpha-
lipoic acid protects rat cortical neurons against cell death induced by amyloid and hydrogen
peroxide through the Akt signalling pathway. Neurosci. Lett. 312, 125-128.
Zhou Q, Lam PY, Han D, Cadenas E (2008) c-Jun N-terminal kinase regulates mitochondrial
bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons. J.
Neurochem. 104, 325-335.
Zhou Q, Lam PY, Han D, Cadenas E (2009) Activation of c-Jun-N-terminal kinase and decline
of mitochondrial pyruvate dehydrogenase activity during brain aging. FEBS Lett. 583, 1132-
1140.
60
CHAPTER THREE
ASTROCYTIC METABOLIC AND INFLAMMATORY CHANGES
AS A FUNCTION OF AGE
SUMMARY – This study examines age-dependent metabolic-inflammatory axis in primary astro-
cytes isolated from brain cortices of 7-, 13-, and 18 month-old Sprague Dawley male rats. Astro-
cytes showed an age-dependent increase in mitochondrial oxidative metabolism respiring on glu-
cose and/or pyruvate substrates; this increase in mitochondrial oxidative metabolism was accom-
panied by increases of COX3/18SrDNA values, thus suggesting an enhanced mitochondrial bio-
genesis. Enhanced mitochondrial respiration in astrocytes limits the substrate supply from astro-
cytes to neurons; this may be viewed as an adaptive mechanism to altered cellular inflammatory-
redox environment with age. These metabolic changes were associated with an age-dependent
increase in hydrogen peroxide generation (largely ascribed to an enhanced expression of NOX2)
and NFκB signaling in the cytosol as well as its translocation to the nucleus. Astrocytes also dis-
played augmented responses with age to inflammatory cytokines, IL-1β and TNFα. Activation of
NFκB signaling resulted in increased expression of nitric oxide synthase 2 (inducible nitric oxide
synthase), leading to elevated nitric oxide production. IL-1β and TNFα treatment stimulated mi-
tochondrial oxidative metabolism and mitochondrial biogenesis in astrocytes. It may be surmised
that increased mitochondrial aerobic metabolism and inflammatory responses are interconnected
and support the functionality switch of astrocytes, from neurotrophic to neurotoxic with age.
61
INTRODUCTION
Brain aging is accompanied by a hypometabolic state that involves decreased glucose uptake,
reduced expression and translocation to the plasma membrane of neuronal glucose transporters
GLUT3 and GLUT4, and an imbalance of insulin (the PI3K/Akt pathway)- and c-Jun N-terminal
kinase (JNK) signaling; these changes impinge on mitochondrial metabolism and neuronal sur-
vival (Jiang et al. 2013; Yin et al. 2013). Astrocytes have a supportive function for neurons in
the central nervous system: neurons harbor a strong aerobic metabolism, while astrocytes primar-
ily rely on the ATP derived from glycolysis with lactate extrusion as the end point (Dienel &
Hertz 2001; Bolañ os et al. 2010). Lactate diffuses from astrocytes and is taken up subsequently
by neurons through high-affinity monocarboxylate transporter 2 (MCT2) to serve as a key me-
tabolite for neuronal aerobic metabolism and thus meeting the large energy demands in neuronal
activity (Magistretti 2011; Suzuki et al. 2011). Age-dependent glial fibrillary acidic protein
(GFAP, a marker of astrocytes) expression and astrocyte activation have been reported, but little
is known about the astrocytic metabolic state, especially because astrocyte activation requires
energy. An in vivo magnetic resonance spectroscopy study revealed reduced neuronal mitochon-
drial metabolism and increased glial mitochondrial metabolism with age in human brains
(Boumezbeur et al. 2010).
Astrocytes are intimately involved in age-dependent inflammatory responses: (Campuzano
et al. 2009): they sense and amplify inflammatory signals from microglia and initiate a series of
inflammatory responses that involve NFκB activation and the genes under its control (Zhang et
al. 2013). The transcription factor NFκB is redox-sensitive, i.e. responsive to H
2
O
2
modulation,
and is also stimulated by pro-inflammatory cytokines such as TNFα and IL-1β (Li & Verma
2002). Persistent activation of NFκB engages MAPK activation (Guma et al. 2011) and stress-
62
responsive JNK signaling was especially involved in insulin resistance and inflammation (Han et
al. 2013) and in aging and neurodegenerative diseases (Mehan et al. 2011; Jiang et al. 2013). As-
trocytes surround neurons and synapses and the question arises on how the inflammatory re-
sponses generated by astrocytes propagate to neurons and affect their functions. Cytokines them-
selves and diffusible redox species, such as H
2
O
2
and nitric oxide are intercellular signals that
impinge on neuronal function. Excessive amount of nitric oxide (
.
NO) produced by upregulation
of inducible nitric oxide synthase (iNOS) was implicated in several central nervous system (CNS)
disorders (Brosnan et al. 1994; Luth et al. 2001) and iNOS expression was controlled by NFκB
signaling and JNK signaling (Wang et al. 2004). NADPH oxidase (NOX) enzymes are widely
expressed in different cell types in the CNS and are major sources of oxidants (Sorce & Krause
2009). H
2
O
2
derived from NOX, especially NOX2, fulfill some physiologic functions such as
host defense and cellular signaling, but excessive oxidative stress also contributes to chronic in-
flammation in aging and various neurodegenerataive disorders (Park et al. 2007). Taking into
account of their diffusibility, it may be surmised that
.
NO and H
2
O
2
could act as intercellular me-
diators released from astrocytes and compromise neuronal functions in aging.
Because of the tight association between astrocytes and neurons in structural proximity,
metabolic coupling and inflammatory responses (Belanger & Magistretti 2009), it is intriguing to
hypothesize that they switch from being neurotrophic to neurotoxic in aging and neurodegenera-
tive diseases. Primary astrocytes from senescence-accelerated mouse (SAM) showed elevated
oxidative stress and reduced neuroprotective capacity (Garcia-Matas et al. 2008). This study is
aimed at exploring the mechanistic basis of the age-dependent metabolic - inflammatory axis in
astrocytes. This was assessed on primary astrocytes derived from brain cortices of rats of differ-
63
ent ages in terms of the age-dependent cellular metabolic shifts that are associated with inflam-
matory responses and how inflammatory cytokines modulate energy metabolism.
EXPERIMENTAL PROCEDURES
Isolation and culture of primary astrocytes
Primary astrocytes were isolated from brain cortices of Sprague Dawley male rats of 6-7
month, 12-13 month, and 18 month-old. Rats from different age groups were sacrificed on the
same day, and brains were rapidly dissected in ice-old HBSS. Cortices were subject to Trypsin
digestion and repetitive trituration to extract glia cells. Cells were then filtered and plated into
poly-D-lysine coated flasks in Dulbecco’s Modified Eagle’s Medium/F12 culture medium sup-
plemented with 20% fetal bovine serum, 0.5 U/mL penicillin, and 0.5 mg/mL streptomycin, with
medium renewal every other day during the first week and 2 times a week starting second week.
Once reached confluence after 2-3 weeks, cells were placed onto an orbital shaker for 4 h to
shake off microglia and yield enriched astrocytes for experiments.
Cytosolic and Nuclear Fraction isolation
Cytosolic and nuclear fractions were isolated from primary astrocytes using NE-PER
TM
Nu-
clear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA) follow-
ing manufacturer’s instructions.
DNA isolation and quantification
64
Total DNA from primary astrocytes was prepared using Wizard Genomic DNA Purification
Kit (Promega Corporation, Madison, WI, USA) and following the manufacturer’s instructions.
The relative copy numbers of mitochondrial and nuclear DNA were determined by real-time
PCR with primers specific to the COX3 (mitochondrial) and 18SrDNA (nuclear) genes, 100 ng
DNA, and SYBRGreen PCR master mix (Bio-Rad, Hercules, CA, USA) on an iCycler real-time
PCR machine (Bio-Rad).
Metabolic flux analyses
Primary astrocytes were cultured on Seahorse XF-24 or Seahorse XF-96 (Seahorse BioSci-
ences, Billerica, MA, USA) plates at a density of 75,000 cells/well and 20,000 cells/well respec-
tively. On the day of metabolic flux analysis, media was changed to Krebs-Henseleit buffer
(KHB), pH 7.4, supplemented with 25 mM glucose and/or 1 mM pyruvate and incubated at 37° C
in a non-CO
2
incubator for 1 h. All medium and injection reagents were adjusted to pH 7.4 on
the day of assay. Using the Seahorse XF (Seahorse BioSciences) metabolic analyzer, 3 baseline
measurements of oxygen consumption rate (OCR) were sampled prior to sequential injection of
mitochondrial inhibitors. Three metabolic determinations were sampled following addition of
each mitochondrial inhibitor prior to injection of the subsequent inhibitors. The mitochondrial
inhibitors used were oligomycin (4 M), FCCP (carbonyl cyanide 4-(trifluoromethoxy)- phenyl-
hydrazone) (1 M), and rotenone (1 M). OCR was automatically calculated and recorded by the
Seahorse software. After the assays, protein level was determined for each well to confirm equal
cell density per well.
Measurement of mitochondrial membrane potential
65
Cells were harvested, washed with PBS, and stained with 30 nM of TMRM for 30 min, then
washed with PBS. TMRM signal, which measures mitochondrial membrane potential, was ana-
lyzed with a flow cytometer (BD Biosciences). Data were collected from 10,000 cells from each
sample and analyzed with the software WinMDI.
Measurement of H
2
O
2
and nitrite
H
2
O
2
generation and release rate by primary astrocytes was determined by the Amplex Red
/Peroxidase Assay kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.
NO production was measured as total NO
2
-
present in the medium and detected by DAN assay
using Nitrate/Nitrite Fluorometric Assay Kit (Cayman Chemical, Ann Arbor, MI, USA).
Western blot analysis
Cytosolic and Nuclear Fraction from primary astrocytes were solubilized in SDS sample
buffer, separated by SDS/PAGE, and transferred onto PVDF membranes. Using appropriate an-
tibodies, the immunoreactive bands were visualized with an enhanced chemiluminescence rea-
gent. The blots were quantified using UN-SCAN-IT gel 6.1 (Silk Scientific, Inc., Orem, UT,
USA).
Statistical analysis
Data are reported as means ± SEM of at least 4-5 independent experiments. Significant dif-
ferences between mean values were determined by student t-test.
66
RESULTS
Age-dependent astrocytic metabolic shift
Astrocytes mainly rely on anaerobic glycolysis for energy and its aerobic (mitochondrial
metabolism of pyruvate) is generally much weaker than that of neurons, which rely almost en-
tirely on aerobic glycolysis. As mentioned above, lactate generated during anaerobic glycolysis
in astrocytes supports neuronal oxidative metabolism, i.e., oxidation of lactate to pyruvate and its
further mitochondrial metabolism to satisfy neuronal energy demands (Magistretti 2011; Suzuki
et al. 2011).
Astrocyte metabolic profiles were assessed with the Extracellular Flux analyzer: astrocytes
isolated from 7-, 13-, and 18 month-old Sprague Dawley rat cortices displayed an age-dependent
increase in OCR (Oxygen Consumption Rate) reflecting mitochondrial respiration (Table 1; Fig.
1). ECAR (Extra Cellular Acidification Rate), reflecting anaerobic glycolysis (i.e., lactate for-
mation) did not change with age (not shown). Fig. 1A shows a time course of astrocytes respiring
on pyruvate (OCR): basal respiration in astrocytes from 18 month-old rat (192 ± 9 pmoles/min)
cortices was ~50% higher than that in astrocytes from 7 month-old cortices (130 ± 6 pmoles/min);
ATP turnover (following oligomycin addition) was also higher in the astrocytes from older rats.
The most striking increase (~two fold) was in maximal respiratory capacity (following the addi-
tion of FCCP); this resulted in a much higher reserve capacity (OCR
MAXIMAL RESPIRATORY CAPACITY
– OCR
BASAL RESPIRATION
) (238 ± 29 pmoles/min) in astrocytes from 18 month-old rat cortices (Fig.
1A, Table 1). Similar trends in maximal respiratory capacity and reserve capacity were observed
with astrocytes respiring on glucose and glucose + pyruvate (Table 1). The increased astrocytic
aerobic metabolism may be a consequence of augmented mitochondrial biogenesis, as indicated
by the increasing COX3/18SrDNA values with age (Fig. 1B). Tetramethylrhodamine methyl es-
67
Fig. 1 Age-dependent increases in astrocytic aerobic metabolism
(A) Representative figure of changes in astrocytic aerobic metabolism with age. Primary astro-
cytes were cultured on Seahorse XF-24 plates, and Oxygen Consumption Rate (OCR) was measured in
the presence of pyruvate as the solely important substrate. A more comprehensive and quantitative com-
parison of astrocytic aerobic metabolism in the presence of different substrates was shown in Table 1.
(B) Age-dependent increase in mitochondrial biogenesis was determined by the ratio of
COX3/18SrDNA, assayed by real-time PCR. (C) Age-dependent increase in mitochondrial membrane
potential was determined by TMRM staining using FACS. (D) Quantification of the increase in mito-
chondrial membrane potential with age. *p < 0.05.
ter (TMRM) staining (Fig. 1C) revealed 35% and 51% increases in mitochondrial membrane po-
tential of astrocytes from 13- and 18 month-old rats compared to astrocytes from 7 month-old
rats (Fig. 1D).
68
Table 1
Bioenergetic Parameters of Primary Astrocytes
_______________________________________________________________________________________________________
______________
OCR (pmoles/min)
____________
______________
Age (months)
____________
7 13 18
_______________________________________________________________________________________________________
_____________________________________________
Pyruvate
____________________________________________
Basal respiration 130 ± 6 122 ± 5 192 ± 9*
OXPHOS-induced respiration 72 ± 6 85 ± 3* 97 ± 9*
H+ leak-induced respiration 58 ± 11 36 ± 6 96 ± 8
Maximal respiratory capacity 224 ± 18 307 ± 9* 430 ± 21*
Non-mitochondrial respiration 42 ± 3 35 ± 2 71 ± 6
Reserve capacity 94 ± 11 185 ± 2* 238 ± 29*
_____________________________________________
Glucose
____________________________________________
Basal respiration 108 ± 11 113 ± 4 117 ± 12
OXPHOS-induced respiration 69 ± 7 79 ± 5 83 ± 7
H+ leak-induced respiration 40 ± 4 34 ± 2 34 ± 11
Maximal respiratory capacity 184 ± 23 226 ± 11* 266 ± 20*
Non-mitochondrial respiration 31 ± 9 34 ± 1 16 ± 12
Reserve capacity 76 ± 11 113 ± 5* 150 ± 9*
_______________________________________
Glucose + Pyruvate
________________________________________
Basal respiration 206 ± 6 263 ± 4* 222 ± 6
OXPHOS-induced respiration 139 ± 8 176 ± 7* 154 ± 7
H+ leak-induced respiration 68 ± 2 86 ± 4 68 ± 1
Maximal respiratory capacity 398 ± 11 503 ± 18* 532 ± 5*
Non-mitochondrial respiration 48 ± 8 59 ± 10 31 ± 10
Reserve capacity 191 ± 17 240 ± 21* 310 ± 10*
_______________________________________________________________________________________________________
*p < 0.05
69
Age-dependent increases in astrocytic H
2
O
2
generation and NOX expression
Effective cellular sources of H
2
O
2
are the electron leak of the mitochondrial respiratory
chain, inflammatory- and non-inflammatory NADPH oxidases, and monoamine oxidase associ-
ated with the outer mitochondrial membrane. Whereas the latter generates directly H
2
O
2
upon the
two-electron reduction of O
2
, the mitochondrial respiratory chain and NADPH oxidases generate
H
2
O
2
upon disproportionation of O
2
.–
. The rate of H
2
O
2
release from primary astrocytes in-
creased with age (Fig. 2A): there was a 50% increase in the H
2
O
2
release rate by astrocytes iso-
lated from 13 month- and 18 month-old rats relative to astrocytes from 7 month-old rats. To fur-
ther investigate the sources of increased H
2
O
2
release, primary astrocytes from different aged rats
were treated with mitochondrial respiratory chain inhibitor antimycin A, monoamine oxidase
(MAO) inhibitor deprenyl, and NOX inhibitor diphenyleneiodonium (DPI) (Fig. 2A). H
2
O
2
re-
lease from astrocytes was insensitive to antimycin A and deprenyl, whereas treatment with di-
phenyleneiodonium elicited a 50% decrease in H
2
O
2
release rate; hence, the reduced H
2
O
2
re-
lease rate was defined DPI-sensitive H
2
O
2
release. The substantial increase in DPI-sensitive
H
2
O
2
release with age was most prominent in astrocytes from 13 month-old rats, with a 79% in-
crease compared to astrocytes from 7 month-old rats (Fig. 2B). This indicates that the increase in
H
2
O
2
release from older astrocytes was likely due to increased NOX activity. NOX2 and NOX4
are the two main isoforms of NOX in astrocytes, the former induced by inflammation and the
latter is usually considered the constitutive NOX isoform. The DPI-sensitive H
2
O
2
formation was
associated with an age-dependent increase in NOX2 expression (60% and 100% increases in as-
trocytes from 13 month- and 18 month-old rats respectively) (Fig. 2C). NOX4 expression slight-
ly increased with age, especially in astrocytes from 13 month-old rats, despite no statistically
significance (Fig. 2D). Of note, data in Fig 1D shows an age-dependent increase in mitochondri-
70
al membrane potential: this condition is always associated with a decreased efflux of H
2
O
2
from
mitochondria and thus it strengthens the notion that NOX activity is the major source of H
2
O
2
in
astrocytes and that it increases with age.
Fig. 2 Age-dependent increases in Astrocytic H
2
O
2
generation and NOX expression
(A) H
2
O
2
release rate in the astrocytic medium was determined by Amplex Red assay. Pri-
mary astrocytes were treated with antimycin A, deprenyl, or DPI at 50 μM for 1 h to determine the
contribution to H
2
O
2
generation from different subcellular sources. (B) Increased DPI-sensitive H
2
O
2
release rate with age. DPI-sensitive H
2
O
2
release rate was defined by subtraction of H
2
O
2
release rate
with DPI treatment from the basal release rate. Expressions of (C) NOX2 and (D) NOX4 were de-
termined by Western Blot. *p < 0.05.
71
Age-dependent changes in the redox-sensitive NFκB transcription factor
NFκB is a redox-sensitive transcription factor; in addition to receptor engagement and
phosphorylation of the IKK complex, the latter phosphorylates I B with the consequent release
of free NFκB and its translocation to the nuclei. Hence, NFκB remains sequestered in the cytosol
by IκBα under basal conditions, and translocates to the nucleus upon stimulation such as LPS,
TNFα, IL-1β, where it induces expression of genes involved in inflammatory responses such as
iNOS, TNFα, IL-1β. H
2
O
2
promotes NF B activation at least at two levels: H
2
O
2
enhances
phosphorylation of the IKK complex (either via stimulation of protein kinase D or inhibition of
the phosphatase PP2A); H
2
O
2
enhances the phosphorylation of I B at Tyr
42
and serine phos-
phorylation of p65 thus facilitating the dissociation of NF B (Storz & Toker 2003; Takada et al.
2003; Storz et al. 2004; Loukili et al. 2010).
The basal levels of NFκB in the cytosol of astrocytes from older rats was over two-fold of
those from young rats (Fig. 3A). Upon stimulation with IL-1β, IκBα is rapidly degraded, thus
resulting in free NFκB. Therefore, the ratio of cytosolic IκBα/NFκB was used to assess the sensi-
tivity of the cytosolic NFκB pathway, with lower values indicating more available NFκB for
translocation to the nucleus: data in Fig. 3B indicated that astrocytes isolated from 13 month- and
18 month-old rats expressed a better responsiveness to IL-1β induction, reflected by a substantial
decrease in IκBα/NFκB ratio compared with 7 month-old astrocytes (50% and 48% decrease
compared with 73%). Constitutive NFκB levels in the nucleus of astrocytes increased with age,
with 36% increase at 13 month-old and 150% increase at 18 month-old compared with those
from 7 month-old rats (Fig. 3C,D). Upon IL-1β treatment, astrocytes displayed robust increase in
NFκB nuclear translocation with astrocytes from older rats showing even larger increase of
NFκB in the nuclear fraction as compared with younger rats (56% increase at 13 month-old and
72
more than three-fold of expression at 18 month-old) (Fig. 3C,D). Treatment with TNF elicited
similar trends but with more robust changes, i.e., higher than those elicited by IL-1 and several
fold higher than the constitutive NF B nuclear levels (Fig. 3C,D).
Fig. 3 Age-dependent activation of NFκB pathway
(A) Age-dependent increase in cytosolic NFκB p65 level. (B) Age-dependent increases
in the sensitivity of cytosolic NFκB signaling in response to 100 ng/ml IL-1β treatment. The
sensitivity was determined by the ratio of IκBα and NFκB level indicating the freely available
NFκB. (C) Age-dependent increases in NFκB p65 in the nuclear fractions of primary astrocytes
at basal level and in response to 100 ng/ml IL-1β and 50 ng/ml TNFα treatments. TBP (Tata
Box Binding Protein) was used as a loading control for nuclear fraction. (D) Quantification of
NFκB p65 in the nuclear fractions. *p < 0.05, **p < 0.01.
73
IL-1 induces activation of iNOS and JNK expression in astrocytes – Effect of age
iNOS (inducible nitric oxide synthase; NOS2) is largely responsible for inflammation-
related long-lasting production of nitric oxide (
.
NO). iNOS expression was hardly detectable un-
der basal conditions but substantially increased in response to IL-1β treatment: astrocytes from
13 month- and 18 month-old rats showed stronger iNOS expression than younger counterparts
(Fig. 4A). Accordingly,
.
NO basal generation (measured as NO
2
–
in the medium) in astrocytes
from older rats was ~40% higher than that in astrocytes from 7 month-old rats (Fig. 4B). Howev-
er, increased expression of iNOS upon treatment of astrocytes with IL-1 resulted in a large in-
crease in NO
2
–
formation, with values 2.0- and 2.4-fold higher in astrocytes from older rats than
those from 7 month-old rats (Fig. 4B). Of note, treatment with IL-1 elicited a minor increase
(47%) in NO
2
–
levels in astrocytes from 7 month-old rats (Fig. 4B). The increased expression of
iNOS and, as a corollary, of
.
NO generation is apparently at odds with the higher metabolic rates
(OCR data in Table 1) observed in astrocytes as a function of age, especially considering that a
major function of
.
NO –after activation of soluble guanylate cyclase– is the reversible inhibition
upon binding to cytochrome oxidase (complex IV).
The constitutive levels of JNK activation (pJNK/JNK values) were prominent in astrocytes
from older rats: statistically significant higher pJNK/JNK values in astrocytes from older rats
compared with those from 7 month-old rats. These values were increased upon treatment with
IL-1: pJNK/JNK values were 2.4- and 2.1-fold higher in astrocytes from 18- and 13 month-old
rats than those in astrocytes from 7 month-old rats (Fig. 4C).
JNK activation is apparently at odds with the reported age-dependent increase in rat brain
and its impact on neuronal metabolism: active (bisphosphorylated) JNK translocates to mito-
chondria where it is docked on the outer mitochondrial membrane; this triggered a phosphoryla-
74
tion cascade that resulted in inhibition of the pyruvate dehydrogenase complex (upon phosphory-
lation of the E
1
subunit) and the detrimental consequence on energy metabolism (Zhou et al.
2008; Zhou et al. 2009; Jiang et al. 2013). However, these experiments were performed in pri-
mary cortical neurons and it does not seem to apply to astrocytes. Here we demonstrated that
JNK is activated in primary astrocytes upon IL-1β treatment, and there is increased JNK activity
with age both constitutively and in the presence of inflammatory cytokines. Activated JNK could
be involved in the degradation of IκB, thus enhancing NFκB signaling and are also involved in
the induction of proinflammatory cytokine genes coding for TNFα, IL-6, and others (Mehan et al.
2011). JNK and p38 but not ERK were upregulated upon treatment of rat astrocytes with IL-1
and TNF (Thompson & Van Eldik 2009).
75
Fig. 4 IL-1 induces activation of iNOS and JNK expression in astrocytes – Effect of age
(A) Astrocytic iNOS expression induced by 100 ng/ml IL-1β was determined by Western Blot. (B) Ni-
trite in the medium of primary astrocytes was determined by DAN assay as described in the Experimental Proce-
dures. (C) Basal JNK activity in primary astrocytes increased with age. 100 ng/ml IL-1β increased JNK activity,
and there was an age-dependent increase in JNK activity in the presence of 100 ng/ml IL-1β. *p < 0.05.
76
Cytokine treatment increased astrocytic aerobic metabolism
It is well established that aging is associated with increased release of inflammatory cytokines
including IL-1β and TNF . Data in Fig. 1 indicate an age-dependent increase of mitochondrial
oxidative metabolism in astrocytes and data in Figs. 3 and 4 indicate that the total constitutive
levels of NFκB increase in cytosol and nucleus and that translocation to the latter is strongly
stimulated by the cytokines IL-1 and TNF . Hence, the question as to the effect of cytokines on
astrocyte metabolism (OCR) needs to be addressed: primary cultured astrocytes treated with dif-
ferent concentrations IL-1β and TNFα showed dose-dependent increases in mitochondrial respi-
ration (Fig. 5). At the highest IL-1 concentration (1000 ng/ml), basal respiration was increased
3.7-fold (from 45.7 pmoles/min in control to 171.4 in IL-1-supplemented astrocytes) and the
reserve capacity 2.2-fold (from 51.4 pmol/min in control to 114.2 in IL-1-supplemented astro-
cytes) (Fig. 5A). TNF at the highest concentration (500 ng/ml) exerted similar effects: 70% in-
crease in basal respiration and a more robust (2.1-fold) increase in astrocytic reserve capacity
(Fig. 5B). The effects of these cytokines on COX3/18SrDNA values (reflecting mitochondrial
biogenesis) is shown in Fig. 5C: IL-1 increased COX2/18SrDNA values ~79% and TNF
~52%. This indicates that cytokines may enhance mitochondrial respiration (oxidative metabo-
lism) by increasing mitochondria number.
77
Fig. 5 Cytokine treatment increased astrocytic
aerobic metabolism
Primary astrocytes were cultured on Sea-
horse XF-96 plates and treated with escalating
concentrations of (A) IL-1β and (B) TNFα, and
Oxygen Consumption Rate (OCR) was measured.
(C) Increases in mitochondrial biogenesis by in-
flammatory cytokines (IL-1β, 100 ng/ml; TNFα,
50 ng/ml) were determined by the ratio of
COX3/18SrDNA, assayed by real-time PCR. *p <
0.05.
78
DISCUSSION
Although microglia are generally considered the resident immune cells in the brain, the im-
pact of astrocytes on inflammation cannot be understated. First, astrocytes outnumber microglia
in the brain; second, astrocytes detect and amplify inflammatory signals from microglia, espe-
cially by self-propagation of the cytokine cycle to generate large amount of cytokines within a
short period of time and, last but not least, astrocytes are in close proximity to neurons and syn-
apses, so they directly affect neuronal functions.
A salient feature of this study is the age-dependent astrocytic metabolic shift: astrocytes rely
primarily on ATP derived from glycolysis and the final product, pyruvate, is reduced to lactate,
which supports energy demands in neurons when released from astrocytes (Dienel & Hertz 2001;
Bolañ os et al. 2010; Magistretti 2011; Suzuki et al. 2011). Because of this neurotrophic support
of astrocytes, it is interesting that mitochondrial oxidative metabolism in astrocytes is enhanced
with age, especially considering the general energy deficit or hypometabolic state associated
with brain aging. However, few studies actually distinguished astrocytic metabolism from neu-
ronal metabolism in aging. Furthermore, astrocytes mainly rely on anaerobic glycolysis for ener-
gy, and its oxidative metabolism is generally much weaker than that of neurons, so its alterations
with age could be easily masked if mitochondrial metabolism measures are performed on the
whole brain. Astrocytes are generally considered neurotrophic inasmuch as providing neurons
with energy substrates and recycling neurotransmitters. However, data here showed that astro-
cytes turn “selfish” with age, by utilizing energy substrates for their own metabolism rather than
distributing to neurons. An argument supporting this increase in "energy-efficient metabolism in
astrocytes" is that inflammatory reactions in astrocytes in response to infection or other stressors
are metabolically expensive events (Johnson et al. 2012) and may stimulate mitochondrial me-
79
tabolism for the energy support. This notion is support by the finding that IL-1β and TNFα stim-
ulate mitochondrial metabolism, which is likely to be the scenario in aging too. Aging is usually
accompanied by recurrent injury, invasion, and other insults causing chronic inflammation. As-
trocytes amplify inflammatory signals rather than standing in the first line of defense as micro-
glia, therefore their sustained production of inflammatory mediators require a steady supply of
ATP, which could be supported by oxidative metabolism rather than glycolysis. Increased mito-
chondrial metabolism was postulated to result from mitochondrial biogenesis but not higher mi-
tochondrial quality and inflammatory cytokines were shown to induce mitochondrial biogenesis
to support metabolic functions and cell viability (Piantadosi & Suliman 2012). An increase in
reactive astrocytes is a hallmark of brain aging and neurodegenerative diseases. It may be sur-
mised that increased mitochondrial aerobic metabolism and inflammatory responses support the
functionality switch of astrocytes, from neurotrophic to neurotoxic with age. Strategies that
modulate substrate metabolism in astrocytes would be innovative approaches to address impaired
neuronal function and neurodegeneration with age.
It has been shown that TNFα, IL-1β, and IL-6 expression increased in rat brain cortex and
striatum during aging, and the expression of cytokines was mostly attributed to astrocytes, but
not in microglia or neurons (Campuzano et al. 2009). NFκB is the master regulator in inflamma-
tion. The activation of canonical NFκB pathway follows binding of IL-1β and TNFα to their re-
ceptors, activation of IKK complex, degradation of IκBα, and translocation of cytosolic NFκB to
the nucleus, where it initiates transcription of a set of inflammation-related genes. Activation of
NFκB pathway along with MAPK was found to account for increased chemokines CCL2/MCP-1
and CCL7/MCP-3 in rat astrocytes treated with IL-1β and TNFα (Thompson & Van Eldik 2009).
Constitutive levels of NFκB in the cytosol were substantially increased in aging (Fig. 3A), which
80
serves as a free pool ready to enter nucleus and induce transcription of inflammatory genes. The
most determining evidence for NFκB activation with age was the elevation of the constitutive
level of NFκB in the nucleus (Fig. 3C,D). However, the sensitivity of NFκB signaling to cyto-
kines was increased in aging. Specifically, upon stimulation by IL-1β, young astrocytes showed
moderate activation of cytosolic NFκB signaling (assessed by decreased IκBα/NFκB ratio),
whereas astrocytes from old rats exhibited a much stronger response to IL-1β (Fig. 3B). In sup-
port of this, upon IL-1β stimulation, older astrocytes had a larger increase in nuclear transloca-
tion of NFκB than young astrocytes. H
2
O
2
has been shown to play an important role in NFκB
activation and signaling (Oliveira-Marques et al. 2013) and this study suggests that the age-
dependent increase in H
2
O
2
generation by astrocytes is largely driven by an increased NOX2 ex-
pression, the NOX isoform typically induced by inflammation (Sorce & Krause 2009) and slight
increases in NOX4, the constitutive isoform. In this context, NADPH oxidases were elevated and
activated in brains from Alzheimer’s disease and Parkinson’s disease patients (Park et al. 2005).
Activation of NOX enzymes in astrocytes exposed to toxic stimuli such as Amyloid β was found
to induce neuronal damage (Abramov et al. 2004; Abramov & Duchen 2005).
In this study experimental model,
.
NO is produced by iNOS in the astrocytes, which is regu-
lated by NFκB at a transcriptional level.
.
NO is involved in the regulation of a broad spectrum of
pathophysiological processes in the brain, with major impacts on signaling via cGMP, regulation
of mitochondrial function (upon reversible binding to cytochrome oxidase), and multiple S-
nitrosylation targets associated with cellular functional responses.
.
NO derived from astrocytes
resulted in glia-induced neuronal death (Bal-Price & Brown 2001). This study showed that
.
NO
released from astrocytes increased substantially with age both constitutively and in the presence
of inflammatory cytokines (Fig. 4B). The increase in
.
NO production upon IL-1β stimulation is
81
even more pronounced at older ages, which, in turn, resulted from the age-dependent increased
sensitivity of NFκB signaling. It is conceivable that
.
NO is exported from astrocytes and elicit
damage (in neurons) at sites distant from its generation. Although the elevated
.
NO levels by as-
trocytic iNOS may predict mitochondrial damage and cell toxicity, results from this study sug-
gest that astrocytes may be resistant to
.
NO toxicity. Astrocytes were more resistant to
.
NO tox-
icity than neurons due to their elevated GSH synthesis (Gegg et al. 2003) and displayed a much
lower sensitivity than neurons, when both exposed to a steady flux of
.
NO, due to a higher capac-
ity to recover GSH through glutathione reductase (Yap et al. 2010). These arguments, however,
cannot bridge the discrepancy between the age- and iNOS-dependent
.
NO generation (Fig. 4A,B)
and the age-dependent increase in mitochondrial oxidative metabolism (Fig. 1). In this regard, it
was reported that iNOS stimulated hepatic mitochondrial biogenesis by a mechanism entailing
association of iNOS to the outer mitochondrial membrane, its binding and S-nitrosylation of
HHsp60 and Hsp70, thus promoting Tfam accumulation in mitochondria (Suliman et al. 2010).
ACKNOWLEDGEMENTS – Supported by NIH grant RO1AG016718
82
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86
CHAPTER FOUR
THE METABOLIC-INFLAMMATORY PHENOTYPE IN OLD ASTROCYTES
EFFECT OF LIPOIC ACID
SUMMARY – Aging astrocytes display increased oxidative (mitochondrial) metabolism and up-
regulation of inflammatory responses involving NFκB signaling as well as inflammatory media-
tors H
2
O
2
and NO. Lipoic acid showed beneficial effects in terms of restoring brain energy me-
tabolism in Fischer 344 rat brain cortices. R-(+)-Lipoic acid treatment of primary astrocytes –
isolated from 24 month-old Fischer 344 rat brain cortices– induced a shift of metabolic pheno-
types in old astrocytes: lipoic acid suppressed oxidative metabolism but augmented anaerobic
glycolysis. Lipoic acid treatment diminished H
2
O
2
generation, which was associated with de-
creased NOX4 levels. Lipoic acid suppressed redox-sensitive NFκB cytosolic signaling and its
nuclear translocation, which were induced by IL-1β stimulation. Reduced NFκB signaling result-
ed in decreased expression of iNOS, which –along with decreased level of nNOS– lead to atten-
uation of NO generation. The effects of lipoic acid may be interpreted in the context of brain ag-
ing, where reactive astrocytes deprive neurons of metabolic intermediates and release neurotoxic
inflammatory mediators. Lipoic acid seems to reverse the metabolic and inflammatory pheno-
types of old astrocytes to that of young and neurotrophic astrocytes.
87
INTRODUCTION
Brain aging entails a significant decline in energy metabolism, consisting of decreased glu-
cose uptake, reduced neuronal glucose transporters, an imbalance of insulin/JNK signaling, and
impaired bioenergetics, which are mainly attributed to neurons (Jiang et al. 2013). Astrocytes are
generally considered neurotrophic by providing neurons with lactate as an important energy in-
termediate, which is supported by a robust anaerobic glycolysis (Dienel & Hertz 2001; Belanger
et al. 2011; Magistretti 2011). However, enhanced mitochondrial metabolism with age was
shown in primary astrocytes isolated from Sprague Dawley rats, which could be viewed as neu-
rotoxic by limiting the substrate supply to neurons.
Enhanced astrocytic mitochondrial metabolism was suggested to involve inflammatory re-
sponses, initiated by pro-inflammatory cytokines such as TNFα and IL-1β. Increased inflamma-
tion is inherent in brain aging and neurodegenerative diseases, with astrocytes playing essential
roles in sensing and amplifying inflammatory signals from microglia. Astrocytes displayed an
increase in H
2
O
2
release with age, which was attributed to increased expression of NADPH oxi-
dases but not to mitochondrial respiration. Furthermore, astrocytes showed age-dependent in-
crease in redox-sensitive NFκB signaling, evidenced by increased expression and nuclear trans-
location of NFκB P65, resulting in the up-regulation of iNOS expression and increase in NO
production. Increased inflammatory responses from astrocytes could easily propagate to neurons,
considering the close proximity of the two necessary for metabolic coupling and the diffusibility
of molecules such as H
2
O
2
and NO. Therefore, strategies that modulate inflammatory responses
and associated metabolic status would be innovative approaches to address impaired neuronal
function with age and neurodegeneration.
88
R-(+)-lipoic acid (1, 2-dithiolane-3-pentanoic acid) was shown to exert beneficial effects on
neuronal functions with age by its insulin-like effect, via thiol-disulfide exchange reactions. Insu-
lin and its receptors are widely distributed in the CNS, and insulin receptors are present in astro-
cytes as well as neurons (Unger et al. 1991). However, the role of insulin signaling in astrocytes
is less known and is presumed to be different than that in neurons, just as they differ in metabolic
pathways. Generally, astrocytes do not express insulin-sensitive glucose transporters such as
GLUT3 and GLUT4 that are usually associated with neurons. Astrocytes possess PFKFB3,
which is a potent stimulator of glycolysis, but is constantly degraded in neurons (Bolañ os et al.
2010). PFKFB3 catalyzes the formation of fructose-2, 6-bisphosphate (F2, 6P2), which is an al-
losteric activator of 6-phosphofructo-1-kinase (PFK1), a master regulator of glycolysis. The ac-
tivity of PFKFB3 was shown to be regulated by insulin signaling at both expression level (Duran
et al. 2009) and phosphorylation at Ser
483
(Deprez et al. 1997). It is therefore hypothesized that
insulin signaling might regulate astrocytic glycolysis through PFKFB3 rather than the uptake of
glucose. Insulin signaling was also shown to inhibit lipopolysaccharide-induced nitric oxide syn-
thase expression in primary astrocytes (Li et al. 2013). IGF1 gene delivery to astrocytes reduced
their inflammatory responses to LPS (Bellini et al. 2011). The current study was aimed at inves-
tigating the effects of lipoic acid on old astrocytes that have turned neurotoxic with age and ex-
amining its ability to reverse enhanced mitochondrial metabolism and augmented inflammatory
responses in primary astrocytes.
EXPERIMENTAL PROCEDURES
Isolation and culture of primary astrocytes
89
Primary astrocytes were isolated from brain cortices of Fischer 344 male rats of 24 month-
old. Rat brains were rapidly dissected in ice-old HBSS. Cortices were subject to Trypsin diges-
tion and repetitive trituration to extract glia cells. Cells were then filtered and plated into poly-D-
lysine coated flasks in Dulbecco’s Modified Eagle’s Medium/F12 culture medium supplemented
with 20% fetal bovine serum, 0.5 U/mL penicillin, and 0.5 mg/mL streptomycin, with medium
renewal every other day during the first week and 2 times a week starting second week. Once
reached confluence after 2-3 weeks, cells were placed onto an orbital shaker for 4 h to shake off
microglia and yield enriched astrocytes for experiments.
Cytosolic and Nuclear Fraction isolation
Cytosolic and nuclear fractions were isolated from primary astrocytes using NE-PER
TM
Nu-
clear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA) follow-
ing manufacturer’s instructions.
Metabolic flux analyses
Primary astrocytes were cultured on Seahorse XF-24 or Seahorse XF-96 (Seahorse BioSci-
ences, Billerica, MA, USA) plates at a density of 75,000 cells/well and 20,000 cells/well respec-
tively. On the day of metabolic flux analysis, media was changed to Krebs-Henseleit buffer
(KHB), pH 7.4, supplemented with 25 mM glucose and/or 1 mM pyruvate and incubated at 37° C
in a non-CO
2
incubator for 1 h. All medium and injection reagents were adjusted to pH 7.4 on
the day of assay. Using the Seahorse XF (Seahorse BioSciences) metabolic analyzer, 3 baseline
measurements of oxygen consumption rate (OCR) were sampled prior to sequential injection of
mitochondrial inhibitors. Three metabolic determinations were sampled following addition of
90
each mitochondrial inhibitor prior to injection of the subsequent inhibitors. The mitochondrial
inhibitors used were oligomycin (4 μM), FCCP (carbonyl cyanide 4-(trifluoromethoxy)- phenyl-
hydrazone) (1 μM), and rotenone (1 μM). OCR was automatically calculated and recorded by the
Seahorse software. After the assays, protein level was determined for each well to confirm equal
cell density per well.
Measurement of mitochondrial membrane potential
Cells were harvested, washed with PBS, and stained with 30 nM of TMRM for 30 min, then
washed with PBS. TMRM signal, which measures mitochondrial membrane potential, was ana-
lyzed with a flow cytometer (BD Biosciences). Data were collected from 10,000 cells from each
sample and analyzed with the software WinMDI.
Measurement of Cell viability
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-cell viability was as-
sessed by measuring the ability of the cells to reduce MTT. After the lipoic acid treatment, the
medium was removed, and the cells were washed two times with PBS and then replaced with
HEPES-buffered toxicity medium containing 5 mM HEPES, 154 mM NaCl, 4.6 mM KCl, 2.3
mM CaCl
2
, 1.1 mM MgCl
2
, 33 mM glucose, 5 mM NaHCO
3
, 1.2 mM Na
2
HPO
4
, pH 7.4, and 0.5
mg/ml of MTT (Sigma Aldrich). The cells were incubated for 90 min in the above media, and
after incubation the resulting formation of formazan crystals was measured by dissolving in
DMSO and reading the absorbance at 490 nm in a microplate spectrophotometer.
Measurement of H
2
O
2
and nitrite
91
H
2
O
2
generation and release rate by primary astrocytes was determined by the Amplex Red
/Peroxidase Assay kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.
NO production was measured as total NO
2
-
present in the medium and detected by DAN assay
using Nitrate/Nitrite Fluorometric Assay Kit (Cayman Chemical, Ann Arbor, MI, USA).
Western blot analysis
Cytosolic and Nuclear Fraction from primary astrocytes were solubilized in SDS sample
buffer, separated by SDS/PAGE, and transferred onto PVDF membranes. Using appropriate an-
tibodies, the immunoreactive bands were visualized with an enhanced chemiluminescence rea-
gent. The blots were quantified using UN-SCAN-IT gel 6.1 (Silk Scientific, Inc., Orem, UT,
USA).
Statistical analysis
Data are reported as means ± SEM of at least 4-5 independent experiments. Significant dif-
ferences between mean values were determined by student t-test.
RESULTS
Lipoic acid induces a shift of metabolic profile in primary astrocytes
Astrocytes normally rely on anaerobic glycolysis for energy production and their mitochon-
drial oxidative phosphorylation is usually suppressed. However, astrocytes isolated from 7-, 13-,
and 18 month-old Sprague Dawley rat cortices displayed an age-dependent increase in OCR
(Oxygen Consumption Rate) reflecting mitochondrial respiration. Here the effects of lipoic acid
92
on astrocytic metabolic profiles were assessed with the Extracellular Flux analyzer: lipoic acid
decreased mitochondrial respiration in a dose-dependent manner (Fig. 1A). Both basal respira-
tion and maximal respiratory capacity decreased upon lipoic acid treatment. Anaerobic glycoly-
sis was measured by monitoring extracellular pH change, reflected by ECAR (Extracellular
Acidification Rate), induced by lactic acid formation. Astrocytic anaerobic glycolysis was signif-
icantly up-regulated upon the treatment by 20 μM lipoic acid, but not by other doses (Fig. 1B).
OCR/ECAR ratio was used as an index reflecting relative activity of oxidative metabolism and
anaerobic glycolysis. 20 μM lipoic acid substantially decreased OCR/ECAR ratio in old astro-
cytes, inducing a shift of metabolic profile towards stronger anaerobic glycolysis relative to aer-
obic metabolism (Fig. 1C). Of note, this shift in metabolic profile driven by 20 μM lipoic acid
was not accompanied by cytotoxicity, determined by MTT assay (Fig. 1D).
93
Fig. 1 Lipoic acid induces a shift of metabolic profile in primary astrocytes
(A) Dose-dependent changes in astrocytic aerobic metabolism upon lipoic acid treatment
for 24 hr. Primary astrocytes were cultured on Seahorse XF-24 plates, and Oxygen Consumption
Rate (OCR) was measured. (B) Changes in astrocytic anaerobic glycolysis upon lipoic acid treat-
ment. Extracellular Acidification Rate (ECAR) was used to assess astrocytic glycolysis. (C)
OCR/ECAR ratio was decreased by 20 μM lipoic acid treatment. (D) Cell viability did not change
following 20 μM lipoic acid treatment for 24 hr.
94
Lipoic acid increases anaerobic glycolysis in primary astrocytes via its insulin-like effect
The effect of lipoic acid on astrocytic glycolysis was Akt-dependent: specific PI3K inhibitor
LY294002 abrogated its stimulating effect on glycolysis (Fig. 2A). LY294002 alone substantial-
ly decreased anaerobic glycolysis in primary astrocytes, highlighting the role of PI3K/Akt path-
way in the regulation of astrocytic glycolysis (Fig. 2A). Importantly, the effect of lipoic acid was
surmised to be upstream of PI3K/Akt pathway, likely on the IRS (insulin receptor substrate). To
verify that notion, lipoic acid was shown to increase Akt phosphorylation at Ser
473
(active form)
and IRS phosphorylation at Tyr
608
(active form) (Fig. 2B). As a potent inhibitor of PI3K,
LY294002 abrogated the effect of lipoic acid on downstream Akt phosphorylation but not the
upstream IRS phosphorylation (Fig. 2B). As an essential regulator of glycolysis, PFKFB3 was
activated upon phosphorylation at Ser
483
by lipoic acid treatment. LY294002 was able to abolish
its phosphorylation induced by lipoic acid, indicating that lipoic acid activated PFKFB3 by its
insulin-like effect (Fig. 2B). Lipoic acid exerted no effects on the levels of other components of
astrocytic glycolysis, including lactate dehydrogenase (LDH), glucose transporter GLUT1, and
monocarboxylate transporter MCT1/4 (Fig. 2C-F).
95
Fig. 2 Lipoic acid increases anaerobic glycolysis in primary astrocytes via its insulin-like effect
(A) ECAR was significantly increased by 20 μM lipoic acid treatment for 24 hr, but decreased by
LY294002 inhibition. (B) The effects of lipoic acid and LY294002 on IRS, Akt, and PFKFB3 activity.
Activities were measured in terms of the phosphorylation levels (active forms), probed by Western Blot.
Lipoic Acid has no statistically significant effect on the level of (C) LDH, (D) GLUT1, (E) MCT1 and (F)
MCT4 in primary astrocytes.
96
Lipoic acid decreases mitochondrial membrane potential and H
2
O
2
production
Mitochondrial membrane potential of primary astrocytes –measured by TMRM staining and
analyzed by FACS (Fig. 3A)– was decreased by 51%, 55%, and 59% with lipoic acid of 20 µ M,
50 µ M, and 200 µ M respectively (Fig. 3B). As mitochondrial respiration and membrane poten-
tial were decreased by lipoic acid, H
2
O
2
production normally increases (Murphy 2009). However,
H
2
O
2
production was shown to be inhibited by lipoic acid at 20 µ M (Fig. 3D). This could be ex-
plained by lipoic acid-induced decrease in NADPH oxidase 4 (NOX4) levels (Fig. 3C). NOX4 is
the constitutive form of NADPH oxidase; NADPH oxidases were shown to be the main source
of H
2
O
2
production in astrocytes.
Fig. 3 Lipoic acid decreases mitochondrial membrane potential and H
2
O
2
production
(A) Lipoic acid decreased astrocytic mitochondrial membrane potential meas-
ured by TMRM staining and FACS analysis. (B) Quantification of mitochondrial mem-
brane potential relative to control. (C) The level of NOX4 was decreased by lipoic acid
treatment. (D) 20 μM lipoic acid decreased astrocytic H
2
O
2
production. H
2
O
2
release
rate into the astrocytic medium was measured by Amplex Red assay. *p < 0.05
97
Lipoic acid suppresses NFκB signaling
Activated NFκB pathway is highly involved in brain aging and neurodegenerative diseases
(Sanguino et al. 2006; Vromman et al. 2013). As the master regulator of inflammation, NFκB is
normally sequestered in the cytosol by IκB, but translocates to the nucleus following disassocia-
tion from IκB in response to the stimulation by IL-1β (Fig. 4). Nuclear NFκB initiates expression
of genes involved in inflammatory responses such as iNOS, TNFα, and IL-1β. Lipoic acid treat-
ment decreased NFκB nuclear translocation likely by retaining NFκB in the cytosol (Fig. 4).
Fig. 4 Lipoic acid suppresses NFκB signal-
ing
(A) Cytosolic NFκB P65 level was
decreased in response to 100 ng/ml IL-1β
treatment, 20 μM lipoic acid moderately re-
tained P65 in the cytosol. (B) Lipoic acid
decreased nuclear translocation of NFκB P65
induced by IL-1β treatment. Nuclear fractions
of astrocytes were isolated and probed with
NFκB P65 antibody, TBP (Tata Box Binding
Protein) was used as a loading control for
nuclear fraction. *p < 0.05
98
Lipoic acid decreases NO production
Neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) are the
major nitric oxide synthases in astrocytes. iNOS accounts for the sustaining production of high
level of NO and its expression was substantially induced by IL-1β stimulation (Fig. 5A). Lipoic
acid was shown to reduce its expression levels significantly (Fig. 5A). nNOS accounts for consti-
tutive production of NO and its expression was decreased by 20 μM lipoic acid (Fig. 5B). JNK is
involved in inflammation and activation of iNOS (Wang et al. 2004). JNK activity was up-
regulated by IL-1β and down-regulated by lipoic acid treatment (Fig. 5E). NO production was
assessed by nitrite and/or nitrate concentrations in the medium of primary astrocytes. IL-1β po-
tently increased nitrite concentration as well as nitrite + nitrate concentration in the medium, but
they were reduced substantially by lipoic acid (Fig. 5C-D).
99
Fig. 5 Lipoic acid decreases NO production
(A) 20 μM Lipoic Acid decreased iNOS expression induced by 100 ng/ml IL-1β treatment (B) 20 μM
Lipoic Acid decreased constitutive nNOS level in primary astrocytes. Lipoic acid decreased NO production in-
duced by IL-1β treatment, and NO production was measured in terms of nitrite (C) and nitrite+nitrate (D) level in
the astrocytic medium. (E) JNK activity was substantially increased by IL-1β treatment, but suppressed by 20 μM
lipoic acid. *p < 0.05
100
DISCUSSION
The hypometabolism in brain aging is largely ascribed to neurons, as they are the major bio-
energetic cell types in the brain. In line with that, brain neuronal glucose transporter GLUT3 de-
creases substantially with age (Jiang et al. 2013). However, another substantial energy source for
neurons is lactate generated by astrocytes. This astrocyte-neuron metabolic coupling was ex-
plained in a model in which glutamate uptake by astrocytes stimulates anaerobic glycolysis that
produces lactate to be transported to neurons (Magistretti et al. 1999). This notion is also sup-
ported by the fact that astrocytes normally harbor strong anaerobic glycolysis but suppressed mi-
tochondrial metabolism compared with neurons (Bolañ os et al. 2010). With age, however, astro-
cytes utilize energy substrates for their own mitochondrial metabolism rather than distributing to
neurons. In the current study, lipoic acid was shown to reverse this trend by inducing a metabolic
shift in 24 month-old astrocytes: lipoic acid suppressed mitochondrial oxidative metabolism but
augmented astrocytic anaerobic glycolysis (Fig. 1A-C). Lipoic acid seems to turn the metabolic
phenotype of old astrocytes into that of young astrocytes and renders astrocytes more neurosup-
portive.
The effect of lipoic acid on astrocytic anaerobic glycolysis was shown to involve PFKFB3,
which was suggested to be regulated by insulin signaling (Fig. 2). PFKFB3 is responsible for
generating fructose-2, 6-bisphosphate (F2, 6P2), which is an allosteric activator of 6-
phosphofructo-1-kinase (PFK1), a master regulator of glycolysis. Following respiration inhibi-
tion, astrocytes compensate energy production by stimulating glycolysis, involving activation of
PFKFB3 and increased amount of F2, 6P2 (Almeida et al. 2004). This adaptive response is not
seen in neurons due to the near absence of PFKFB3. However, the effect of lipoic acid on astro-
cytic anaerobic glycolysis is not likely due to its inhibition of respiration and the consequent ac-
101
tivation of PFKFB3. In contrast with the inhibition on mitochondrial respiration, the enhance-
ment of anaerobic glycolysis by lipoic acid is not dose-dependent, but lipoic acid exerts the most
prominent effect at 20 μM (Fig. 1A-B). This suggests that the stimulating effect of lipoic acid
on astrocytic glycolysis could not be secondary to its inhibitory effect on respiration, but rather
due to other mechanisms, possible its insulin-like effect. In support of that, insulin signaling is
activated by moderate oxidants involving tyrosine phosphorylation of insulin receptor
(Storozhevykh et al. 2007; Cheng et al. 2010). In this study, moderate concentration of lipoic
acid could act as a mild stressor that activates insulin signaling and stimulates astrocytic glycoly-
sis.
The anti-inflammatory effects of lipoic acid seem to center on NFκB: NFκB signaling is re-
dox-sensitive and there are multiple components that are sites for redox modulation by H
2
O
2
and
Trx, especially the ones with accessible cysteine residues (Brigelius-Flohe & Flohe 2011). The
phosphorylation of IKK and IκB is promoted by H
2
O
2
(Takada et al. 2003; Storz et al. 2004;
Loukili et al. 2010). O
2
.–
and H
2
O
2
generated by NOX are likely involved in the assembly and
recruitment of signaling molecules such as IRAK and TRAF6, to IL-1R, leading to NFκB activa-
tion. Therefore the effects of lipoic acid on NFκB signaling may result from a direct interaction
between lipoic acid and redox-sensitive molecules such as NFκB (Nishi et al. 2002), but more
likely, from modulation of cellular redox status (Fig. 3; Fig. 5). However, those effects are not
likely due to the direct interaction between lipoic acid and H
2
O
2
/O
2
.–
, but possibly due to its indi-
rect effect on the cellular antioxidant system. Lipoic acid has been shown to regenerate GSH
(Busse et al. 1992; Suh et al. 2004) and induce Nrf2-controlled Phase 2 antioxidant enzymes.
Furthermore, NADPH oxidase was shown to be the primary source of H
2
O
2
in astrocytes and
lipoic acid substantially decreased NOX4 expression in old primary astrocytes.
102
Overproduction of NO by up-regulation of iNOS is implicated in multiple neuropathologies
(Brosnan et al. 1994). iNOS expression is affected by NFκB and JNK signaling (Wang et al.
2004). NO released from astrocytes was shown to increase with age. Lipoic acid effectively re-
duces NO production in old primary astrocytes by reducing iNOS as well as nNOS expression
and inactivates JNK (Fig. 5). This is interpreted as a neuroprotective effect, as NO, like H
2
O
2
, is
an important diffusible molecule that could affect neuronal function. NO derive from astrocytes
was shown to cause glia-induced neuronal death (Bal-Price & Brown 2001).
The anti-inflammatory effects of lipoic acid were observed in different experimental models:
lipoic acid attenuated LPS-induced inflammatory responses by activation of PI3K/Akt signaling
(Zhang et al. 2007), reduced age-dependent vascular oxidative stress and inflammation (Li et al.
2010), decreased Th1-mediated inflammation in LPS-induced uveitis (Merida et al. 2013), and
inhibited NFκB activation in human T cells (Suzuki et al. 1992).
Overall, results from this study demonstrated that lipoic acid effectively reversed the meta-
bolic phenotypes of 24 month-old astrocytes to that of young astrocytes that consist of robust
anaerobic glycolysis but suppressed mitochondrial metabolism. The anti-inflammatory effects of
lipoic acid are achieved by suppressing redox-sensitive NFκB signaling and reducing diffusible
messengers such as H
2
O
2
and NO. The effect of lipoic acid on astrocyte anaerobic glycolysis is
likely to result from activated PFKFB3, regulated by insulin signaling. Its anti-inflammatory ef-
fect could also involve interplay between insulin signaling and JNK signaling. As astrocytes
switch from neurotrophic to neurotoxic with age, lipoic acid may serve as a strategy to alter as-
trocytic metabolic-inflammatory axis, leading to enhanced neuroprotection. This effect, com-
bined with its direct effect on neuronal function, may partly account for the beneficial effects of
lipoic acid in brain aging.
103
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Fig. 1 Coordinated interactions of mito-
chondrial function and redox-sensitive
signaling and transcriptional pathways
CHAPTER FIVE
CONCLUSIONS AND FUTURE DIRECTIONS
Aging is risk factor for a wide array of neurodegenerative diseases, such as Alzheimer’s dis-
ease, Parkinson’s disease, and Huntington's disease. Energy deficit, oxidative and nitrosative
stress, and inflammation are all inherent features of brain aging; this aspects of brain aging
should be viewed as an integrative processes that governs cell function.
Mitochondria integrate energy and redox metabolism as the energy–redox axis of the cell
(Fig. 1): mitochondrial bioenergetics is an important determinant of the steady-state levels of
H
2
O
2
in conjunction with the mitochondrial redox systems, which can be glutathione- and thi-
oredoxin based. The function of the redox systems requires sufficient supply of NADPH to
maintain redox status.
Mitochondrial redox status affects their energy-
transducing capacity via redox modulation on mito-
chondrial proteins. Furthermore, redox-sensitive in-
sulin signaling and JNK signaling are modulated by
mitochondrial oxidants and they in turn impinge on
mitochondrial functions, thus establishing a metabol-
ic triad that is disrupted in brain aging. Brain aging
is characterized by an imbalance of insulin and JNK
signaling: reduced insulin sensitivity and increased
JNK activity are associated with decreased glucose
uptake into the brain, decreased mitochondrial bio-
genesis in neurons, and impaired bioenergetics cou-
pled with increased release of H
2
O
2
. Part of the studies in this dissertation strengthened the no-
tion that imbalance of the insulin- and JNK signaling pathways is a determinant of brain aging,
inasmuch as both signaling pathways are redox regulated by mitochondrion-generated second
messengers and both signaling pathways regulate mitochondrial function.
The interplay among energy metabolism, redox status, and inflammation is manifested in
primary astrocytes isolated from rats of different ages. As a complete different cell type from
neurons, astrocytes normally perform anaerobic glycolysis to support neurons with metabolic
107
substrate lactate and amplify inflammatory signals to protect against insults. In brain aging, how-
ever, astrocytes switch from neurotrophic to neurotoxic. Increased H
2
O
2
production from astro-
cytic NADPH oxidases is associated with activation of redox-sensitive NFκB signaling resulting
in increased iNOS expression and NO generation. Chronic inflammatory responses require sus-
tainable energy support from mitochondria and trigger mitochondrial biogenesis and enhanced
respiration which restricts substrate supply to support neurons. Of note, neurons are especially
vulnerable to energy deprivation and oxidative and nitrosative stresses (H
2
O
2
and NO from as-
trocytes) due to limited regenerative capacity.
In our studies, R-(+) lipoic acid was shown to restore the energy deficit and attenuate the in-
flammatory responses associated with brain aging. Exogenous lipoic acid exerted effects –under
a general mechanistic umbrella encompassing thiols/disulfide exchange– different from the
lipoamide occurring as a metabolic cofactor of mitochondrial -ketoacid dehydrogenases. The
insulin-like effect of lipoic acid in restoring energy metabolism was partly accomplished by re-
storing the imbalance of Akt/JNK signaling, augmenting glucose uptake, and increasing mito-
chondrial biogenesis. Lipoic acid does not directly associate with mitochondria but acts on re-
dox-sensitive pathways by thiol/disulfide exchange mechanisms that are sensitive to the cell's
redox environment. This was viewed in terms of a triad encompassing mitochondrial function,
signaling pathways, and transcriptional pathways. Fig. 2 shows the sites of lipoic acid action in a
brain aging model (it summarizes evidence in chapter 1 and chapter 2).
Fig. 2 Sites of lipoic acid action in brain
aging
Lipoic acid was shown to activate
IR, IRS, Akt, GLUT translocation, and
inactivate GSK3β. The effect of lipoic
acid on synaptic plasticity was demon-
strated in a triple transgenic mouse model
of Alzheimer’s disease.
108
JNK AKT
OVERACTIVE IIS MAY LEAD TO
• INCREASED STRESS SENSITIVITY
• DECREASED LIFESPAN
REDUCED IIS MAY RESULT IN
• METABOLIC DYSFUNCTION
JNK activity
IIS activity
Growth
Stress
sensitivity
Decreased
lifespan
Stress
protection
Lifespan
extension
Insulin
resistance
Diabetes
Karpac & Jasper (2009)
Trends Endocrinol Metab 20, 100-106
THE BALANCE BETWEEN
SURVIV AL AND DEATH SIGNALS
DETERMINES THE FATE OF NEURONS
EXCESSIVE JNK ACTIVITY LEADS TO
• INSULIN RESISTANCE
• NEURODEGENERATION
The anti-inflammatory properties of lipoic acid in old primary astrocytes were likely due to
its reaction with redox-sensitive NFκB signaling or through its indirect effects on the redox sta-
tus of astrocytes. Lipoic acid was shown to suppress astrocytic aerobic metabolism but increase
anaerobic glycolysis that could be beneficial for neurons by providing more lactate as substrates.
In summary, lipoic acid elicits metabolic and inflammatory changes in old astrocytes and shifts
them from neurotoxic back to neurotrophic.
Future directions of the work in this dissertation would surround energy-redox status in brain
aging and therapeutic agents. Not limited in the brain but even at a systemic level, insulin signal-
ing and JNK signaling should be well-balanced. Overactive insulin signaling may lead to in-
creased stress sensitivity and decreased lifespan, whereas reduced insulin signaling may result in
metabolic dysfunction; conversely, excessive JNK activity leads to insulin resistance and neuro-
degeneration (Fig. 3). Therefore developing redox modulators as therapeutic agents to target re-
dox-sensitive insulin and JNK pathways and maintain them at an appropriate balance would be
promising for treating metabolic diseases and promoting healthy aging.
Fig. 3 The balance between
survival and death signals de-
termines the fate of neurons.
The balance of Insulin
signaling and JNK signaling
should be well-maintained to
optimize healthspan and
lifespan.
109
Although the beneficial effects of lipoic acid are observed in the brain, its effects on periph-
eral tissues cannot be excluded. Lipoic acid was shown to improve cardiovascular systems, liver,
adipocytes, and skeletal muscles by stimulating eNOS expression, increasing GSH synthesis, and
augmenting glucose uptake and metabolism. Hence, lipoic acid is also likely to improve brain
functions indirectly by optimizing energy and redox homeostasis at a systemic level.
The mechanism of astrocytes switching from neurotrophic to neurotoxic in aging and age-
related diseases could be partly explained by the alterations in their metabolic-inflammatory axis,
but still requires further investigations. The effects of lipoic acid as a redox modulator on meta-
bolic and inflammatory components of old astrocytes seem to switch their phenotype to that of
young astrocytes; these are likely ascribed to the thiol-disulfide exchange reactions, but still re-
mains unresolved. Despite that, development of therapeutic agents that target astrocytes and im-
prove their neurotrophic functions could serve as an innovative strategy for the treatment of neu-
rodegenerative diseases in aging. As astrocytes have easy access to blood vessels, they are the
first target for therapeutic agents that cross blood brain barrier. Therefore, therapeutic agents tar-
geting astrocytes would be very efficient and effective.
Abstract (if available)
Abstract
Aging is risk factor for diseases in both peripheral and central nervous system. Studies in this dissertation are aimed at investigating brain aging in the context of mitochondrial energy‐redox axis and inflammation. ❧ The progress of a hypometabolic state inherent in brain aging was studied in an animal model consisting of Fischer 344 rats of young, middle, and old ages. Dynamic microPET scanning demonstrated a significant decline in brain glucose uptake at old ages, which was associated with a decrease in the expression of insulin‐sensitive neuronal glucose transporters GLUT3/4 and of microvascular endothelium GLUT1. Brain aging was associated with an imbalance of the PI3K/Akt pathway of insulin signaling and JNK signaling and a downregulation of the PGC1α‐mediated transcriptional pathway of mitochondrial biogenesis that impinged on multiple aspects of energy homeostasis. R−(+)‐lipoic acid treatment increased glucose uptake, restored the balance of Akt/JNK signaling, and enhanced mitochondrial bioenergetics and the PGC1α‐driven mitochondrial biogenesis. It may be surmised that impairment of a mitochondria‐cytosol‐nucleus communication is underlying the progression of the age‐related hypometabolic state in brain
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Jiang, Tianyi
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Core Title
Energy metabolism and inflammation in brain aging: significance of age-dependent astrocyte metabolic-redox profile
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Molecular Pharmacology and Toxicology
Publication Date
07/08/2015
Defense Date
06/16/2014
Publisher
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astrocytes,brain aging,cytokines,FDG-PET,hydrogen peroxide,Inflammation,insulin signaling,JNK signaling,lipoic acid,mitochondria,mitochondrial bioenergetics,mitochondrial biogenesis,NFκB,nitric oxide,OAI-PMH Harvest,PGC1α,SIRT1
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