Neurochem Res (2010) 35:1880–1915
DOI 10.1007/s11064-010-0307-z
ORIGINAL PAPER
Cellular Stress Responses, Mitostress and Carnitine
Insufficiencies as Critical Determinants in Aging
and Neurodegenerative Disorders: Role of Hormesis
and Vitagenes
Vittorio Calabrese • Carolin Cornelius •
Anna Maria Giuffrida Stella • Edward J. Calabrese
Accepted: 21 October 2010 / Published online: 13 November 2010
 Springer Science+Business Media, LLC 2010
Abstract The widely accepted oxidative stress theory of
aging postulates that aging results from accumulation of
oxidative damage. A prediction of this theory is that,
among species, differential rates of aging may be apparent
on the basis of intrinsic differences in oxidative damage
accrual. Although widely accepted, there is a growing
number of exceptions to this theory, most contingently
related to genetic model organism investigations. Proteins
are one of the prime targets for oxidative damage and
cysteine residues are particularly sensitive to reversible and
irreversible oxidation. The adaptation and survival of cells
and organisms requires the ability to sense proteotoxic
insults and to coordinate protective cellular stress response
pathways and chaperone networks related to protein quality
control and stability. The toxic effects that stem from the
misassembly or aggregation of proteins or peptides, in any
cell type, are collectively termed proteotoxicity. Despite
the abundance and apparent capacity of chaperones and
other components of homeostasis to restore folding equi-
librium, the cell appears poorly adapted for chronic pro-
teotoxic stress which increases in cancer, metabolic and
neurodegenerative diseases. Pharmacological modulation
of cellular stress response pathways has emerging impli-
cations for the treatment of human diseases, including
neurodegenerative disorders, cardiovascular disease, and
Special issue article in honor of Dr. Abel Lajtha
V. Calabrese (&)
C. Cornelius
A. M. G. Stella
Department of Chemistry, University of Catania, Viale Andrea
Doria, 95100 Catania, Italy
e-mail: calabres@unict.it
E. J. Calabrese
Environmental Health Sciences Division, School of Public
Health, University of Massachusetts, Amherst, MA, USA
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cancer. A critical key to successful medical intervention is
getting the dose right. Achieving this goal can be extremely
challenging due to human inter-individual variation as
affected by age, gender, diet, exercise, genetic factors and
health status. The nature of the dose response in and
adjacent to the therapeutic zones, over the past decade has
received considerable advances. The hormetic dose–
response, challenging long-standing beliefs about the nat-
ure of the dose–response in a lowdose zone, has the
potential to affect significantly the design of pre-clinical
studies and clinical trials as well as strategies for optimal
patient dosing in the treatment of numerous diseases. Given
the broad cytoprotective properties of the heat shock
response there is now strong interest in discovering and
developing pharmacological agents capable of inducing
stress responses, including carnitines. This paper describes
in mechanistic detail how hormetic dose responses are
mediated for endogenous cellular defense pathways,
including the possible signaling mechanisms by which the
carnitine system, by interplaying metabolism, mitochon-
drial energetics and activation of critical vitagenes,
modulates signal transduction cascades that confer cyto-
protection against chronic degenerative damage associated
to aging and neurodegenerative disorders.
Keywords Redox state
Cellular stress response

Mitochondrial bioenergetics
Acetylcarnitine
Hormesis

Vitagenes
Introduction
In response to fluctuations of their microenvironment,
cells must continuously activate a series of homeostatic
Neurochem Res (2010) 35:1880–1915
mechanisms that aim at maintaining their bioenergetic,
metabolic, ionic, and genomic integrity. The frontier
between normal fluctuations and potentially pathogenic
perturbations (‘‘stress’’) is not neat, and actually depends
on the capacity of cells to adapt to distinct levels of stress.
Hence, any genetically determined or acquired reduction in
the capacity of cells to adapt to cellular stress is potentially
pathogenic because it lowers the threshold at which
endogenous or exogenous noxious agents cause irrevers-
ible cellular damage, culminating in the irreversible loss
of cells by the activation of programmed cell death
pathways [1].
Maintenance of optimal long-term health conditions is
accomplished by a complex network of longevity assur-
ance processes which are controlled by vitagenes, a group
of genes involved in preserving cellular homeostasis during
stressful conditions [2]. Vitagenes encode for heat shock
proteins (Hsp) Hsp32, Hsp70, the thioredoxin and the
sirtuin protein systems [2]. Dietary antioxidants, such
as polyphenols and L-carnitine/acetyl-L-carnitine, have
recently been demonstrated to be neuroprotective through
the activation of hormetic pathways, including vitagenes.
The hormetic dose–response, challenges long-standing
beliefs about the nature of the dose–response in a lowdose
zone, having the potential to affect significantly the design
of pre-clinical studies and clinical trials as well as strate-
gies for optimal patient dosing in the treatment of numer-
ous diseases [3]. Given the broad cytoprotective properties
of the heat shock response there is now strong interest in
discovering and developing pharmacological agents capa-
ble of inducing stress responses. In this review we discuss
the most current and up to date understanding of the pos-
sible signaling mechanisms by which acetylcarnitine by
activating vitagenes can enhance defensive systems
involved in bioenergetic and stress resistance homeostasis.
The ‘‘Free Radical Hypothesis’’ of Aging
Ageing is typically accepted as being a product of one’s
genetic composition and the cumulative effects of lifestyle
practices and exposures. Humans reach a peak of growth
and development around the time of their mid 20s. Starting
at what is commonly referred to as ‘‘middle age’’, the
operations of the body become more susceptible to wear
and tear, and there is a general decline in physical and
possibly mental performance. During the latter half of life,
an individual is more prone to experiencing problems with
a range of bodily functions and to developing various
chronic or fatal diseases. The cardiovascular, digestive,
excretory, nervous, reproductive and urinary systems are
particularly vulnerable, and the most common age-related
ailments include Alzheimer’s, cancer, arthritis, diabetes,
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and heart disease. In several developed countries, the life
expectancy, especially in females, is now into the 80s, with
a maximal lifespan being reported of approximately
120 years. What dictates or regulates ‘‘why we age’’ and
‘‘why the ageing body loses functional capacity’’ has been
a matter of debate for centuries. It seems likely that several
factors work together in the ageing process, or that one
particular factor is the primary culprit in a given individual.
One of the most studied and accepted hypotheses for the
molecular basis of aging has been the oxidative stress
theory of aging. The origins of the free radical theory of
aging go back to the mid twentieth century, when it was
discovered that oxygen free radicals, traditionally thought
to be too reactive to exist in biological systems, are formed
in situ in response to radiation and oxygen poisoning are
responsible for the associated toxicities [4, 5] (Table 1).
Noting that radiation ‘‘induces mutation, cancer and aging’’
[6, 7] and, drawing on the rate-of-living hypothesis [8],
Denham Harman first conceptualized the free radical the-
ory of aging and suggested that oxygen free radicals
(specifically hydroxyl, OH•, and hydroperoxyl, HO•2)
formed endogenously as by-products from normal oxy-
genutilizing metabolic processes can play an essential role
in the aging process [6]. Later, Harman added a slight
modification to this theory to bring special attention to role
of the mitochondria in the aging process because these
organelles are a major site of reactive oxygen species
(ROS) production [7]. The interest in the free radical the-
ory was at the beginning limited by the persistent doubt
about the existence of oxygen free radicals in biological
systems [7]. Then, for more than 50 years, numerous
reports have examined the links between oxidative stress,
longevity, and age-related disease [9]. Continuing to today,
numerous studies have shown that oxidative damage
increases with age in many organisms and that many forms
of ROS may be the culprits of accumulated oxidative
damage. Thus, Harman’s original hypothesis has been
refined in such a way to address the role of all forms of
ROS in regulating aging and now is generally termed the
oxidative stress theory of aging [10]. The basis of this
theory is that a chronic state of oxidative stress exists in all
cells of aerobic organisms even under normal physiological
conditions because of an imbalance between pro-oxidants
and antioxidants [11]. This imbalance leads to the accu-
mulation of oxidative damage to cellular macromolecules
that increases during aging and contributes to a progressive
decline in the function of cellular processes. Under this
mechanistic framework, the regulation of oxidative stress
may directly control the aging process [12, 13] (Table 1).
During the last few years, cellular oxidant/antioxidant
balance has become the subject of intense study, particu-
larly by those interested in brain aging and in neurode-
generative mechanisms [14]. Several lines of evidence
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1882 Neurochem Res (2010) 35:1880–1915

Table 1 Trends in the free radical theory of aging

1950 1960 1970 1980 1990 2000
O2•--dependent Age-dependent increase Increased life span in transgenic
production of H2O2 of oxidative damage flies
in mitochondria

Free radical theory Identification of Discovery of Age-dependent increase of Age-dependent CuZnSOD overexpression
of oxygen toxicity CuZnSOD MnSOD oxidative damage to increase of oxidative does not extend life span
Gerschman (1954) McCord and Fridovich proteins Stadtman damage to DNA in mouse Epstein (2000)
Fridovich (1969) (1972) (1987) Ames (1990)
Free radical theory Mitochondrial Decreased life span in Mt-catalase
of aging Harman theory of CuZn null flies Phillips overexpression extends
(1956) aging Harman and Hilliker (1989) life span in mouse
(1972) Rabinovitch (2005)
CuZn homozygous null
mice exhibit decreased
life span Huang (2005)

suggest that accumulation of oxidative molecular damage
is a causal factor in senescence. The direct evidence for this
hypothesis is that overexpression of antioxidative genes for
Cu, Zn-superoxide dismutase and catalase in transgenic
Drosophila melanogaster prolongs the life span, retarding
the age-associated accumulation of oxidative damage [12].
Among the correlative evidence supporting the involve-
ment of oxidative stress are the following: (1) oxidative
damage to DNA and proteins increases exponentially with
age, and concomitantly, the rates of mitochondrial O•- 2 and
H2O2 generation as well as the susceptibility of tissues to
experimentally induced oxidative stress are increased; (2)
experimental regimens that extend life-span, such as
caloric restriction in mammals and reduction of metabolic
rate in insect decrease the accumulation rates of oxidative
damage; (3) mitochondria make two rather contradictory
contributions to cell survival [15]. The classically recog-
nized function is the synthesis of ATP for energizing
endergonic reactions, the other is generation of reactive
oxygen species which may compromise the long-term
survival of cells and constitute a major underlying cause of
the aging process. Indeed, these two rather conflicting
functions are part of the same process, namely mitochon-
drial respiration. Physiologically, electron transport chain
consumes up to 90% of the oxygen taken up by a cell, of
which *1% is transformed in superoxide under normal
physiological conditions [2, 16]. The rate of mitochondrial
electron transfer from semi-reduced Q to molecular O2 to
form superoxide is proportional to the product of the
concentrations of semi-reduced Q and O2. It is estimated
that the under pathophysiological conditions, superoxide
production can occur at 2–10% of the uninhibited rates.
Superoxide dismutases, in turn, generate the more stable
hydrogen peroxide before it is transformed by catalase in
water or, depending on the presence of metals, into highly
reactive hydroxyl radicals. Thus, SOD2, which directs
H2O2 out of the mitochondrion, accomplishes two func-
tions: the classical scavenging of superoxide, as well as its
signalization as ROS-dependent signaling pathways. More
than 95% of the O2 taken up by the human body is used by
mitochondrial cytochrome oxidase which adds four elec-
trons to oxygen to generate a molecule of water. Cyto-
chrome oxidase normally does not release reactive oxygen
species into its surroundings. However, a number of
investigations have indicated that brain mitochondria
undergo oxidative stress damage and a decrease of cyto-
chrome c oxidase activity during aging [17]. It has been
postulated that this complex may act as a bottleneck, cre-
ating a situation of ‘‘electron traffic jam’’ upstream, which
would alter the redox state of oxidoreductases in the
electron transfer chain, and increase their autoxidizability
and rate of superoxide generation. A finding that lends
credibility to this hypothesis is that cytochrome c oxidase
activity is directly correlated with the average life-span in
different species [17]. Oxidative damage to key intracel-
lular targets such as DNA or proteins is an important fea-
ture of the normal cellular aging process in the brain, and
several studies have shown that oxidative damage to DNA
or protein extracted from brain tissue increases with age
[14]. Oxidative damage to DNA has been shown to be
extensive and could be a major cause of the degenerative
diseases related to aging such as cancer [18]. Activation of
persistent DNA damage response (DDR) signaling is
associated with the induction of a permanent proliferative
arrest known as cellular senescence, a phenomenon
intrinsically linked to both tissue aging as well as tumor
suppression. The DNA damage observed in senescent cells
has been attributed to elevated levels of reactive oxygen
species (ROS), failing DNA damage repair processes, and/
or oncogenic activation. It is not clear how labile molecules

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such as ROS are able to damage chromatin-bound DNA to
a sufficient extent to invoke persistent DNA damage and
DDR signaling. Recent evidence suggests that the nucle-
otide pool is a significant target for oxidants and that oxi-
dized nucleotides, once incorporated into genomic DNA,
can lead to the induction of a DNA strand break-associated
DDR that triggers senescence in normal cells and in cells
sustaining oncogene activation. Evasion of this DDR and
resulting senescence is a key step in tumor progression
[19]. With respect to this, it has been proposed that DNA
damage is a major factor underlying neuronal degeneration
in normal aging as well as the basis of the neurodegener-
ative conditions associated with Alzheimer’s disease (AD)
[20]. Levels of the oxidized nucleotide 8-hydroxy-deoxy-
guanosine (8-OH-dG) a biomarker of DNA damage, have
also been shown to accumulate with aging. In several
tissues, including brain and muscle, levels of 8-OH-dG
in mtDNA exceed that of nuclear DNA (nDNA) some
16-fold.
Mecocci et al., found that 8-OH-dG significantly cor-
relates with increases in levels of a 7.4-kb deletion in
human brain [21]. Experimental studies using lymphocytes
from humans or mice, or employing transgenic mouse
models that harbor a defined reporter gene, demonstrate
that mutations increase with age [22]. Considering that
nearly 50% of the human genome is transcribed (with 1%
representing protein coding sequences), only a few random
spontaneous mutational events, such as point mutations,
deletions/insertions, or chromosomal rearrangements,
could have profound effects on the entire gene regulatory
circuitry [22].
The idea that DNA damage contributes to the ageing
process and particularly to age-related disease is supported
by the following considerations: (1) DNA is subject to
continuous modification from both endogenous reactive
chemicals, such as reactive oxygen species, and exogenous
environmental factors, such as food agents, industrial
genotoxins, and ultraviolet and ionizing radiation; (2) DNA
is intrinsically unstable, experiencing spontaneous base
loss and deamination at high frequency. Despite the exis-
tence of an array of protective DNA repair systems, there is
evidence that several forms of DNA damage, such as strand
breaks, alkali-labile sites (i.e. abasic lesions), base modi-
fications (most notably, 8-oxo-2-deoxyguanosine) and
cross-links (particularly protein–DNA), accumulate with
age, although the findings depend on the measurement
technique employed and the tissue or organ studied; (3)
there is evidence that both antioxidant defense mechanisms
and DNA repair responses decline with age, mouse species
displaying different life expectancies possessed a repair
potential that correlated with maximal life span. However,
when the organism’s body size was taken into account, the
correlation of DNA repair capacity with life expectancy
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disappeared [23, 24]; (4) studies that involve exogenous
exposure of rodents to a DNA-damaging agent have
revealed that such treatments result in reduced life span,
yet, importantly, without affecting (i.e. accelerating) nor-
mal ageing and (5) potentially most compelling, inherited
disorders originating from defects in DNA damage
response genes give rise to phenotypes that resemble the
normal ageing process. This connection is perhaps best
exemplified by the human segmental progerias Werner
syndrome and Cockayne syndrome. Finally, recent studies
using genetically defined DNA repair mutant mouse
models suggest that ‘‘ageing and end-of-life fitness are
determined both by stochastic damage, which is the cause
of functional decline, and genetics, which determines the
rates of damage accumulation and decline [25]. Moreover,
experimental evidence indicates that intrinsic DNA repair
levels associate with longevity. For instance, EBV-
immortalized lymphoblastoid cell lines from centenarians
possess higher than normal poly(ADP)ribose polymerase 1
(PARP1) activity, presumably reflective of an enhanced
DNA strand break response. On the other hand, the rate of
production of pro-oxidants, which are responsible for the
majority of intracellular damage, has been found in several
different species to inversely correlate with maximal life
expectancy [25]. Consistently, it has been demonstrated
that although DNA repair enzymes are present and active
in senescent post-mitotic tissues such as brain, changes in
the structure and function of ‘‘old’’ chromatin somehow
decreases the capacity of the DNA repair enzymes present
in the nucleus to repair oxidatively damaged DNA. There
are several enzymes systems that have been found to repair
damage to DNA caused by oxidising species. These
include endonucleases, exonucleases, thymine glycol gly-
colases and DNA polymerases. DNA polymerases thus far
detected in mammalian brain (alpha, beta, delta and epsi-
lon), undergo age-dependent changes in activity, but it is
not known which cell types contain which polymerases,
and the ability of nuclei from different brain regions
to repair specific types of oxidative DNA damage is
unknown. In addition, recent evidence indicates that
genetic instability, such as telomere loss, somatic and
mitochondrial DNA mutations, increases with age [23].
An increase in protein oxidative damage, as indicated by
the loss of protein sulfhydryl groups and by a decline in the
activity of enzymes, such as glutamine synthetase and
glucose-6-phosphate dehydrogenase has been demon-
strated to occur in brain during aging [26]. A number of
experimental evidence indicate that increased rate of free
radical generation and decreased efficiency of the repara-
tive/degradative mechanisms, such as proteolysis, both are
factors which primarily contribute to age-related elevation
in the level of oxidative stress and brain damage. With
respect to this, it has been suggested that decreases in
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levels of enzymes which ordinarily protect neuronal cells
against oxidative stress with age may be responsible for
increased levels of free-radical damage in the brain, or that
these enzymes themselves are susceptible to inactivation
by free radical molecules which increase with age in the
brain [27]. During aging a number of enzymes accumulate
as catalytically inactive or less active forms. The age-
related changes in catalytic activity are due in part to
reactions of proteins with oxygen and/or nitrogen free
radical species produced during exposure to ionizing
radiation or to metal ion catalyzed oxidation systems. The
levels of oxidized proteins in brain extracts of rats of dif-
ferent ages increase progressively with age, and in old rats
can represent 30–50% of the total cellular protein [26].
These evidence provides strong evidence that there is a
linkage between the age-dependent accumulation of oxi-
dized proteins and the loss in brain physiological functions.
It has recently been proposed that a primary mechanism
leading to neuronal cell death in ageing and common
neurodegenerative disorders is interference with protea-
some function [28]. The major neurodegenerative diseases,
Alzheimer disease (AD), Parkinson’s Disease (PD), amy-
trophic lateral sclerosis (ALS), Huntington’s disease (HD)
and Freidreich’s ataxia (FA) are all associated with the
presence of abnormal proteins [16, 29]. The origin of HD
and FA involve specific genetic defects that lead to pro-
duction of abnormal proteins [30], whereas AD, ALS, and
PD have been described mostly as sporadic, although
familial types of AD, ALS and PD are recognized.
Examples are the rare mutations in synuclein or parkin that
cause familial PD, and the 2% of ALS cases associated
with mutation in the gene encoding copper-zinc containing
superoxide dismutase (CuZnSOD) [16, 30–32]. Even in the
more common sporadic version of PD, ALS and AD
abnormal proteins are present to a significant extent [33].
Thus the senile plaques typical of AD contain not only
b-amyloid but a wide range of other proteins. Most of them
are oxidized and nitrated. Similar damage has been
described for proteins in the Lewy bodies in sporadic PD.
Oxidative as well as nitrosative protein damage are also
elevated in ALS. In non-dividing cells, such as the great
majority of neurones in the adult brain, the protein content
of cells is approximately constant. Since protein synthesis
is continuous, there must be an equilibrium between syn-
thesis and degradation [34]. A variety of conditions of
neurodegenerative diseases often lead to post-translational
modifications of proteins, including oxidation and nitra-
tion, which might be involved in the pathogenesis of
neurodegenerative diseases. Redox proteomics, a subset of
proteomics, has made possible the identification of spe-
cifically oxidized proteins in neurodegenerative disorders,
providing insight into a multitude of pathways that govern
behavior and cognition and the response of the nervous
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system to injury and disease [35]. Oxidized protein levels
in the CNS tend to increase with age consistently with
several reports that proteasome activity decreases with age
and in neurodegenerative disorders [36, 37]. Importantly,
the accumulation of oxidation products in particular
regions of the brain seems to be related to specific cogni-
tive defects [38]. In addition, studies on the induction of
HSPs response, a cytoprotective mechanism to counteract
oxidative damage, has showed a regional specificity indi-
cating that different brain areas might undergo oxidation
differently and react to protect themselves based on the
strength of the insult [39, 40]. Studies from our laboratory,
using a redox proteomics approach to identify the oxida-
tively modified proteins in different brain regions of
senescent compared to adult rats, have demonstrated that
most of oxidized proteins are involved in energy metabo-
lism pathways, including ATP production, glycolysis and
Krebs cycle. In addition, protein oxidation also affected
components of the cell involved in cell structure, signal
transduction, as well as cellular stress response, such as
Hsp70 [41–44].
Studies on isoprostanes, end product of lipid peroxida-
tion that can be measured in CSF and urine in various
neurodegenerative disorders, suggest that lipid peroxida-
tion is an early stage in these disease processes. Similarly,
another end product of lipid peroxidation, the aldehyde
4-hydroxy-trans-nonenal (HNE), which is highly neuro-
toxic, avidly binds to proteins, and HNE-protein adducts
are demonstrable in senile plaques and tangles in AD,
tissues from ALS patients, and lewy bodies in PD [45, 46].
There are two broad outcomes of lipid peroxidation:
structural damage of membrane integrity and the genera-
tion of aldehydic byproducts, particularly a, b-unsaturated
aldehydes, including 4-hydroxynonenal (HNE) and acro-
lein. These aldehydes react quickly with nucleophilic
sulfhydryl groups and are toxic to primary hippocampal
neuron cultures [45] and are elevated in vulnerable areas of
the brain in LAD [45] and MCI. Current evidence suggests
that HNE and acrolein are by-products of oxidative damage
to polyunsaturated fatty acids (PUFAs). HNE and acrolein
are by-products of x-6 PUFAs, whereas 4-hydroxyhexenal
is derived from x-3 PUFAs [45]. Docosahexaenoic acid
(DHA), an x-3 PUFA, and arachidonic acid (ARA), an x-6
PUFA, are the predominant PUFAs in gray matter. In
addition, numerous studies demonstrate that acrolein and
HNE are highly reactive, resulting in the disruption of
normal cellular processes and activities through a Michael
addition to key amino acids such as lysine, cysteine, and
histidine [46]. Modification of proteins by HNE can result
in a reduction in the activity of several critical enzymes,
including lactate dehydrogenase and ATP synthase [45].
Acrolein can impair metabolic processes by inhibiting
glucose uptake in primary neuronal cultures [45]. In vitro
Neurochem Res (2010) 35:1880–1915
studies also support acrolein’s role in the disruption
of glucose metabolism by demonstrating that acrolein
when incubated with pyruvate dehydrogenase results in
decreased activity [45]. Lovell et al. [47] showed that the
activity of glutathione transferase, a detoxification enzyme
for HNE and acrolein, was significantly decreased in the
HPG of LAD subjects.
In spite of all above mentionated evidence, however,
current literature data showing the effects of alterations in
the antioxidant defense system on the life span of mice (as
well as various invertebrate models) seriously call into
question the classical interpretation of the oxidative stress
theory of aging, in particular the role of oxidative damage
as a major factor underlying the mechanism of the aging
process. The oxidative stress theory of aging proposes a
causal relationship between reactive oxygen species and
aging, predicting that manipulations that alter oxidative
stress/damage will alter aging. While it is clear that oxi-
dative damage increases with age, its role in the aging
process is, however, uncertain. Examining the relationship
between ROS and aging in genetic model organisms, such
as Caenorhabditis elegans, summarizing experiments using
long-lived mutants, mutants with altered mitochondrial
function, mutants with decreased antioxidant defenses,
worms treated with antioxidant compounds, and worms
exposed to different environmental conditions, there is
strong indication that, while there is frequently a negative
correlation between oxidative damage and lifespan, there
are many examples in which they are uncoupled. Neither is
resistance to oxidative stress sufficient for a long life nor
are all long-lived mutants more resistant to oxidative stress.
Similarly, sensitivity to oxidative stress does not neces-
sarily shorten lifespan and is in fact compatible with long
life. Furthermore, recent finding obtained from Clk mutants
of Caenorhabditis elegans, characterized by slow physio-
logic rates, delayed development, and increased life span,
challenge the hypothesis that decreased metabolism leads
to increased longevity through a decreased production of
reactive oxygen species. In fact, although mitochondrial
function is decreased in the Clk mutants, ATP levels are
normal or increased, suggesting decreased energy utiliza-
tion, with no evidence for systematically increased resis-
tance to oxidative stress or decreased oxidative damage.
This phenotype suggests that increased life span may be
achieved by decreasing energy expenditure, without
decreased production of ROS [48]. Overall, the data in
C. elegans indicate that oxidative damage can be dissoci-
ated from aging, at least in experimental situations [49]. If
one consider as gold standard for determining whether
changes in the aging process occur, alteration in life span,
studies from mice with genetic manipulations in their
antioxidant defense system designed to directly address
this prediction have, with few exceptions, shown no change
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in life span. However, when these transgenic/knockout
mice are tested using models that develop various types of
age related pathology, they show alterations in progression
and/or severity of pathology as predicted by the oxidative
stress theory: increased oxidative stress accelerates
pathology and reduced oxidative stress retards pathology.
These contradictory observations might mean that (1)
oxidative stress plays a very limited, if any, role in aging
but a major role in health span and/or (2) the role that
oxidative stress plays in aging depends on environment. In
environments with minimal stress, as expected under
optimal husbandry, oxidative damage plays little role in
aging. However, under chronic stress, including patholog-
ical phenotypes that diminish optimal health, oxidative
stress/damage plays a major role in aging. Under these
conditions, enhanced antioxidant defenses exert an ‘‘anti-
aging’’ action, leading to changes in life span, age-related
pathology, and physiological function as predicted by the
oxidative stress theory of aging [13].
The Mitochondrial Theory of Aging
Mitochondria are membrane-enclosed organelles found in
eukaryotic cells where account for the generation of ATP
as a source of chemical energy. The synthesis of ATP
occurs through the respiratory or electron transport chain
(ETC.) which is located at the inner mitochondrial mem-
brane and consists of five protein complexes (Complexes
I–V). Mitochondrial respiration accounts for approximately
90% of cellular oxygen consumption. Most of the oxygen
that is consumed is reduced to water through four con-
secutive one-electron reductions. During this process, a
small proportion of the oxygen molecules (1–2%) are
converted to superoxide anion radicals. The main sites of
superoxide production in the respiratory chain have been
recognized to be complexes I and IV [50] and recent
studies have identified at least nine submitochondrial
reactive oxygen species (ROS) generating sites [51]. The
superoxide that is formed then dismutates either sponta-
neously or enzymatically through the action of superoxide
dismutase to form hydrogen peroxide [52]. Hydrogen
peroxide then can diffuse throughout the cell and decom-
pose to form noxious hydroxyl radicals, which can injure
the cell through interactions with macromolecules. Because
of these processes, mitochondria are a major source for
physiological or endogenous production of ROS [2].
Although much attention has been focused on the harmful
effects of ROS, it now has become apparent that mitoc-
hondrially generated ROS are also involved in the regula-
tion of intracellular signal transduction pathways leading to
cellular activities such as proliferation [2]. In addition to
supplying ATP, mitochondria are required for numerous
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other cellular functions including biosynthesis of heme,
cholesterol and phospholipids [51], as well as initiation of
the apoptotic process [2]. Mitochondria are organelles
which, according to the endosymbiosis theory, evolved
from purpurbacteria approximately 1.5 billion years ago.
One of the features of mitochondria is that they have their
own genome [52–54]. Mitochondria replicate and tran-
scribe their DNA semiautonomously. Like nuclear DNA,
mitochondrial DNA (mtDNA) is constantly exposed to
DNA damaging agents. Regarding the repair of mtDNA,
the prevailing concept for many years was that mtDNA
molecules suffering an excess of damage would simply be
degraded to be replaced by newly generated successors
copied from undamaged genomes. However, evidence now
clearly shows that mitochondria contain the machinery to
repair the damage to their genomes caused by certain
endogenous or exogenous damaging agents. Harman [6, 7]
proposed that free radicals are involved in the aging pro-
cess and subsequently suggested that mitochondria-derived
ROS may contribute to cellular aging [7]. Experimental
support for mitochondrial involvement in cellular senes-
cence was initially provided by treatment of IMR-90
fibroblasts with N-tertbutyl hydroxylamine, an antioxidant
that is recycled by the mitochondrial electron-transport
chain. N-tert-butyl hydroxylamine extends fibroblast rep-
licative capacity and delays age-related changes in mito-
chondrial function, including reducing ROS production,
preservation of mitochondrial membrane potential, and
increasing the cellular GSH/GSSG ratio [51]. More
recently, it was demonstrated that mitochondria derived
ROS play an important and direct role in the shortening of
telomeres and the onset of senescence [51]. It has been
proposed that mitochondrial dysfunction induces mito-
chondrial biogenesis, thus increasing the number of sites in
the cell for the production of ROS, which accelerates
telomere shortening [2, 50].
It has been proposed that accumulation of mtDNA
during life is a major cause of age-related disease and this
is because of its high mutagenic propensity. The lack of
introns and protective histones, limited nucleotide excision
and recombination DNA repair mechanisms, location in
proximity of the inner mitochondrial membrane which
expose it to an enriched free radical milieu, are all factors
contributing to a 10-fold higher mutation rate occurring in
the mtDNA than in the nDNA [16]. Moreover, a large body
of evidence indicates that mtDNA mutations increase as a
function of age reaching the highest levels in brain and
muscle. More than twenty different types of deletions have
been documented to accumulate in aging human tissues.
The first report on an age-related increase in a mtDNA
deletion, was called the ‘‘common deletion’’ and was found
in elderly brain and in Parkinson’s disease [16]. This
deletion has been described to occur between 13-bp
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Neurochem Res (2010) 35:1880–1915
sequence repeats beginning at nucleotides 8470 and 13447,
removing almost a 5-kb region of mtDNA between ATPase
8 and the ND5 genes. The deletion is thought to occur
during replication of the mtDNA, the absent sequence
encoding for six essential polypeptides of the respiratory
chain and 5 tRNAs. It has been associated with several
clinical diseases, such as chronic progressive external
ophthalmoplegia and Kearns Sayre syndrome. Several age-
related disorders have been shown to be linked to higher
levels of mtDNA mutations than age-matched controls. In
the CNS a 17 times higher levels of the common deletion in
the striatum of patients with Parkinson’s disease have been
demonstrated, compared to age-matched controls. Evi-
dence also exists indicating higher levels of this deletion in
patients with Alzheimer’s disease which parallel increased
levels in the oxidized nucleotide 8-OH-dG [50]. Over 100
mutations of mitochondrial DNA (mtDNA) have been
associated with human disease [55]. The phenotypic
manifestation of mtDNA mutations is extremely broad,
from oligosymptomatic patients with isolated deafness,
diabetes, ophthalmoplegia, etc., to complex encephalo-
myopathic disorders that may include dementia, seizures,
ataxia, stroke-like episodes, etc. The genotype variants are
also wide, with rearrangements (deletions, duplications)
and point mutations affecting protein coding genes, tRNAs
and rRNAs. There are some broad genotype/phenotype
correlations but also substantial overlap. The pathogenetic
mechanisms involved in the expression of mtDNA muta-
tions are still not yet fully understood. More recently,
mutations of nuclear genes encoding subunits of the
respiratory chain, particularly those of complex I, have
been identified. These predominantly, but not exclusively,
involve infant onset disease with early death. Recently it
has become clear that the function of the respiratory chain
may be impaired by mutations affecting other mitochon-
drial proteins or as a secondary phenomenon to other
intracellular biochemical derangements. Examples include
Friedreich ataxia where a mutation of a nuclear encoded
protein (frataxin), probably involved in iron homeostasis in
mitochondria, results in severe deficiency of the respiratory
chain in a pattern indicative of free radical mediated
damage [16, 31, 56, 57].
Mutations of nuclear encoded proteins involved in
cytochrome oxidase assembly and maintenance have been
characterised and, as predicted, are associated with severe
deficiency of cytochrome oxidase and, most frequently,
Leigh syndrome. Defects of intracellular metabolism, with
particularly excess-free radical generation including nitric
oxide or peroxynitrite, may cause secondary damage to the
respiratory chain. This is probably of relevance in Hun-
tington disease, motor neuron disease (amyotrophic lateral
sclerosis) and Wilson disease. These disorders seem to
have defective oxidative phosphorylation as a common
Neurochem Res (2010) 35:1880–1915
pathway in their pathogenesis and it may be that treatments
designed to improve respiratory chain function may ame-
liorate the progression of these disorders [55]. These
finding establishes the relationship between age-associated
accumulation of mtDNA mutations and bioenergy dys-
function as a key feature of the aging process, at least in
tissues predominantly composed by postmitotic cells, such
as CNS and skeletal muscle. Relevant to mitochondrial
bioenergetics, in fact, is the finding of a significant
decrease in state 3/state 4 ratio, which has been observed to
occur in brain during aging [57]. Since this ratio relates to
the coupling efficiency between electron flux through the
electron transport chain and ATP production, an increase in
state 4 would results in a more reductive state of mito-
chondrial complexes and, consequently, to an increase in
free radical species production. A decrease in state 3/state 4
respiration during aging has been found associated with a
significant decrease in cardiolipin content in brain mito-
chondria [58]. This loss could play a critically important
role in the age-related decrements in mitochondrial func-
tion, and appears to be associated with both quantitative
and qualitative region-specific protein changes which are
parallel to structural changes, such as decrease of the inner
membrane surface, smaller as well as sparser cristae,
decreased fluidity and increased fragility. Modifications in
cardiolipin composition it is recognized to accompany
functional changes in brain mitochondria which include all
proteins of the inner mitochondrial membrane that gener-
ally require interaction with cardiolipin for optimal cata-
lytic activity. Acetylcarnitine fed to old rats increased
cardiolipin levels to that of young rats and also restored
protein synthesis in the inner mitochondrial membrane, as
well as cellular oxidant/antioxidant balance, suggesting
that administration of this compounds may improve cel-
lular bioenergetics in aged rats [59]. Interestingly, caloric
restriction, a dietary regimen that extends life-span in
rodents, maintains the levels of 18:2 acyl side chains and
inhibits the cardiolipin composition changes [60] In addi-
tion, caloric restriction showed to retards the aging asso-
ciated changes in oxidative damage, mitochondrial oxidant
generation and antioxidant defenses observed during aging
[2, 61].
Recent evidence suggests that calorie restriction and
specifically reduced glucose metabolism induces mito-
chondrial metabolism to extend life span in various model
organisms, including Saccharomyces cerevisiae, Dro-
sophila melanogaster, Caenorhabditis elegans and possi-
bly mice. In conflict with Harman’s free radical theory
of aging, these effects may be due to increased formation
of reactive oxygen species within the mitochondria causing
an adaptive response that culminates in subsequently
increased stress resistance assumed to ultimately cause a
long-term reduction of oxidative stress. This type of
1887
retrograde response has been named mitochondrial hor-
mesis or mitohormesis, and may in addition be applicable
to the health-promoting effects of physical exercise in
humans and, hypothetically, impaired insulin/IGF-1-
signaling in model organisms. Consistently, abrogation of
this mitochondrial ROS signal by antioxidants impairs the
lifespan-extending and health-promoting capabilities of
glucose restriction and physical exercise, respectively. In
summary, ROS are essential signaling molecules (Fig. 1)
which are required to promote health and longevity. Hence,
the concept of mitohormesis provides a common mecha-
nistic denominator for the physiological effects of physical
exercise, reduced calorie uptake, glucose restriction, and
possibly beyond [62].
Energy Thresholds in Brain Mitochondria: Implication
for Age-Related Disorders
Mitochondria contains essential mechanisms of energy
production, signalization, biosyntheses, and apoptosis and
the coordinated orchestration and control of these specific
events is a critical determinant in cell physiology [50].
However significant morphological and functional differ-
ences exist in mitochondria isolated from different tissues
associated with different energy demands as well as pref-
erences in the type of energy substrates, especially in
organs that present with highly specialized functions.
Consistent with this notion, in human heart, mouse and rat
mitochondria, have been identified approximately 600–700
proteins with a strong diversity in their expression levels
among tissues; this suggests quite a large regulatory and
composition diversity of the mitochondrial proteome,
which could explain the observed structural and functional
heterogeneity along with the consequent diversity in the
control of mitochondrial respiration. These differences in
the control of oxidative phosphorylation might reflect
physiological variations in the OXPHOS machinery, which
could allow the efficiency and the rate of ATP production
to adapt to the tissue-specific energy demand. This, oxi-
dative phosphorylation capacity presents an high degree of
variability depending on (1) the mitochondrial content, (2)
the amount of respiratory chain complexes, and (3) their
intrinsic activity: these factors combine together to result in
a greater level of functional diversity [62]. Human cells
contain from a few hundred to more than a thousand
mitochondria; each mitochondrion in turn has 2–10 copies
of mtDNA, thus, several thousands copies of the mito-
chondrial genome can be present within a single cell.
Importantly, unique to mtDNA is that it is inherited
exclusively through the mother, and may exist in many
different copies in the oocyte cytoplasm. This implies that
no mtDNA recombination occurs at fertilization and only a
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1888
Fig. 1 Major sites for mitochondrial ROS production and the
principle of preconditioning. The ubiquinone (Q) cycle is initiated
when one electron from ubiquinol (QH2) is donated to the Rieske-
iron sulfur (R-FeS) protein and the second electron is donated to
cytochrome b. The intermediate moiety is the free radical ubisem-
iquinone (Qo), which can donate electrons to molecular oxygen to
generate superoxide. Mitochondrial electron transport chain generates
sequential accumulation of mutations from the maternal
lineage account for mtDNA variations. Moreover, mtDNA
is particularly prone to mutation, being estimated as 10
times greater then nuclear DNA [52], owing to the absence
of protective proteins (such as histones) and of a high-
efficiency repair system. Thus, mutant and wild-type
(normal) mtDNA can coexist within a cell in any propor-
tion and this situation is termed heteroplasmy. It is
becoming increasingly clear that the mitochondrial genome
may play an essential role in the pathogenesis of neuro-
degenerative diseases, and evidence for mitochondria being
a site of damage in neurodegenerative disorders is based in
part on observed decreases in the respiratory chain com-
plex activities in Parkinson’s, Alzheimer’s, and Hunting-
ton’s disease. Such defects in respiratory complex
activities, possibly associated with oxidant/antioxidant
imbalance, are thought to underlie defects in energy
metabolism and induce cellular degeneration [53]. The first
hint that mitochondria play a role in human disease did not
emerge until 1958, when a Swedish patient was identified
who had symptoms of severe perspiration, polydipsia,
polyphagia, weight loss and weakness [63]. Biochemical
investigation showed a basal metabolic rate 200% above
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Neurochem Res (2010) 35:1880–1915
superoxide at complex I, II, and III. Complex I and II generate
superoxide within the mitochondrial matrix. Complex III can generate
superoxide in both the intermembrane space and the matrix. Release
of superoxide from complex III into the cytosol is followed by
conversion to H2O2 and subsequent activation of oxidant-dependent
(redox) signaling pathways which results in precondition
normal, very low weight (37 kg) and basal temperature
reaching 38C. Moreover, she had a partially uncoupled
respiration which accounted for her generation of excessive
heat and high calorie consumption. Although the primary
etiologic event in Luft’s disease remains to be identified,
the disease is associated with release of mitochondrial
calcium stores, abnormal calcium cycling and sustained
stimulation of loosely coupled respiration. The discovery
of Luft’s disease cleared the path for fertile investigations
and since 1960s, over 120 human mitochondrial diseases
have been discovered, many of which involve selected
populations in the central nervous system consisting of
postmitotic, highly energy-dependent cells. Many of these
diseases have been associated with specific inherited
mitochondrial DNA mutations and respiratory chain defi-
ciencies. Point mutations may involve either the RNA or
protein-encoding genes, and rearrangements may take the
form of deletions or duplications. In the presence of het-
eroplasmy there is a critical ratio of mutant to wild-type
mitochondrial genomes that is necessary before the disease
becomes both biochemically and clinically apparent. As
might be expected mtDNA disorders are phenotypically
diverse given the ubiquitous presence of mitochondria and
Neurochem Res (2010) 35:1880–1915
the variation in the levels of heteroplasmy in the body. Yet
many have predominant neurologic and muscular symp-
toms, including dementia, seizures, ataxic syndromes,
peripheral neuropathies, and progressive myopathy. Post-
mitotic tissues typically show increased levels of mutant
mitochondria due to the inability of these tissues to select
against cells containing mutant mtDNA genomes. CNS
imaging of patients with mtDNA disorders often reveals
moderate degrees of cerebral or cerebellar atrophy that are
consistent with neurodegeneration, often comparable with
the pathologies associated with the brain in senescence or
in dementia [64].
Several mechanisms have been proposed to explain the
variability of the phenotypic expression of a mtDNA
mutation, such as sporadic mutation or mitotic segrega-
tion. However all these hypotheses incorporate a unique
feature of mitochondrial genetics and pathologies; that of
the heteroplasmic concept of mtDNA mutations. Levels
of mutation can vary considerably between mitochondria,
cells, and even tissues within the same individual. Con-
sequently, the expression of a mutation in the mtDNA can
be thought of as a function of the degree of the hetero-
plasmy. In general, whether or not a metabolic defect
expresses itself as a recognizable clinical disease will
depend upon the extent to which it affects the metabolic
pathway in question and this can lead to a threshold
expression of the disease state [65]. One of the most
important features recognized in mitochondrial diseases is
the existence of a threshold in the degree of a mito-
chondrial deficit for the expression of the disease, and
these were shown by Wallace [66, 67] to be related to the
balance between normal and mutant mtDNA. Consistent
to this, only 10% of wild type DNA is enough to maintain
a normal respiratory rate, whereas it must be achieved
80–90% deletion of mtDNA before complex IV activity is
compromised [50, 68]. This indicates a threshold in the
heteroplasmy of the mutation around 90% exists before a
pathological consequence become manifest. Below this
threshold the flux of respiration and of ATP synthesis are
at a level which does not compromise normal metabolism.
As consequence, there are at least four levels at which
threshold effects occur in mitochondrial metabolism, with
respect to their possible involvement in the pathogenesis
of neurodegeneration. The first is the expression of the
heteroplasmy of mtDNA at the level of a given enzymatic
step, whereby mitochondria from patients might exhibit a
particular ratio of defective DNA compared to normal
DNA. The second is the threshold effect observed in the
mitochondrial metabolism as a result of a decrease in a
given mitochondrial activity. The third may occur in the
expression of defective mitochondria with respect to the
whole cellular metabolism. The fourth, is the fact that
the control coefficient of a given step may vary depending
1889
on different types of mitochondria, which leads to the
observation that the threshold value for a given complex
of the electron transport chain can vary according to the
threshold in the energy demand of different tissues.
According to the metabolic control theory [69–71] the
importance of the control of a given isolated step on
the global flux depends on several parameters, including
the absolute content of enzyme, its intrinsic kinetic
parameters and associated regulations, the architecture of
the metabolic network, the concentration of intermediate
substrates, and the respective steady state [70]. Further-
more, at each level, single threshold effect will reinforce
the others, as interpreted by the Double threshold
hypothesis [50, 72], whose predictions are now beginning
to be documented. Consistent with this, metabolic control
analysis (MCA) of complex I activities in mitochondria
has revealed some interesting findings concerning mito-
chondrial heterogeneity in the brain. When the relative
contribution of individual mitochondrial respiratory chain
complexes to the control of NAD-linked substrate (glu-
tamate and malate) oxidative phosphorylation in mito-
chondria from various rat brain regions was investigated,
it was found that complex I possessed a higher control
over oxidative phosphorylation in synaptic mitochondria
than in non-synaptic mitochondria. Using rotenone or
myxothiazol and KCN to titrate out complex I, III and IV
activities, respectively, while measuring oxidative phos-
phorylation parameters, the highest flux control coeffi-
cients were found with complex I in mitochondria of
synaptic origin. This demonstrates that respiratory chain
complexes are involved in the control of mitochondrial
respiration. However, the distribution of the control
indices of these complexes may be different, depending
on the tissue from which the mitochondria were isolated.
Brain mitochondria show lowest oxidative phosphoryla-
tion efficiency with complex I substrates, measured as
P/O ratios, compared to mitochondria from heart and liver
[72]. Data from studies of rat brain mitochondria of non
synaptic origin have shown that thresholds exist whereby
complex activities need to be reduced by at least 60%
before major changes in ATP synthesis and oxygen con-
sumption occur. Interestingly, in synaptic mitochondria,
titration of various complexes with specific inhibitors
generated threshold curves showing that complex I, III
and IV activities had to be decreased 25, 80 and 70%,
respectively before major changes in rates of oxygen
consumption and ATP synthesis were observed [70–72].
Thus mitochondria of synaptic origin complex I activity
has a major control of oxidative phoshorylation, such that
when a threshold of 25% inhibition is exceeded, energy
metabolism is compromised, and reduction in ATP syn-
thesis ensues. Moreover, glutathione depletion abolishes
the threshold for complex I providing experimental
123
1890
evidence that antioxidant status, which is critically
involved in neurodegenerative disorders and in particular
in PD, maintain energy thresholds in mitochondria
[71, 72].
According to the metabolic control theory, at molecular
level, a critical step in the expression of a threshold for a
clinical disease is the impact that a localized defect in a
given point on the global flux of a metabolic network
[50, 73–75].
Mitochondrial dysfunction is characteristic of several
neurodegenerative disorders [50] and also of the aging
process [55]. Parkinson’s disease is characterized by a
selective decrease in dopamine in the striatum caused by a
degeneration of dopaminergic neurons in the zona com-
pacta of the substantia nigra [54, 59, 66]. Complex I
(NADH:ubiquinone oxidoreductase, EC 1.6.5.3) activity is
reduced by 35–40% in substantia nigra homogenates in
postmortem studies of Parkinson’s disease patients [64].
However, recent evidence has suggested that a complex I
deficiency may be widespread in the parkinsonian brain, as
the occurrence of a mutation in mitochondrial DNA in
idiopathic Parkinson’s disease [71], and an associated
reduction in complex I activity in mitochondria from the
frontal cortex [39, 71] have been reported. This evidence
supports the hypothesis that complex I deficiency plays a
central role in the initial etiology of the disease [16, 31].
The cause of the complex I deficiency is unknown, how-
ever, followup studies on Parkinson’s disease patients
injected with fetal tissue enriched in dopaminergic cells
shows that an environment exists in the parkinsonian brain
that induces the classical disease pathology in the trans-
planted cells [76]. It is not known if complex I activity in
these cells is reduced following transplantation or if a
complex I defect in surrounding cells induces the neuro-
degenerative condition. However, synaptosomes show that
complex I deficiencies can result in large amounts of glu-
tamate being released that may be potentially excitotoxic to
surrounding cells [77]. Dopamine neurons are known to
possess glutamate receptors, therefore, existing complex I
deficiencies that lead to increased glutamate release may
increase excitotoxic events in the parkinsonian brain [77].
The significance of energy threshold theory applied
to mitochondrial dysfunction is, is relevant to described
decrease of 40% in complex I activity in Parkinson’s dis-
ease patients [72], which would correspond to a 35–40%
inhibition of ATP synthesis and respiration in the synaptic
mitochondria model. Titration of complex I activity in
synaptosomes, followed by depolarization with KCl or
4-aminopyridine, reduces ATP concentrations and leads to
significant amounts of Ca2?-independent glutamate
release [77]. These results indicates that a 40% decrease in
complex I activity in the substantia nigra in PD patients can
induce excess release of excitotoxic glutamate following
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Neurochem Res (2010) 35:1880–1915
action potentials. This is corroborated by the finding that
reduction of complex I activities in synaptosomes from
aged rats resulted in a faster release of glutamate, com-
pared to synaptosomes from adult rats, suggesting that
nerve terminals from aged animals, as opposed to younger
rats, are more vulnerable to mitochondrial dysfunction and
damage [78]. Further refinements of this notion point to the
nature of complex I assembly in brain mitochondria.
Mammalian complex I is, in fact, composed of 45 different
subunits that assemble together at the inner mitochondrial
membrane [50]. The assembly of this complex is compli-
cated partly because of its large size and partly because its
regulation by two genomes. Seven of the complex I sub-
units are encoded by the mitochondrial (mt) DNA and the
remaining by the nuclear genes. The nuclear DNA-encoded
subunits are synthesized in the cytosol and are imported
into the organelle. The mtDNA-encoded subunits must
assemble with the nuclear encoded subunits to generate the
functional holoenzyme [50]. The multimeric holoenzyme is
L-shaped with a hydrophobic membrane arm embedded in
the mitochondrial inner membrane and perpendicular to it
is a hydrophilic peripheral arm which protrudes into the
mitochondrial matrix [72]. The 14 most conserved subunits
(which form the fully functional complex I in bacteria)
form the ‘core’ structure. The ‘core’ structure is also
considered to be the minimal structure required for the
functionality of the enzyme [72]. The function of most of
the additional 31 supernumerary subunits is not yet clear
but some are hypothesized to stabilize or to protect the
complex from ROS damage. Several of these subunits may
have an additional function. For example, the subunits
NDUFA13 [also known as GRIM-19] and NDUFS1 have
been implicated to have a role in apoptosis [72].
Assembly of complex I subunits is an intricately elab-
orate process, however emerging evidence indicates that
complex I assembly is not sequential but is modular which
means that the subunits are not incorporated one by one
into the complex but instead several discrete assembly
intermediates are assembled independently and are joined
in several steps to form a peripheral arm and a membrane
arm, with peripheral arm assembled independently of the
membrane arm; sub-assemblies of the nuclear DNA-
encoded subunits and the mtDNA-encoded subunits can be
formed independently. In addition to this, complex I
assembly is a highly dynamic process in which existing and
newly synthesized subunits can be exchanged, so that a
complex I transition between assembled and intermediate
forms may constitute a mechanism which promotes the
turnover of subunits, some of which may be oxidatively
damaged [72]. Considering that an increased number of
complex I subunits become oxidatively damaged in the
brains of patients with Parkinson’s disease [79], it is con-
ceivable to hypothesize that a decreased rate of protein
Neurochem Res (2010) 35:1880–1915
import and/or assembly which slows the exchange rate of
pre-existing complex I subunits for new subunit import
may underlie a pathogenic basis of the disease. This may
lead to increased oxidative damage, complex I deficiency
and subsequent overt disease. The complex I assembly
process is even more complicated by the fact that in
addition to a perfect assembly, the nuclear transcription,
translation, processing, export of the subunits and then
their import into the mitochondria, insertion into the
membrane, stabilization and activation of the enzyme,
every step requires a precise coordination. In light of these
events it is reasonable that chaperons, which assist the
correct assembly of other polypeptide containing structures
in vivo, although are not a component of the final func-
tional structure, may contribute to pathogenesis of the
disease. This possibility is corroborated by the evidence
that mutations in the assembly chaperones that lead to the
inability to assemble a properly functioning complex I and
thus lead to complex I deficiency diseases, have been
reported [55, 59]. Contrarily to the numerous complex III
and complex IV assembly chaperones known, so far only
three candidate complex I assembly factors have been
found. Two of these, the NDUFAF1 (the human homo-
logue of fungal protein CIA30) and a paralogue of complex
I subunit B17.2 known as B17.2-like (B17.2L) are well
established and well studied complex I assembly chaper-
ones [80, 81]. An emerging area of investigation is the
supramolecular organization into ‘respirasomes’ of indi-
vidual electron transport chain complexes. The five com-
plexes (complexes I–V) of the oxidative phosphorylation
(OXPHOS) system of mitochondria can be extracted in the
form of active supercomplexes. Single-particle electron
microscopy has provided 2D and 3D data describing the
interaction between complexes I and III, among I, III and
IV and in a dimeric form of complex V, between two ATP
synthase monomers. The stable interactions are called su-
percomplexes which also form higher-ordered oligomers.
Cryo-electron tomography provides new insights on how
these supercomplexes are arranged within intact mito-
chondria. In the past, two models have been proposed for
the organization of the respiratory chain. The random
collision model and the solid state model. According to the
first model, complex I–IV moves freely by lateral diffusion
in the inner mitochondrial membrane and electron transfer
is based on random collisions of the involved components.
Ubiquinone and cytochrome c have a diffusion rate that is
faster than the movements of the membrane embedded
complexes [72]. In spite of this elaboration, the solid model
of the electron transport chain complex organization is
more widely accepted now, which promotes the preferen-
tial associations and specific aggregation of the complexes.
This stems from immunoprecipitation as well as flux con-
trol experiments, which point to the supramolecular
1891
organization of the complexes. Organization into super-
complexes has the advantage of improving stabilization of
individual complexes, but at the same time mutations
leading to a loss of complex III prevents supercomplex
formation, thus leading to a secondary loss of complex I.
Finally this model is sustained by the finding that the
different complexes are present in vivo at defined stoi-
chiometries, and single particle electron microscopy has
provided the 3D maps of the supercomplexes. The su-
percomplexes have been identified and characterized from
a broad range of organisms using BN-PAGE and sucrose
gradient centrifugation. The supercomplex I–III, charac-
terized in Arabidopsis, consists of complex I and dimeric
complex III. The supercomplex III–IV consisting of
dimeric complex III and one to two copies of monomeric
complex IV, has been characterized in yeast. The largest
supercomplex from bovine heart, consists of complex I
plus complex III2 plus complex IV1–4 and is termed the
‘respirasome’. The supramolecular organization of the
oxidative phosphorylation complexes into respirasomes
may be of functional importance as they may enhance
electron transfer rates, reduce diffusion distances of sub-
strates by substrate channeling and increase the stability
of the complexes. Although little is known about super-
complexes in the brain, the different energy thresholds
and flux control coefficients described above may be
related to a range of supercomplex formations in brain
mitochondria [82]. Formation of supercomplexes I–III has
been demonstrated to be facilitated by lipid component in
the inner membrane, in particular cardiolipin, which
appears critical for the stability of supercomplexes [83].
As a consequence, exposure of mitochondria to reactive
oxygen species can oxidatively damage cardiolipin with
an impact on supercomplexes stability. This may be an
underlying cause of aging and age-related disorders like
Parkinson’s disease, where oxidative damage can induce
reduction in mitochondrial energetic capacity due to dis-
sociation of supercomplexes, with pathological implica-
tions [72]. Consistent to this, progress by structural
investigations, paralleled by insights into the functional
roles of respirasomes including substrate channelling and
stabilization of individual complexes have revealed car-
diolipin an important factor involved in the structural
stability of respirasomes which in turn is required to
maintain cells and tissues in a healthy state. Defects in
cardiolipin remodeling cause devastating diseases like
Barth syndrome, a disease associated with a mutation in
human TAZ gene, which results in production of an
altered gene product, Tafazzin, a putative phospholipid
acyltransferase. Patients suffering from Barth syndrome
exhibit defects in cardiolipin, associated with mitochon-
drial morphological abnormalities and respiratory chain
dysfunction [83].
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Redox Regulation of Mitochondrial Dynamics Fusion
and Fission: Role in Cell Survival
Mitochondria are the major source of energy (ATP and
NAD?) production in eukaryotic cells, and produce cor-
respondingly large amounts of superoxide anion radical
through abstraction of an electron from oxygen in the so-
called electron transport chain at the inner mitochondrial
membrane [50]. Mitochondrial function is essential to
neuronal processes such as energy production, Ca2? regu-
lation, maintenance of plasma membrane potential, protein
folding by chaperones, axonal and dendritic transport and
the release and re-uptake of neurotransmitters at synapses.
Mitochondria help neurons to satisfy the high energy
demand which is required for proper neuronal function and,
unlike other cell types, cannot switch to glycolysis when
oxidative phosphorylation becomes limited. Furthermore,
cells in response to changing energy demands continually
adjust the rate of mitochondrial fission and fusion, to
facilitate the distribution of mitochondria [2, 61].
Mitochondria may produce superoxide in relatively
constant amounts, or may elicit spontaneous or environ-
mentally-induced ‘‘superoxide flashes’’ [2]. This is con-
sistent with the general notion that in the mitochondrial
respiratory chain complexes I and III are the main sites of
superoxide production [2]. In this context, any impairment
in the electron transfer process determines that upstream
carriers become more reduced, thus filling Q cycle electron
pool. The cytochrome (cyt)2 bc1 complex (EC 1.10.2.2)
(cyt bc1 complex), localized in the inner membrane of
mitochondria, couples the oxidation of a substrate quinol
(QH2) with the generating proton motive force across the
energy transducing membrane system, where the energy is
ultimately stored in the form of ATP (Fig. 1). Cytochrome
bc1 complexes contain four redox-active metal centers,
arranged in two separate chains. The ‘‘high potential
chain’’ consists of the Rieske iron-sulfur [2Fe2S] cluster
(Fig. 1) and cyt c1. The ‘‘low potential chain,’’ which binds
to the cyt b subunit of ubiquinol oxidizing complexes,
consists of two b-type hemes, cyt bL and bH labeled for
their relatively lower and higher electrochemical potentials
[84]. Three enzymatic binding sites participate in catalysis
on the cyt bc1 complex, the quinol oxidase (Qo) site,
quinol reductase (Qi) site, and a docking site for soluble cyt
c on cyt c1. The Qo site is located toward the positively
charged side of the membrane, where protons are released
during turnover of the enzyme (Fig. 1). The Qi site is
located toward the negatively charged surface of the
membrane, where protons are taken up during catalysis.
The water-soluble cyt c docking site is located on the
c-type cyt representing the terminal electron carrier within
the cyt bc1 complex on the positive side of the membrane.
Cyt bc1 complex catalysis is thought to occur by a
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Neurochem Res (2010) 35:1880–1915
‘‘Q-cycle’’ mechanism (Fig. 1). Conceivably, a key reac-
tion in the Q-cycle is the ‘‘bifurcated’’ electron transfer at
the Qo site, although the exact mechanism is still contro-
versial [84]. In the bifurcated reaction, QH2 is oxidized at
the Qo site, with one electron being transferred through the
high potential chain to reduce cyt c, and the other electron
is transferred through the low potential chain, to reduce a
quinoid species (Q or SQ, depending on the state of the
two-electron gate) at the Qi site. Two turnovers of the Qo
site are required to reduce a Qi site Q to a QH2 (Fig. 13).
Under some conditions, the Q-cycle can be short-circuited
by various ‘‘bypass’’ reactions, some of which yield the
physiologically deleterious superoxide [2]. The bypass
reactions are typically observed in vitro under ‘‘partially
inhibited’’ conditions, e.g. in the presence of antimycin A,
or under high proton motive force, where it is possible that
the [2Fe2S] cluster can oxidize QH2 to a semiquinone
(SQ), but processing of electrons by the low potential chain
is hindered, resulting in the accumulation of a SQ inter-
mediate, which in turn can reduce O2 to superoxide [2].
Physiologically, electron transport chain consumes up to
90% of the oxygen taken up by a cell, of which *1% is
transformed in superoxide under normal physiological
conditions [85]. The rate of mitochondrial electron transfer
from semi-reduced Q to molecular O2 to form superoxide
is proportional to the product of the concentrations of semi-
reduced Q and O2. It is estimated that the under patho-
physiological conditions, superoxide production can occur
at 2–10% of the uninhibited rates. Superoxide dismutases,
in turn, generate the more stable hydrogen peroxide before
it is transformed by catalase in water or, depending on the
presence of metals, into highly reactive hydroxyl radicals.
Thus, SOD2, which directs H2O2 out of the mitochondrion,
accomplishes two functions: the classical scavenging of
superoxide, as well as its signalization as ROS-dependent
signaling pathways. The binding of CO to cytochromes
localized within complex IV (i.e., cytochrome a3) pro-
motes reduction in the respiratory carriers in the cyto-
chrome bc1 region of complex III, associated with
increased leakage of superoxide and H2O2 production
(Fig. 1).
Much of the superoxide produced is efficiently con-
verted to hydrogen peroxide via the activity of superoxide
dismutases including cytoplasmic (SOD1) and mitochon-
drial (SOD2) forms of the enzyme. Being a highly reactive
free radical, superoxide can damage molecules (DNA,
proteins and lipids), and so its conversion to hydrogen
peroxide protects cells. However, in the presence of even
very low concentrations of Fe2? or Cu?, hydrogen perox-
ide can generate hydroxyl radical which is a potent inducer
of membrane lipid peroxidation [33]. In addition, nitric
oxide (which is generated in response to activation of the
enzyme nitric oxide synthase by Ca2?/calmodulin) can
Neurochem Res (2010) 35:1880–1915
interact with mitochondrial superoxide to generate the
highly reactive free radical peroxynitrite [41, 86]. Methods
have been developed to detect and quantify oxidative
damage to proteins, lipids and DNA, with antibodies that
selectively recognize proteins modified by the lipid per-
oxidation product 4-hydroxynonenal [86] and by nitration
[41, 86] being particularly useful. In addition, fluorescent
probes for imaging relative levels of overall mitochondrial
redox status and superoxide have been used to elucidate
roles for mitochondrial ROS in a range of physiological
and pathological processes [87]. The damaging effects of
excessive production of ROS are believed to contribute to a
wide range of diseases including cancers, cardiovascular
disease and inflammatory conditions such as arthritis [88].
Neurons may be particularly vulnerable to mitochondrial
ROS because of their high energy demands, their excit-
ability, and the fact that they are postmitotic and are
therefore in most cases not replaceable [88]. However,
lower subtoxic levels of mitochondrial ROS can activate
signaling pathways that protect cells against injury and
disease [2, 86].
Recent evidence suggests that changes in mitochondrial
activity can trigger morphological adaptations of the
mitochondrial network, and clinical studies further indicate
that molecular defects affecting its dynamics lead to
pathology. This suggests the existence of a link between
energy status and organellar network configuration. With
respect to this, a balance between fusion and fission events,
mediated by specific proteins that could participate in the
modulation of energy production, occurs controlling
mitochondrial configuration [89]. In physiological settings,
mitochondria are known to continuously undergo fission
and fusion (known as mitochondrial dynamics) to generate
smaller organelles or rather elongated structures, respec-
tively. Through this process, termed mitochondrial bio-
genesis, formation of new mitochondria occur, a process
associated to repair of defective mitochondrial DNA,
through redistribution of mitochondria to sites with high
energy demand. In neurons, the fission/fusion machinery
proteins, regulated by a machinery involving large dyn-
amin-related GTPases, maintain mitochondrial integrity
and insure their presence at critical sites that demand high
concentrations of ATP, especially the synapse [90]. These
proteins includes Drp1 and Fis1, acting as fission proteins,
and Mitofusin (Mfn) and Opa1, operating as fusion pro-
teins. Given the critical role of mitochondria in neurons,
impaired mitochondrial dynamics is increasingly impli-
cated in neurodegenerative diseases including Parkinson,
Alzheimer and Huntington diseases [91].
Dysfunction in mitochondrial dynamics can result from
either genetic mutations in fission- or fusion related genes
or from posttranslational changes to the fission or fusion
proteins. Hereditary mutations in the mitochondrial fusion
1893
GTPases optic atrophy-1 (OPA1) and mitofusin-2 (Mfn2)
occur in Charcot-Marie-Tooth Disease (CMT), a peripheral
neuropathy characterized by muscle weakness and axonal
degradation of sensory and motor neurons, and hereditary
motor and sensory neuropathy type VI, clinically similar to
CMT with the addition of optic atrophy and visual
impairment, as well as in Autosomal Dominant Optic
Atrophy, a disease characterized by progressive loss of
vision and degeneration of the optic nerve and retinal
ganglion cells. In addition, there is evidence that increased
mitochondrial fission occur in Parkinson’s disease (PD)
models through two proteins, PTEN-induced kinase 1
(PINK1) and Parkin, which are mutant in familial forms of
PD. Furthermore, mutant huntingtin, the disease-causing
protein in Huntington’s disease (HD), alters mitochondrial
morphology and dynamics [91]. Rotenone, a pesticide and
inducer of PD symptoms, and amyloid-b (Ab) peptide,
which is causally linked to Alzheimer’s disease (AD), are
able to initiate mitochondrial fission [92].
Posttranslational modifications caused by nitrosative/
oxidative stress may sustain the pathogenesis of the more
common sporadic cases in neurodegeneration, which
appear to result from complex interactions of multiple
genes and/or their products with environmental factors.
Recent studies have identified three post-translational
modifications that seem to regulate mitochondrial fission
and fusion proteins: phosphorylation, sumoylation and
ubiquitination [92]. Protein Kinase A (PKA, cAMP-
dependent protein kinase) phosphorylates Drp1, which
results in a significant decrease in GTPase activity and
inhibition mitochondrial fission. Interestingly, mitosis-
promoting factor (MPF, Cdk1/cyclin B) phosphorylates
Drp1 at Ser585 in rats during mitosis. Unlike phosphory-
lation by PKA, Cdk1/cyclin B phosphorylation seems to
stimulate mitochondrial fission during mitosis. Through
this versatile mechanism, phosphorylation of Drp1 at
different amino acids causes opposite effects [93, 94].
Sumoylation is another means of Drp1 regulation. SUMO1
(small ubiquitin-related modifier-1) interacts with Drp1
and associates with mitochondria at fission sites before and
after fission [94]. Overexpression of SUMO1 leads to
mitochondrial fragmentation and apoptosis, probably
because SUMO1 protects Drp1 from degradation, stabi-
lizing Drp1 and enhancing its binding to mitochondria.
Ubiquitination has also been documentated to be related
with mitochondrial dynamics [94]. There is evidence that
MARCH-V or MARCH5, a mitochondrial outer membrane
protein, ubiquitinates Drp1, thus promoting fission. Finally,
the finding that Bax and Bak, two pro-apoptotic regulators,
interact with mitochondrial fission and fusion GTPases,
lends support to the functional link between mitochondrial
dynamics and apoptosis. Bax associates with Drp1 and
Mfn2 on mitochondria during apoptosis. Furthermore,
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1894
neurons under nitrosative stress exhibit Bax foci on mito-
chondria undergoing mitochondrial fission. Inhibition of
Drp1 function delays mitochondrial fission, which results
to be associated with Bax foci formation and neuronal cell
death [94].
An additional potentially deleterious effect of increased
ROS levels is chronic mitochondrial fission. Nitrosative
stress causes profound mitochondrial fission in neurons
prior to the onset of neuronal loss in an animal model of
stroke and treatment with antioxidants has been shown to
reduce mitochondrial fission [94]. Amyloid-beta peptide,
thought to be a key mediator of AD pathogenesis, engenders
S-nitrosylation and thus hyperactivation of the mitochon-
drial fission protein Drp1. This activation leads to excessive
mitochondrial fragmentation, bioenergetic compromise,
and synaptic damage in models of AD [90]. Expression of
Mfn or dominant-negative Drp1 partially prevented mito-
chondrial fission and neuronal cell death by nitric oxide,
which is known to be largely caspase independent. Another
study found that oxidative stress promoted mitochondrial
fission in cerebellar granule neurons and that Mfn2
expression was protective [94]. Whether oxidative stress
directly regulates the mitochondrial fission and fusion
GTPases is currently unclear. However, given that mito-
chondrial integrity is essential to cellular homeostasis, when
the energetic demand is low, excess mitochondria are
unnecessary and can generate excessive reactive oxygen
species [95]. In addition, if uncoupled, they can consume
ATP, thus their elimination by autophagy is an efficient
cytoprotective response. Furthermore, uncoupled or unsta-
ble mitochondria may release ROS, and various apoptosis-
promoting factors, including cytochrome c, AIF, SMAC/
DIABLO, thus promoting damage to neighboring mito-
chondria [96].
Another important event in mitochondrial biology is
mitochondrial biogenesis, which represents a phenomenon
relying on a spatiotemporally coordinated synthesis and
import of approximately 1000 nuclear-encoded proteins,
some of which are assembled with mitochondrial-encoded
proteins within newly synthesized inner and outer mito-
chondrial phospholipid membranes. The replication of
mitochondrial DNA, as well as the mitochondrial fusion and
fission mechanisms, also must be synchronized with these
processes [97]. Oral acetylcarnitine supplementation
increases mitochondrial content, as well as nuclear tran-
scripts of factors involved in mitochondrial biogenesis
(PGC-1a, NRF-1, TFAM). This event results associated to
increased levels of mitochondrial transcripts for COX I,
ATP6 and ND6, as well as 16S rRNA. In the same condi-
tions, stimulation of vitagenes, in particular HO-1, lead to
production of carbon monoxide which, according to the
model proposed by Piantadosi [98], is able to bind to the-
reduced heme a3 in the cytochrome c oxidase complex,
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Neurochem Res (2010) 35:1880–1915
inducing a parallel increase in the formation of superoxide
from complex III. Superoxide, in turn, induces MnSOD
overexpression with formation of hydrogen peroxide and
consequent signalization of the so called preconditioning
(Fig. 1). As signaling molecule H2O2 activates protein
kinase B (Akt), which deactivates glycogen synthase
kinase-3b, allowing the nuclear translocation of Nrf2.
Nuclear Nrf2 binds to antioxidant response elements in the
HO-1, MnSOD, and NFR-1 gene promoters, thus amplify-
ing the initial signal and driving the transcription of TFAM
and other genes that have promoter binding sites for NRF-1
[2, 3, 41, 86, 99]. These genes are involved in the control of
mitochondrial transcription and protein synthesis, mito-
chondrial protein import, and oxidative phosphorylation
[99]. Consistent with this notion, acetylcarnitine was found
to reverses the age-related decrease in the activity of com-
plex III and oxidative phosphorylation through complexes
III and IV, and increases the amount of cytochrome b and
aa3 hemes in mitochondria isolated from old rats [97]. Of
interest, both cytochrome b and aa3 proteins are encoded by
the mitochondrial genome, suggesting that acetylcarni-
tine enhances either mtDNA transcription, the stability of
mitochondrial mRNA, or mitochondrial protein synthesis
[97].
Hormesis
Hormesis is a dose response phenomenon characterized by
a low dose stimulation and a high dose inhibition (Fig. 2).
It may be graphically represented by either an inverted
U-shaped dose response or by a J- or U-shaped dose
response. The term hormesis was first presented in the
published literature in 1943 by Southam and Ehrlich who
reported that low doses of extracts from the Red Cider tree
enhanced the proliferation of fungi with the overall shape
of the dose response being biphasic. However, credit for
experimentally demonstrating the occurrence of hormesis
goes to Hugo Schulz (1888) [100] who reported biphasic
dose responses in yeast following exposure to a large
number of toxic agents. The work of Schulz inspired a
large number of investigators in diverse fields to assess
whether such low dose effects may be a general feature of
biological systems. In fact, similar types of dose response
observations were subsequently reported by numerous
researchers assessing chemicals [101] and radiation
[102–113] with investigators adopting different names such
as the Arndt-Schulz Law, Huppe’s Rule, and other terms to
describe these similar dose response phenomena. Despite
the rather substantial historical literature concerning hor-
metic dose responses, this concept had a difficult time
being incorporated into routine safety assessment and
pharmacological investigations, principally because it
Neurochem Res (2010) 35:1880–1915
Fig. 2 Dose-response curve. Quantitative features of hormesis
(1) required more rigorous evaluation in the low dose zone,
(2) failure of investigators to understand its clinical sig-
nificance (3) failure to appreciate the quantitative features
of the hormetic dose response (4) failure to understand the
limitations of its implications for commercial applications
in agriculture as well as medicine, (5) because of the pre-
dominant interest in responses at relatively high doses
during most of the twentieth century as well as (6) the
continuing, yet inappropriate, tendency to associate the
concept of hormesis with the medical practice of home-
opathy [114–116]. However, from the late 1970’s
[117, 118] there has been a growing interest in hormetic-
like biphasic dose responses across the broad spectrum of
biomedical sciences. This resurgence of interest resulted
from a variety of factors, including the capacity to measure
progressively lower doses of drugs and chemicals, the
adoption of cell culture methods which has permitted more
efficient testing of numerous doses and the need to
re-examine the validity of linearity at low dose modelling
of cancer risks due to their enormous cost implications for
regulations, as well the astute observations of independent
investigators and their capacity to generalize their findings
across biological systems [117, 119].
What has emerged from these research initiatives from
highly diverse biomedical areas is the recognition that
hormetic dose responses were common and highly gener-
alizable, being independent of biological model, endpoints
measured and chemical class and/or physical agent studied
[110–113, 120–123]. This was an unexpected finding as
hormetic responses were often considered by many in the
so-called mainstream branches of toxicology and pharma-
cology to be paradoxical, not commonly expected and
being of questionable reliability with a lack of capacity for
replication. The casual dismissal of the hormesis concept
during the mid decades of the last century is reflected in the
general absence of the hormesis concept from the leading
toxicological and biomedical textbooks. This situation has
radically changed such that hormesis is now incorporated
1895
Fig. 3 Citations of Hormesis/Hormetic in Web of Science Database
into all leading textbooks of toxicology [124] encyclope-
dias [115, 125] and other leading monographs. In fact,
while the terms hormetic and hormesis were cited only
about 160 times during the entire decade of the 1980’s
within the Web of Science database, in 2009 alone these
terms were cited nearly 2,500 times (Fig. 3).
Of further significance were observations that these
broad ranging dose response relationships also shared the
same general quantitative features. More specifically, the
low dose stimulation which becomes manifested immedi-
ately below the pharmacological and toxicological thresh-
olds is modest in magnitude being at most only about
30–60% greater than the control group response. The width
of the hormetic stimulation is usually about 10–20 fold
starting immediately from the zero equivalent dose (i.e.
estimated threshold) (Fig. 2). The hormetic dose response
may result from either a direct stimulation or via an
overcompensation stimulatory response following disrup-
tion in homeostasis [110, 126]. Regardless of the mode of
action by which the stimulation occurs the quantitative
features of hormetic dose responses are similar. These
observations are based on copious data derived from the
published literature ranging from plants to humans [121,
127], involving numerous receptor systems [114, 128–
130]. These findings have recently led to nearly 60 bio-
medical scientists recommending that biological stress
responses, including those of pre- and post-conditioning, be
integrated within an hormetic context, along with the
adoption of a terminology that would be based within an
interdisciplinary framework [131].
The hormetic dose response confers a new set of inter-
pretations for the dose response. At high doses within a
toxicological setting, the typical endpoints measured indi-
cate cellular damage. However, as the dose decreases
below the threshold the low dose stimulation more likely
represents a manifestation of an adaptive response that
conforms to a measure of biological performance as may
be seen in the cases of modest increases in cognition,
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growth, longevity, bone density and other biomedical
endpoints of interest.
The consistency of the vast array of hormetic findings
suggests strongly that this dose response may be a mani-
festation of the plasticity of biological systems. That
essentially all biological models respond to imposed stress
with the same quantitative features of the dose response is a
central finding within the biological sciences that has not
been previously recognized. These findings suggest that the
hormetic dose response would have been broadly selected
for and highly conserved. This adaptive response not only
enhances survival by conferring resistance to environ-
mental stress but it represents a way to regulate the allo-
cation of biological resources in a manner that ensures
cellular and organismal stability. The consistency of the
hormetic dose response across the vast array of biological
models, at different levels biological organization,
regardless of the type of stress and health status of the
individual, indicate that hormesis provides a quantitative
index of biological plasticity across multiple levels of
biological organization, making it a central biological
element for enhancing survival.
These quantitative features of the hormetic dose response
have important medical implications. Most significantly,
the hormetic dose response imposes constraints upon the
magnitude of a drug to induce a desired effect. For example,
if a drug increased cognitive performance in an elderly
patient by approximately 25–30%, the hormetic model
suggests that this level of performance could not be further
increased using a new drug combination. This concept has
been supported in a variety of studies on hormesis and drug
interaction. Flood [132–135] has demonstrated that the
hormetic response for memory was bounded by the 30-60%
increase even when several drugs were used in combination
which were designed to maximize memory outcome. This
response magnitude constraint has been reported for
immune stimulation, bacterial growth, increases in hair
growth, plant growth, decrease in anxiety, decreases in
tumor incidence and numerous other endpoints [136].
This limitation in the magnitude of the stimulatory
response is a critical implication of the hormesis dose
response concept. It is an observation which is based on
extensive findings and it is a controlling feature which
defines what pharmaceutical companies can expect to
achieve with drugs that are designed to enhance perfor-
mance. However, the limitation in the magnitude of
response is also potentially important with respect to the
capacity to detect a desirable response. This may not be a
particularly important issue when using highly inbred
animal models or cell cultures where experimental condi-
tions can be highly controlled. However, attempting to
measure a low dose hormetic stimulation within the context
of a clinical trial can be problematic. Given the likelihood
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Neurochem Res (2010) 35:1880–1915
of considerable human variation in response to a drug, it is
possible that the test population may have their respon-
siveness distributed over a range of responses that includes
toxicity, optimal response and a group in which the dose is
ineffective. The data from all subjects in such studies
would normally be averaged together leading to a marked
dilution of an overall positive treatment effect in the
optimal response zone subgroup. This suggests a possible
reason why drugs that were very successfully tested in
preclinical studies with highly inbred strains of animals
could and often have failed during the clinical trial. Of
particular note is that investigators may have to modify
doses based upon the sensitivity or susceptibility of the
subjects. Calabrese and Baldwin [137] have shown that the
hormetic dose response is often expressed in the broad
range of subjects independent of their susceptibility. As
expected, those individuals that are very resistant to the
drug or chemical treatment would have their hormetic
response shifted to the right on the dose response graph
whereas those individuals with greater than normal sus-
ceptibility would have their hormetic response shifted to
the left. The hormetic dose response therefore imposes
considerable challenges to the biomedical community that
is interested in the development of drugs that are concerned
with improvements in human performance.
The hormetic dose response can also have undesirable
effects. This may be most readily seen in the case of drugs
that are designed to suppress growth or kill cells or
organisms at higher doses. For example, there is now
substantial evidence that low doses of many antitumor
drugs can stimulate the proliferation of such cells at lower
concentrations [138]. This also been shown to be the case
with antibiotics, including penicillin [139] and streptomy-
cin [139, 140]. This phenomenon has also been reported
with selected cardiac glycosides that have effects on non-
target tissues such as the prostate where it is able to
enhance the proliferation of smooth muscle cells by about
30% with clinically relevant doses [141, 142]. Such a 30%
increase in prostate smooth muscle was considered likely
to impede urination in males. The failure to consider the
possibility of the hormetic response not only can lead to a
lack of recognition of a desirable drug induced response
but it can also result in failure to prevent an adverse effect
of drug treatment.
Since hormetic dose responses have now been widely
reported in biological systems, there is the desire to
develop a new subfield of hormetic mimetics. These mi-
metics are agents that can activate hormetic pathways with
the intention of producing a desirable clinical effect. For
example, it would be desirable to develop agents that can
induce the desirable adaptive hormetic response without
having risks associated with the exposures as in the case of
certain chemical agents and radiation.
Neurochem Res (2010) 35:1880–1915
Mitostress Versus Mithormesis and Neuroprotection
Recent findings have overturned the long-held belief that
mitochondrial ROS have only a negative impact on cell
function and survival. It is now evident that mitochondrial
superoxide and hydrogen peroxide play important roles in
a range of cellular functions, and can also activate sig-
naling pathways that promote cell survival and disease
resistance. Exposure of hippocampal neurons to subtoxic
levels of hydrogen peroxide triggers the release of Ca2?
from the endoplasmic reticulum by causing the opening of
both IP3 and ryanodine receptor channels [143]. Interest-
ingly, superoxide enhances long-term potentiation of
synaptic transmission in hippocampal CA1 neurons by a
mechanism requiring activation of ryanodine receptors
and extracellular regulated kinases [144]. Mitochondrial
ROS may also play roles in recovery from injury. For
example, superoxide can stimulate neurite outgrowth by
directly activating protein kinase C [145]. Moreover,
peroxynitrite can promote the phosphorylation and nitra-
tion of regulatory sites within receptor tyrosine kinases
thereby activating cell survival signaling pathways
including those involving PI3 kinase–Akt and mitogen-
activated protein (MAP) kinases [2]. Nitration of proteins
involved in synaptic vesicle trafficking can enhance glu-
tamate release, suggesting a potential role for superoxide
and peroxynitrite in regulating neurotransmission [2].
Mitochondrial superoxide production, it is known, con-
tributes to damage of neurons in pathological conditions
such as chronic cerebral hypoxia or Alzheimer’s disease
[2]. However, it has been widely reported that transient
exposure of neurons to low levels of superoxide which are
converted into hydrogen peroxide can protect the neurons
against a subsequent exposure to what would have
otherwise been a lethal level of stress. This neuroprotec-
tive effect of a subtoxic increase in cellular oxidative
stress has been termed ‘‘preconditioning’’ and clearly falls
under the paradigm of hormesis [2]. Although the
involvement of oxidants in many signaling pathways is
well documented, the cellular strategies for conferring
pathway specificity to such reactive molecules is still
elusive. Recent studies suggest that cells may spatially
restrict oxidant production to allow microdomain-specific
signaling [146]. The specific molecular mechanisms by
which mitochondrial ROS elicit hormetic responses in
neurons are poorly understood, but emerging evidence
suggests important roles for certain transcription factors
with regulatory effects. For example, NF-kB is activated
in neurons in response to oxidative stress and plays a
pivotal role in the adaptive response that protects the
neurons against more severe oxidative stress, by induc-
ing the expression of protective genes against oxidative
stress including SOD2 and Bcl-2 [147], as well TNF
1897
up-regulates the expression of Mn-SOD via an NF-kB-
mediated mechanism, thereby protecting neurons against
excitotoxic, ischemic and oxidative injuries [148]. While
mitochondrial H2O2 may activate adaptive stress response
pathways in neurons, they may also play neuroprotective
roles by acting on other cell types in the nervous system.
For example, microglial activation, which occurs in
response to increased oxidative stress, can have either
beneficial or detrimental effects on neurons and neural
progenitor cells depending upon the type and amounts of
cytokines and growth factors secreted by the microglia
[149]. In addition, oxidative stress can stimulate angio-
genesis in the brain [2], which is critical to restore normal
neuronal perfusion during the days and weeks after a
stroke. This last effect represents an example of ‘‘trans-
cellular’’ hormesis mediated by ROS [150].
Redox-Dependent Control of the Proteostasis Network:
Relevance to Antidegenerative Strategies
Neurodegenerative disorders are often characterized by the
aggregation and accumulation of misfolded proteins (e.g.
Alzheimer’s disease, Parkinson’s disease, Amyotrophic
lateral sclerosis, Huntington’s disease (HD) and fronto-
temporal dementia). Aggregated proteins are very toxic to
cells in culture and both in vitro and in vivo there is
overwhelming evidence that these aberrant proteins are key
players in neurodegeneration. Protein quality control is a
cellular defense mechanism against misfolded proteins that
prevents aggregate formation under physiological condi-
tions. The presence of accumulated aggregates of mis-
folded proteins in many neurodegenerative disorders,
suggests that protein quality control failed to restore
homeostasis in these pathological conditions. In fact, evi-
dence from observations in cellular disease models, mouse
models, as well as from post mortem patient material
indicates activation of the quality control machinery in
response to the pathological process. In addition, interfer-
ence with protein quality control by genetic or chemical
manipulation often results in aggregate formation and
neurodegeneration. This stresses the importance of proper
quality control in neurodegenerative disorders and indi-
cates that it may provide a target for therapeutic inter-
vention [1–3, 15, 31, 86, 151–153]. A wide range of
cellular defence mechanisms operate as a proteostasis
network, to counteract this effect, including antioxidant
proteins [superoxide dismutase 1and 2 (SOD1, SOD2),
catalase, glutathione peroxidase, thioredoxin reductase,
heme oxygenase (HO-1) and cystationine-b synthase
(CBS)], glutathione, molecular chaperones such as the heat
shock proteins (HSPs) that reduce aggregation and promote
folding of misfolded proteins [154].
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The proteostasis network regulates protein function [1,
155–158], as an integrated biological system evolved to
protect protein fold. As such, proteostasis controls con-
centration, conformation, binding interactions (quaternary
structure), and location of individual proteins making up
the proteome by readapting the innate biology of the cell,
often through transcriptional and translational changes [1].
Proteostasis thus influences specific cellular functions and
enables differentiated cells to change their physiology for
successful organismal development and aging in the face of
constant intrinsic and environmental challenges to prevent
disease onset. Proteostasis is influenced by the chemistry of
protein folding/misfolding and by numerous regulated
networks of interacting and competing biological pathways
that influence protein synthesis, folding, trafficking, dis-
aggregation, and degradation. The composition of the
proteostasis network is regulated by signaling pathways
including (1) the unfolded protein response (UPR) that
increases the production of endoplasmic reticulum (ER)
chaperones and suppresses translation, (2) the heat shock
response (HSR), (3) the ubiquitin proteasome (UPS) for
removal of proteins and (4) the authophagy degradation
pathway that targets long lived proteins and organelles for
removal, together with epigenetic programs [159]. At this
level, regulation of cellular redox state becomes a crucial
mediator of multiple metabolic, signalling and transcrip-
tional processes operating within this integrated network
machinery [2, 41, 152, 153, 156].
ER Pathway
The ER is a highly dynamic organelle with a central role in
lipid and protein biosynthesis. The ER produces the
transmembrane proteins and lipids for most cell organelles
and is responsible for the synthesis of almost all secreted
proteins. Synthesized and correctly folded proteins that
pass the quality control are transported through the secre-
tory pathway to reach their final destination including cell
surface, lysosomes, GA, extracellular space, and the ER.
After translocation to the ER through the Sec61p translo-
con complex channel, nascent proteins are assisted in their
folding by a complex family of chaperones, foldases and
co-factors [160]. The ER also has an important role in Ca2?
storage and signaling. The resting intra-ER Ca2? concen-
tration is three to four orders of magnitude higher than
cytosolic Ca2?. This gradient is generated by the
sarco(endo)plasmic reticulum Ca2? ATPase (SERCA)
proteins, which pump Ca2? into the ER, and the
Ins(1,4,5)P3 and ryanodine receptors that release Ca2?
from the ER [2]. Due to its ability to store and secrete
Ca2?, the ER controls a wide range of cellular processes
such as organogenesis, transcriptional activity, stress
responses, and apoptosis [2]. The translation of proteins is
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Neurochem Res (2010) 35:1880–1915
performed by ribosomes on the cytosolic surface of the ER,
and the unfolded polypeptide chains are translocated into
the ER lumen via the Sec61 complex [161]. In the ER
lumen these chains are often N-glycosylated and folded
into secondary and tertiary structures that are stabilized by
disulfide bonds [162]. Most glycolpolypeptides thus elab-
orated are released from calnexin/calreticulin/ERp57 in a
native, transport competent state, are rapidly deglycosy-
lated and sequestered in transport vesicles that leave the
ER. Glycoproteins presenting not complete folding after a
single step of association with calnexin/calreticulin/ERp57
and polypeptides, undergo an additional folding attempt
characterized by disulfide rearrangement or reshuffling.
Glycopolypeptides released from calnexin which display
major folding defects are finally targeted by BiP/Grp78 and
dislocated across the ER membrane for proteasome-medi-
ated degradation by ER-associated degradation (ERAD)
[163].
ERAD is a mechanism employed by ER protein quality
control system and the calnexin cycle to eliminate mis-
folded or unassembled proteins generated during the fold-
ing process [163]. ERAD impairment is implicated in some
neurodegenerative diseases associated to protein misfold-
ing, such as Huntington’s, Parkinson’s diseases and ALS
[160]. Chaperones, transmembrane proteins and ubiquitin-
associated enzymes selecting target misfolded proteins to
the cytoplasm for degradation by the proteasome system
comprise the ERAD machinery [163], which includes
the ER degradation-enhancing mannosidase-like lectins
(EDEMs), consisting of mannosidase-like proteins directly
involved in recognition and targeting of unfolded proteins
for degradation [163]. Three EDEM homologues, EDEM-
1, -2 and -3 have been identified, and probably their
function is similar, delivering ERAD substrates to the
retrotranslocation channel [160]. EDEM1 associates with
the DERLIN family of proteins [161], which are ER-
membrane proteins components of the retrotranslocation
channel, besides to Sec61p. It is estimated that the protein
concentration within the ER lumen reaches 100 mg/ml, a
concentration highly prone to protein aggregation that is
avoided by an effective folding quality control system
[164]. Conditions that interfere with the ER function con-
sequently lead to abnormal protein folding and to the
accumulation of unfolded/misfolded proteins on its lumen,
a cellular condition referred as ‘ER stress’ [160]. ER stress
can be originated by various cellular alterations, such as
calcium homeostasis disruption, redox changes, reduction
of disulfide bonds, glucose/energy deprivation, viral
infections, accumulation of folding-incompetent products
of mutated genes, and high secretory activity, among others
[2]. To manage protein folding stress, cells activate adap-
tive pathways known as the Unfolded Protein Response
(UPR), which aims to re-establish cell homeostasis [2]. The
Neurochem Res (2010) 35:1880–1915
unique oxidizing environment of the ER and the numerous
protein chaperones present in the organelle are crucial for
the proper folding of proteins and protein complexes [118].
The disulfide bond formation is catalyzed by protein
disulfide isomerase (PDI) [165]. PDI is oxidized by Ero1-a
and -b into a disulfide donor, whereas reduced PDI can
isomerize disulfide bonds in client proteins. Other ER-
resident folding factors include amino acid cis–trans
isomerases, the chaperones GRP94 and Ig heavy chain
binding protein (BiP), N-glycosylation enzymes and the
lectins calnexin and calreticulin that specifically chaperone
N-glycans [165], all of which operate in complex multi-
protein structures [162]. While assisting with folding, these
chaperones and foldases bound client proteins in the ER
until the maturely folded proteins meet all quality control
standards and exit the ER [165]. Thus, the ER is exquisitely
sensitive to alterations in homeostasis, and proteins formed
in the ER may fail to attain correct conformation due to: (1)
lack of chaperones or cellular energy to promote chaper-
one-protein interactions; (2) Ca2? depletion; (3) disruption
of redox state; (4) protein mutations that hamper adequate
folding; and (5) reduction of disulfide bonds [2].
Eukaryotic cells respond to unfolded proteins in their
endoplasmic reticulum (ER stress), amino acid starvation,
or oxidants by phosphorylating the alpha subunit of
translation initiation factor 2 (eIF2a). This adaptation
inhibits general protein synthesis while promoting trans-
lation and expression of the transcription factor ATF4 [2].
Atf4(-/-) cells are impaired in expressing genes involved
in amino acid import, glutathione biosynthesis, and resis-
tance to oxidative stress [2]. Under low stress conditions,
GRP78 is associated with several different ER proteins
including PERK (PKR-like ER kinase), IRE-1 (inositol
requiring element-1) and ATF6 (activating transcription
factor 6). When cells are under oxidative and metabolic
stress, GRP78 is recruited to oxidatively damaged and
misfolded proteins, and PERK, IRE-1 and ATF-6 are
mobilized to serve the complementary roles in the UPR.
PERK suppresses protein translation by phosphorylating
eIR2a, and also phosphorylates and thereby activates the
transcription factor Nrf2 resulting in the production of
antioxidant and phase 2 enzymes. IRE-1 activation results
in the production and nuclear translocation of the tran-
scriptional regulator XBP-1 which, in turn, induces the
expression of GRP78 and GRP94. During the UPR, ATF-6
is cleaved to generate an active transcription factor which
translocates into the nucleus and induces the expression of
several cytoprotective proteins. Examples of stimuli that
activate the UPR include nutrient deprivation, altered
protein glycosylation, oxidative stress and increased in-
tralumenal Ca2? levels.
Until recently, the endoplasmic reticulum (ER) was
viewed as a protein synthesis factory that was not involved
1899
in signal transduction processes or cellular stress responses.
We now know that the ER plays pivotal roles in regulating
cellular Ca2? homeostasis and transduces Ca2?-mediated
signals from extracellular signals (excitatory neurotrans-
mitters and growth factors) and signals emanating from
internal organelles including mitochondria and the nucleus
[166]. When stimulated by IP3 or Ca2? itself, Ca2? is
released from the ER through opened IP3 receptor and
ryanodine receptor channels, respectively. An ER mem-
brane Ca2? ATPase is responsible for transporting Ca2?
into the ER. Excessive ER stress and dysregulation of ER
Ca2? homeostasis is believed to contribute to the dys-
function and death of neurons in ischemic stroke, Parkin-
son’s disease and AD [162]. The ER is also an important
sensor of cellular stress. In response to energetic, oxidative
and ionic (particularly Ca2?) stress, protein synthesis may
be decreased, and signals are sent from the ER to the
nucleus that result in the up-regulation of the expression of
genes encoding ER protein chaperones such as GRP78 and
GRP94, and antioxidant enzymes [163]. The ER plays
important roles in hormetic responses to stress in neurons.
Bcl-2 can decrease ER Ca2? uptake, resulting in enhanced
Ca2? signaling and activation of cyclic AMP response
element binding protein, thereby promoting the regenera-
tion of injured axons [167]. Moreover, ER-mediated sig-
nals associated with the UPR enhance antioxidant defenses
and inhibits caspase-dependent cell death. Thus, mild ER
stress can elicit multiple hormetic responses in neurons. A
better understanding of adaptive responses to ER stress
may lead to novel therapeutic approaches for both acute
and chronic neurodegenerative conditions. As evidence, it
has been reported that, by activating ryanodine receptors in
the ER, paraxanthine (the major metabolite of caffeine) can
prevent the degeneration of dopaminergic neurons in a
model of Parkinson’s disease [2].
Induction of ER protein chaperones is another thera-
peutic approach that is supported by experimental data
showing that GRP78, GRP94 and ORP150 [168] can pro-
tect neurons against excitotoxic and ischemic death. Mild
ER stress responses may be elicited by factors that are
known to be neuroprotective including mild energetic
stress (dietary energy restriction and 2-deoxyglucose
treatment and exercise [169]. Adaptation to ER stress is
mediated by the activation of an integrated signal trans-
duction pathway known as the unfolded protein response
(UPR). In neurodegeneration, either in mouse models or in
post-mortem brain from ND patients, activation of the UPR
correlates with the amount of aggregated proteins [170].
Moreover, the UPR has generally been viewed as a
homeostatic mechanism designed to avoid the accumula-
tion of toxic protein aggregates. However, evidence exists
that deletion of X Box binding protein-1 (XBP-1), a key
UPR transcription factor that regulates genes involved in
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protein folding and quality control, by disrupting UPR, can
prevent neurodegeneration [171]. Although counterintui-
tive, this finding can be explained in several integrated
ways: (1) mild, UPR stimulates the adaptive up-regulation
of chaperones, thus resulting cytoprotection, whereas pro-
longed UPR activation results in Bax-mediated activation
of pro-apoptotic pathways depending on c-Jun N-terminal
kinase (JNK) stimulation [172]. In addition, ablation of
CHOP, a transcription factor involved in the UPR, restores
motor function in a mouse model of Charcot Marie Tooth
1B neuropathy, possibly via inhibition of ER stress-
induced apoptosis [173]; (2) inhibition of the ER-associ-
ated degradation (ERAD) pathway consequent to the
ablation of XBP-1 might explain the compensatory stim-
ulation of autophagy; (3) accumulation of unfolded or
misfolded proteins due to the absent UPR leads to
exhaustive ER stress, which in turn induces autophagy via
activation of protein kinase C h (PKC h) [174]; (4) dis-
turbance of the UPR induces oxidative stress and depletion
of glutathione, which activate UPR-dependent cell death
[172], (5) oxidative stress can also stimulate autophagy by
activating ATG4, a cysteine redox-sensitive protease.
Thus, at sublethal levels, ROS can act as signaling mole-
cules for the induction of cytoprotection and can even
prolong life span, as described in C. elegans under glucose
restriction [175].
Autophagy Pathway
Autophagy is the cellular pathway of ‘‘self digestion,’’ a
regulated lysosomal pathway for the degradation and
recycling of long-lived proteins and organelles. During
autophagy, cytoplasmic constituents are sequestered into
double-membraned autophagosomes, which are delivered
to lysosomes and degraded. This process generates nucle-
otides, amino acids, and fatty acids, which are recycled for
ATP generation and macromolecular synthesis [176].
While the UPR controls the degradation of smaller units
of unfolded or misfolded proteins via transcription of
chaperones, larger aggregates are detoxified via autophagy.
Autophagy involves the encapsulation of cargo on a dou-
ble-membrane vesicle to form the autophagosome. Auto-
phagosomes fuse with lysosomes to form the autolysosome
where the cargo is degraded. In addition, hybrid interme-
diates called amphisomes are formed when autophago-
somes fuse with the endosomes or MVEs, before fusing
with lysosomes. The ubiquitin-proteasome and autophagy-
lysosome pathways are the two main routes of protein
clearance in eukaryotic cells [176]. Proteasomes predomi-
nantly degrade short-lived nuclear and cytosolic proteins.
However, substrates need to be unfolded to pass through
the narrow pore of the proteasome barrel, which excludes
the clearance of aggregated proteins observed in many
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Neurochem Res (2010) 35:1880–1915
neurological disorders [160]. In contrast, lysosomal-related
compartments are able to degrade substrates such as pro-
tein complexes and organelles. Increasing evidence indi-
cates that the clearance of proteins through autophagy
correlates with their propensity to aggregate. In light of the
evidence that many aggregate-prone proteins cause many
distinct diseases possibly by gain of function mechanisms,
efforts have been made to explore therapeutic strategies
that reduce the levels of such proteins, at either the syn-
thesis or degradation stage [160]. Recent studies indicate a
crucial role of autophagy in protein quality control, with a
demonstrated function in the maintenance of the homeo-
stasis of the nervous system [172]. Notably, p62 (also
termed sequestosome 1) interplays between polyubiquiti-
nation and the autophagy-degradative mechanisms, thus
shuttling substrates of the proteasome system. Compared to
the latter, autophagy is less selective, but emerging evi-
dence indicates that autophagy is highly regulated and
specific for the different target organelles, such as mito-
chondria (mitophagy) endoplasmic reticulum (reticulo-
phagy), peroxisomes (pexophagy), ribosomes (ribophagy),
granules (crinophagy), and pathogens (xenophagy). Cyto-
sol, cytoskeleton, nuclei (nucleophagy), and protein
aggregates (aggrephagy) can also be removed by
autophagy.
Several upstream regulators of autophagy have been
characterized [176]. The class III PI3 kinase complex,
including Beclin-1/Atg6, is required for generation of pre-
autophagosome structures. The mammalian target of rap-
amycin (mTOR), a nutrient-sensing kinase complex that
regulates cell growth and survival, blocks autophagy dur-
ing nutrient-rich conditions by inhibiting the Atg1 com-
plex, which is involved in the initiation stages of
autophagic activity. Upstream activators of mTOR (NF-kB,
class I PI3 kinase, Akt and NF-kB) suppress autophagy,
whereas inhibitors of this pathway such as PTEN (phos-
phatase and tensin homologue) induce autophagy.
Accordingly, rapamycin, a classical mTOR inhibitor, has
been shown, in vivo, to alleviate neurodegeneration in
models of Huntington’s disease, associated with reduced
neuronal loss, improved survival and degradation of toxic
protein aggregates [172]. Atg proteins regulate steps which
are sequential in the autophagy process, whose core is
organized around two ubiquitin-like conjugation systems,
such as the ubiquitin-like protein Atg12 and the ubiquitin-
like protein LC3-I. When Atg12 is activated, it forms a
covalent bond with Atg5, which then interacts with other
components to form a multimeric complex that translocates
to membranes of early autophagosomes. Alternatively,
LC3 is cleaved and then conjugated to phosphatidyletha-
nolamine (PE) via Atg7 and Atg3 (another ubiquitin carrier
protein (E2)-like protein), generating LC3-II. The uncon-
jugated LC3-I remains in the cytosol while the conjugated
Neurochem Res (2010) 35:1880–1915
LC3-II form targets to the autophagosomal membrane after
the formation of the active Atg12–Atg5 complex [177]. As
elongation of the autophagosomal membrane occurs,
cytoplasmic proteins and organelles are sequestered into
double-membraned autophagosomes, which are delivered
to lysosomes and degraded [176]. Autophagy is critical for
the maintenance of neuronal homeostasis and contributes
to basal elimination of misfolded proteins in the nervous
system. Brain specific ablation of the essential autophagy-
related genes atg5 and atg7 leads to spontaneous neuro-
degeneration with pathological features closely resembling
Alzheimer’s and Parkinson’s disease consisting of neuro-
logical/motor dysfunction, accumulation of poly-ubiquiti-
nated protein aggregates, neuronal loss and premature
death [160]. Similar to UPR, a dual role in cell survival or
cell death has been attributed to autophagy. Autophagic
degradation of cellular materials generates amino acids and
fatty acids, which can be used for protein synthesis and
ATP generation during stressful conditions such as star-
vation, as well as removal of protein aggregates (which can
trigger apoptosis) and damaged mitochondria (source of
apoptotic proteins and toxic ROS) result to be protective
mechanisms. Conversely, prolonged autophagy can lead
to cell death through excessive self-digestion or activation
of apoptosis. Consistent with this notion, prosurvival
autophagy in response to starvation, growth factor with-
drawal, ischemia/reperfusion injury, and various chemo-
therapeutic drugs [178, 179] has been proven, as well as
autophagic cell death has been observed in response to
hypoxia, oxidative stress, radiation, GF withdrawal, lipo-
polysaccharide, overexpression of smARF, and various
chemotherapeutic drugs [178].
Cellular Stress Response and The Vitagene Network
Protein thiols and, more in general, protein quality control
processes, play a critical role in cellular homeostasis which
is maintained by a highly complex network of molecular
interactions that balances protein biosynthesis, folding,
translocation, assembly/disassembly, and clearance [151].
The ability to ensure proper protein folding is critical for
cellular function and organismal viability. In cells under-
going division, damaged and oxidized proteins can be
sequestered and retained in mother cells, enabling daughter
cells to have a pristine, undamaged proteome. However, in
post-mitotic cells, such as most neurons, protein quality
control must be maintained by other, possibly more com-
plex, mechanisms [152]. Furthermore, particular neuronal
populations are more vulnerable to proteotoxicity while
others are more able to tolerate the misfolding and aggre-
gation of disease proteins. Thus, the cellular environment
must play a significant role in determining whether disease
1901
proteins are converted into toxic or benign forms. The
endomembrane neuronal network divides brain cells into
different subcellular compartments that possess distinct
sets of molecular chaperones and protein interaction
networks [2]. Cells buffer proteotoxic events related to
intracellular protein misfolding via chaperone-mediated
partitioning of nonnative conformers between pathways for
proper folding, inclusion body formation, and degradation.
Chaperones, in fact, act as agonists and antagonists of
disease protein aggregation to prevent the accumulation of
toxic intermediates in the aggregation pathway. Interacting
partners can also modulate the conformation and locali-
zation of disease proteins and thereby influence proteo-
toxicity [152]. Thus, interplay between these protein
homeostasis network components can modulate the self-
association of disease proteins and determine whether they
elicit a toxic or benign outcome [2]. Polypeptides that fail
to fold properly, along with damaged and oxidized mature
proteins, are targeted for degradation by specialized cel-
lular degradation machineries. A failure to prevent the
misfolding and aggregation of one protein can destabilize
the proteome, resulting in uncontrolled aggregation of
other polypeptides. When conformationally challenged
aggregation-prone proteins are expressed, the resulting
unfolded or misfolded proteins are rapidly degraded via the
ubiquitin–proteasome pathway. However, in some cases,
protein aggregation leads to the development of so-called
‘conformational diseases’. Among the conformational
diseases are the human neurodegenerative diseases, such as
Alzheimer’s (AD), Parkinson’s (PD) and Huntington’s
(HD). In AD, dual digestion of amyloid precursor protein
(APP) releases the aggregation-prone peptides that are
collectively termed amyloid-b (Ab). The accumulation and
aggregation of Ab (particularly the highly aggregative
Ab1–42) is associated with AD. Similarly, aberrant
aggregation of a-synuclein is associated with the emer-
gence of PD, whereas Huntington and other CAG triplet
diseases are typified by the accumulation of polyglutamine-
containing aggregates. This also includes prion diseases
such as Creutzfeldt-Jakob disease with accumulation of
misfolded prion protein, type II diabetes with accumulation
of islet amyloid polypeptide, and amyotrophic lateral
sclerosis with aggregated superoxide dismutase-1 [153]. As
one of the most characteristic feature present in most brain
oxidant disorders is the occurrence of extra- or intracellular
fibrillar aggregates containing misfolded proteins with
beta-sheet conformation. These aggregates are composed
of distinct proteins in each neurodegenerative disease. As
already mentioned, toxicity deriving from misfolded pro-
teins or peptides are collectively termed proteotoxicity, and
it has become evident that protein aggregation is tightly
linked to the emergence and development of neurodegen-
erative diseases. Moreover, the mechanisms that have been
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found to counter toxic protein aggregation in different cell
types and different organisms are highly conserved. Thus,
it is likely that proteotoxicity that is associated with the
expression of neurodegeneration-linked aggregation-prone
proteins in any tissue can provide insights into the neuronal
defence mechanisms. Recent data suggest that small olig-
omeric aggregating structures, termed also protofibrils, are
the underlying cause, rather than high-molecular-mass
aggregates as previously assumed. In vitro studies on
fragments of amyloidogenic proteins and synthetic pep-
tides have established that the tendency for a protein to
form amyloid is often limited to a short sequence of the full
protein, known as an SRE (selfrecognition element). SREs
form the core of amyloid fibrils. These amyloidogenic
sequences constitute ‘hotspots’ for aggregation of the
native protein into amyloid fibrils and are often the sites of
mutations leading to early-onset amyloidosis. In the case of
tau-paired helical filaments, which accumulate in the neu-
rofibrillary tangles characteristic of AD and other neuro-
degenerative diseases, it has been shown that only three
residues (Val-Tyr-Lys), are sufficient for fibril formation.
Similarly, short sequences forming the core domain of
various amyloid fibrils have been identified for Ab (amy-
loid b-peptide), calcitonin, IAPP (islet amyloid polypep-
tide), insulin and a-synuclein. It may be possible to delete
residues freely on either side of an SRE while retaining the
ability to form amyloid [154]. Genetic and age-related
factors, as it is known, act as vicious cycle increasing the
amounts of pathogenic proteins in AD where, the increase
in Ab42 levels is caused by: (1) mutations in amyloid
precursor protein or presenilins (for example, c-secretase),
(2) by reactive oxygen species and (3) by reductions in
Ab-degrading enzymes (AbDE), such as neprilysin and
insulin-degrading enzyme, as well as increases in tau
concentrations are influenced by oxidant damage, phos-
phorylation and calcium; in PD, where increased levels of
a-synuclein caused by triplication of its gene or mutations
in parkin, DJ1, ubiquitin carboxy-terminal hydrolase 1
(UCHL1), phosphatase and tensin homologue induced
kinase 1 (PINK1) or leucine-rich repeat kinase 2 (LRKK2),
are associated with proteasome impairment and oxidative
stress; in HD, with polyglutamine expansions in huntingtin
(HTT). The protein aggregation process itself is enhanced
by: increasing protein concentration; the action of trans-
glutaminases; protein chaperone insufficiency; mutations in
a-synuclein (PD) and polyglutamine expansions in hun-
tingtin (HD); and/or post-translational modifications, such
as oxidations induced by, for example, hydrogen peroxide
(H2O2), Fe2? and Cu?, and phosphorylation. Although
the proteins involved can differ, there is considerable
overlap in the mechanisms by which they damage and kill
neurons. Oligomers of Ab, a-synuclein and HTT might
damage and kill neurons by inducing membrane-associated
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Neurochem Res (2010) 35:1880–1915
oxidative stress (MAOS), thereby impairing mitochondrial
function and thus causing degenerative cell death [155].
Cellular stress response is the ability of a cell to coun-
teract stressful conditions (Fig. 4). This phenomenon,
which includes heat shock response (HSR), represents an
ancient and highly conserved cytoprotective mechanism
[2, 3, 14]. Production of heat shock proteins, including
protein chaperones, is essential for the folding and repair of
damaged proteins, serving thus to promote cell survival
conditions that would otherwise result in apoptosis
[15, 16]. The term ‘molecular chaperone’ denotes a large
family of ubiquitous proteins that function as part of an
ancient defense system in our cells. Chaperones promote
cell survival by sequestering damaged proteins and pre-
venting their aggregation. During stressful conditions, such
as elevated temperature, they prevent protein aggregation
by facilitating the refolding or elimination of misfolded
proteins. The stress-induced response to damaged proteins
is helped by a sophisticated regulatory system, which shuts
down most cellular functions and, in parallel, induces the
synthesis of several chaperones and other survival-pro-
moting proteins. Therefore, many of the chaperones are
also called stress or ‘heat shock’ proteins in reference to the
archetype of cellular stress, heat shock. Besides their role
during stress, chaperones have multiple roles under normal
conditions. They promote the transport of macromole-
cules (e.g. proteins or RNA) and participate in remodel-
ling events involving larger protein complexes, including
signaling, transcription, cell division, migration and
differentiation.
Molecular chaperones both in the cytosol (heat-shock
proteins, crystallins, prefoldin, Hsc70,) in the endoplasmic
reticulum (Bip, Grp94, calnexin, calreticulin) form large
complexes and have a large number of co-chaperones to
regulate their activity, binding properties and function
[151]. These chaperone complexes regulate local protein
networks, such as the mitochondrial protein transport
apparatus and the assembly and substrate specificity of the
major cytoplasmic proteolytic system, the proteasome
[156]. Chaperones has been demonstrated to protect the
cell nucleus after stress and to promote the transport of
ribosomal subunits and the mobility of steroid receptors
inside the nucleus and, in addiction, regulate both the
activation and the disassembly of numerous transcrip-
tional complexes, emerging thus as regulators critical
transcriptional networks [157]. Stress-induced nuclear
translocation of chaperones may preserve also nuclear
remodelling capacity during environmental damage, and
thus protect the integrity of DNA. Consistently, there is
significant interest in the discovery and development of
small molecules that modulate heat shock responses and
parallel stress response pathways for therapeutic purposes
[2, 3, 14, 16].
Neurochem Res (2010) 35:1880–1915
Fig. 4 Cell stress responses. Expression of HS genes is induced in
response to physiological and environmental stress conditions
including longevity stimuli, such as fasting, caloric restriction or
acetylcarnitine, and protein conformational diseases. HSF1 can also
be directly stimulated by longevity stimuli such as the histone
deacetylase SIRT1 that directly activates HSF1 by deacetylation, thus
fostering longevity. Cumulating misfolded proteins in response to
proteotoxic environmental stress conditions triggers the cellular stress
response. HSPs that are normally bound to HSF1, maintaining it in a
Cellular stress response (Fig. 5) requires the activation
of pro-survival pathways which, under control of protective
genes called vitagenes [2, 3] produce molecules (heat
shock proteins, glutathione, bilirubin) endowed with anti-
oxidant and anti-apoptotic activities. Generally, molecular
chaperones help hundreds of signaling molecules to keep
their activation-competent state, and regulate various sig-
naling processes ranging from signaling at the plasma
membrane to transcription. In addition to these specific
regulatory roles, recent studies have revealed that chaper-
ones act as genetic buffers stabilizing the phenotypes of
various cells and organisms [82, 158]. Among the cellular
pathways conferring protection against oxidative stress, a
key role is played by the products of vitagenes [2, 3, 14–16,
27, 41]. These include members of the heat shock protein
(Hsp) family, such as heme oxygenase-1 and Hsp72, sir-
tuins and the thioredoxin/thioredoxin reductase system [42,
43, 57, 86]. Recent studies have shown that the heat shock
response contributes to establishing a cytoprotective state
in a wide variety of human diseases, including inflamma-
tion, cancer, aging and neurodegenerative disorders [35].
Given the broad cytoprotective properties of the heat shock
response there is now strong interest in discovering
and developing pharmacological agents capable of induc-
ing the heat shock response [2]. Molecular chaperones are
known to disrupt aggregates but also to promote active
1903
repressed state before stress, are titrate away by damaged or
misfolded proteins with resulting HSF-1 activation. Multi-step
activation of HSF1 involves post-translational modifications, such
as hyperphosphorylation and deacetylation, which allow HSF1 to
trimerize, translocate into the nucleus, and bind to heat-shock
elements (HSEs) in the promoter regions of its target hsp genes.
During aging, a gradual decline in potency of the heat shock response
occur and this may prevent repair of protein damage, leading to
degeneration and cell death
aggregation when the concentration of the aggregating
protein is high. Consistent with this notion, although pro-
tein aggregation is hazardous under certain circumstances,
the creation of apparently less-toxic large aggregates is
protective. This hypothesis is the basis of the therapeutic
potential of heat shock proteins (HSPs), which prevent
protein misfolding and aggregation. Transgenic animal
models of the diseases have demonstrated that induction or
overexpression of HSPs can suppress neuronal dysfunction
and degeneration. Hsp70, in fact it has can reduce the
amount of misfolded and aggregated a-Syn species in vivo
and in vitro, and protect neuronal cells from a-Syn-
dependent neurotoxicity [180], highlighting some of the
intriguing aspects of HSP-based therapy.
The Carnitine System: Physiological Roles,
Insufficiency and Interplay With Mitochondrial
Bioenergetics in Aging and Neurodegenerative
Disorders
Carnitine is present in mammalian cells as free carnitine
and acylcarnitines. While carnitine’s most widely known
function is its involvement in b-oxidation of fatty acids, it
may also have other roles in metabolism. The importance
of acylcarnitines in tissues with high rates of b-oxidation
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1904
Fig. 5 Chemical structures of carnitine and acetylcarnitine molecules
such as heart and muscle is intuitive. However, acylcarni-
tine and carnitine supplementation have resulted in bene-
ficial effects in the treatment of various neurological
diseases, in spite of the fact that fat are not the major fuel
for brain [181]. The acylcarnitine profile has been shown to
be useful in identifying inborn errors of metabolism and to
be altered under different metabolic conditions [182].
Moreover, recent data suggest multifactorial roles for a-
cylcarnitines in neuroprotection. Brain acylcarnitines can
function in synthesizing lipids, altering and stabilizing
membrane composition, modulating genes and proteins,
improving mitochondrial function, increasing antioxidant
activity, and enhancing cholinergic neurotransmission
[183]. The importance of carnitine in brain is emphasized
by carnitine deficiency symptoms, many of which involve
major deleterious effects in brain. Because the brain is
highly reliant on oxidative metabolism, impairment of fatty
acid metabolism and energy production due to lack of
carnitine leads to metabolic encephalopathy [183]. Struc-
turally, the astrocyte swells and mitochondria are expanded
in nerve cells under carnitine deprivation [183]. Fatty acids
become key energy substrates for brain under metaboli-
cally compromised conditions such as fasting or starvation.
Therefore functions of carnitine and acylcarnitines in fatty
acid metabolism, ketosis and buffering of the concentration
ratio of acyl-CoA to free CoA, are significant in brain
metabolism, particularly metabolic disturbances present in
neurological disease [97]. Currently, only a relatively small
subset of acylcarnitines is usually investigated and more
research is needed to exploit the use of acylcarnitines in the
treatment of neurological diseases using a list of acylcar-
nitines encompassing a wide range of these molecules.
L-carnitine (LC) (trimethylamino-b-hydroxybutyrate)
(Fig. 5) is a quaternary amine, naturally present in mam-
malian cells and tissues as both free carnitine and acyl-
carnitines, including acetyl-L-carnitine (ALC). Only LC is
synthesized in the tissue and have biological activity. Its
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Neurochem Res (2010) 35:1880–1915
biological role is to facilitate the transport of fatty acids to
mitochondria for b-oxidation, it facilitates oxidative
metabolism of carbohydrates, increases phosphorylation
oxidative reactions, promotes the excretion of accumulat-
ing organic acids [184–186]. Thus, carnitine functions as
an acyl group acceptor that facilitates mitochondrial export
of excess carbons in the form of acylcarnitines. Whole
body carnitine homeostasis is regulated at multiple levels,
including dietary intake, intestinal absorption, de novo
biosynthesis, and renal reabsorption. Humans obtain car-
nitine from their diet and through endogenous biosynthesis.
Dietary LC derives from the intake of red meats and dairy,
the endogenous synthesis of LC from the aminoacid pre-
cursors L-lysine and L-methionine, mostly in liver and
kidney, trough different metabolic stages for wich ascorbic
acid, niacina, piridossin and iron are necessary [187, 188].
The dietary intake of LC in humans ranges from 1 to
15 lmol/kg body weight/day, whereas the rate of biosyn-
thesis is about 1–2 lmol/kg body weight/day [189]. In
omnivorous humans, animal products provide an estimated
75% of whole body carnitine reserves [190]. Conversely,
vegetarians meet most of their carnitine needs via endog-
enous production [190]. Importantly, typical vegetarian
diets, high in fruits and vegetables and low in fat and
calories, naturally lessens carnitine requirements. By con-
trast, Western diets comprised highly processed foods
which are low in carnitine content and rich in fat and sugar.
Consequently, heavy consumption of these foods can pro-
voke an imbalance between carnitine supply and demand
[97, 191]. Carnitine endogenous biosynthesis is limited by
availability of trimethyllysine, a byproduct of lysosomal
protein degradation. Carnitine reserves can be compro-
mised by depletion of essential micronutrients and/or
metabolites, such as iron, vitamin C, and a-ketoglutarate,
all of which are required cofactors for carnitine biosyn-
thetic enzymes [192].
In the plasma, neither LC nor ALC are bound to proteins
[182]. Concentrations of carnitine and acylcarnitines
change under altered dietary conditions. During starvation
and after eating a high-fat diet, the proportion of carnitine
that is acetylated in liver and kidney significantly increases
associated with an accumulation of several short, medium
and long chain acylcarnitine (LCAC) in skeletal muscle
and a lowering of acylcarnitine levels in the liver and
kidney. Oppositely, a high carbohydrate diet causes very
low levels of ALC in liver. Plasma levels do not directly
correlate with cerebral levels, however starvation causes a
significant increase in mean brain acylcarnitine concen-
tration, with almost all of the increase beeing short-chain
acylcarnitines [97]. Carnitine and its relative esters may be
redistributed to the brain during fasting and the brain may
use them for energy production or, possibly, for the
delivery of acetyl groups [193–196]. Plasma concentration
Neurochem Res (2010) 35:1880–1915
Table 2 Changes in carnitine and acylcarnitine concentrations under
dietary manipulations and alterations in metabolism
Model Diet Carnitine Acylcarnitine
Human Starvation/fasting ; :
High-fat/fat load ; :
Diabetic ketosis ; :
and urinary excretion of acetylcarnitine vary with physio-
logical and pathological states, reflecting mainly the degree
of acetyl-CoA synthesis. In subjects under fasting condi-
tions, acetylcarnitine excretion in the urine increases to
78% of the excreted acylcarnitines [97]. In contrast, in
obese human subjects under fasting conditions, the increase
in urinary excretion of acetylcarnitine is markedly slower
and at a lower level, suggesting a slower formation of
acetyl-CoA derived from fat oxidation (Table 2). In dia-
betic patients with ketosis, acetylcarnitine represented 61%
of the total urine acylcarnitines, and decreased dramatically
upon insulin treatment. The equilibrium between acetyl-
CoA (plus carnitine) and CoA (plus acetylcarnitine)
(acetyl-CoA/CoA ratio) is crucial for mitochondrial
metabolism. The mitochondrial content of endogenous
acetylcarnitine is an indicator of mitochondrial metabolism
of acetyl-CoA (Fig. 6). Acetyl-CoA, derived from pyru-
vate, amino acids, and fatty acids, is reversibly converted
to acetylcarnitine and CoA in the presence of carnitine by
the carnitine acetyltransferase (CAT), a mitochondrial
matrix enzyme attached to the inner membrane. This pro-
cess regenerates free CoASH, which allows fatty acid
oxidation and the tricarboxylic acid (TCA) cycle to pro-
ceed. Acetylcarnitine is transported to the cytosol through
the mitochondrial inner membrane in exchange with car-
nitine by the antiport carnitine acylcarnitine translocase
(CACT), thus providing acetyl groups for the synthesis of
sterols, fatty acids, and ketone bodies [182]. Esters of the
fatty acid of CoA are almost exclusively formed in the
cytosol and they are not transported trough the membranes.
Consequently, under normal conditions, carnitine palmi-
toyltransferase (CPT) 1 and 2, localized, respectively on
the extern and inner surface of inner mitochondria mem-
brane, catalyzes the transfer of acyl groups from acyl-Co-
enzymeA to carnitine to produce acylcarnitines and free
coenzyme A in the cytosol (Fig. 6). Acylcarnitine, trough a
specific carrier protein, then moves in the mitochondria,
where are reconstituted and metabolized as esters of ace-
tilCoA, while the carnitine is transported back in the
cytosol. Ohashi et al. found that acetylcarnitine is trans-
ported by the organic cation transporter 2 (OCTN2), a
homolog of the OCTN1, with high affinity (Km 8.50 lM)
in a saturable manner [188]. The pH profile and Hill
coefficient (0.989) of the OCTN2-mediated acetylcarnitine
1905
uptake were similar to those of carnitine transport [97].
Exogenously administered acetylcarnitine is transported
into mitochondria where the acetyl group favzors the pro-
duction of acetyl-CoA both directly and by activation of
carnitine acetyltransferase (CAT), which is decreased in
aging. The acetyl-CoA acts on the acetylation status of
mitochondrial proteins that increase mitochondrial tran-
scription and protein synthesis. As a result, cytochrome b
content increases, leading to increased activity of electron
transport chain (ETC.) complexes, and stimulating the
oxidative phosphorylation (OXPHOS). This sequence of
events leads to the restoration of the aging-related mito-
chondrial defect [186].
Biological effects of acetylcarnitine relate to (1) the
acetyl unit as acetyl-CoA, (2) the carnitine derived from
the compound, or (3) the intact acetylcarnitine molecule
[97]. Administered acetylcarnitine is transported via the
translocase of the inner membrane to the mitochondrial
matrix where CAT generates acetyl-CoA leaving carnitine.
CAT uses substrate in one direction of the reaction that is
competitive inhibitor respect to the reaction products
obtained in that direction [195]. Therefore, considering the
Km of CAT be 350 lM, if the intramitochondrial con-
centration of acetylcarnitine is higher than this value likely
exogenous acetylcarnitine facilitates the continuous gen-
eration of acetyl-CoA. This, in turn results in an increase of
acetyl-CoA/CoA ratio and in a consequent decrease of the
activity of pyruvate dehydrogenase associated with inhi-
bition of mitochondrial fatty acid b-oxidation and the TCA
cycle. Thus increases in mitochondrial acetyl-CoA may
impact negatively on mitochondrial metabolism. However
consumption of acetyl-CoA in irreversible metabolic
pathways, i.e., lipoic acid, and citrate synthesis within
mitochondria paralleled by corresponding stimulation of
biosynthetic processes within the cytosol, drives the pro-
cess maintaining the mitochondrial content of acetyl-CoA
within a safe range [97]. Acetylcarnitine provides acetyl-
CoA for the synthesis of acetylcholine in the brain and fatty
acids in lipogenic tissues that contain fatty acid synthase,
i.e., brain, liver, and adipose tissue. Citrate generated by
the condensation of acetyl-CoA and oxaloacetate in the
TCA cycle is transported into the cytosol where it is
cleaved by citrate lyase to re-generate cytosolic acetyl-
CoA. One-third of the acetyl-CoA that reaches the cytosol
via citrate in the brain is used for acetylcholine synthesis
[97]. Intravenous administration of acetylcarnitine leads
the acetyl moiety enter the the TCA cycle, as investigated
by 13C NMR spectroscopy, with 13C found in the gluta-
mate, glutamine, and glutathione molecules [97]. Further-
more, carnitine stimulates oxidative metabolism of
pyruvate and branched-chain amino acids, and is involved
in a host of other cellular functions [182]. An increased
acetyl-CoA/CoA ratio decreases the activity of pyruvate
123
1906
Fig. 6 The carnitine system and its interplay with mitochondrial
energetics and metabolism. Carnitine system modulates the metabolic
fate of acetyl-CoA amd hence mitochondrial energetics. Long chain
acyls (LC), traverse the outer mitochondrial membrane via CPT1,
before being internalized through the inner mitochondrial membrane
(IMM) by carnitine translocase (CrTrl) and processed by CPT2, to
AcylCoA in the matrix side of the IMM. B-oxidation process
degrades LC-AcylCoA to one two-carbon fragments of AcetylCoA in
each successive cycle. In conditions where AcylCoA generation
dehydrogenase and inhibits mitochondrial fatty acid
b-oxidation and the TCA cycle. Nuclear acetyl CoA is
either directly imported from the cytosol or synthesized
from acetate by acetyl-CoA synthetase or via the potential
nuclear CAT using acetylcarnitine. The free passage of
acetyl-CoA through the nuclear pore complex facilitates
the traffic of cytosolic acetyl-CoA to the nucleus [97].
Consistent with this notion protein lysine acetylation is
emerging as a key posttranslational modification in cellular
regulation, particularly through the modification of his-
tones and nuclear transcription regulators [196]. It is
already established that transcription of specific nuclear
genes controls the replication and transcription of the
mitochondrial genome. Nuclear respiratory factor-1 (NRF-1)
and mitochondrial transcription factor A (TFAM) are
required for mitochondrial DNA (mtDNA) replication;
together with mitochondrial transcription factors B (TFB1
and TFB2), they stimulate the transcription of both light
and heavy chains of mtDNA [197]. Furthermore, tran-
scription of nuclear DNA recently has been linked to
acetylation and deacetylation of core histone tails at lysine
123
Neurochem Res (2010) 35:1880–1915
exceeds consumption, these molecules can be converted, through
CAT back to acylcarnitine derivatives and exported from the
mitochondria via CrTrl. Supplemented acetylcarnitine is transported
into the mitochondria via CarTrl. Acetyl-CoA formed through the
mass-action of CAT, or synthesized de novo from acetate, becomes
available for tricarboxylic acid cycle (TCA), lipoic acid synthesis,
mitochondrial protein acetylation and energy-dependent processes
modulation
residues, and in addition to this, site-specific acetylation
of non-histone proteins plays an important role in tran-
scriptional regulation. In particular, highmobility group
(HMG)-box proteins are acetylated, and TFAMcontain
twoHMGbox-like domains [97]. Acetylated histone tails
are associated with active chromatin, whereas histone
deacetylation is associated with transcriptional repression
of genes, because the removal of acetyl groups from lysine
residues limits accessibility of the DNA for transcription
[97]. Therefore, histone acetyltransferases and deacetylases
are transcriptional co-regulators. All known acetyltrans-
ferases use acetyl-CoA as a donor for acetylation. Although
the existence of distinct mitochondrial and nucleo-cyto-
solic acetyl-CoA pools has been not univocally demon-
strated, nuclear concentration of acetyl-CoA seems to be
the limiting factor for histone acetylation [198]. This
complex level of regulation of mitochondrial biology is
integrated to the intrinsic function of SIRTs (sirtuins,
SIRT3, 4, and 5) family enzymes, which are nicotin-
amide adenine nucleotide (NAD?)-dependent deacetylase
silent information regulators [2]. Accordingly, proteomic
Neurochem Res (2010) 35:1880–1915
investigations on protein acetylation have identified 388
acetylation sites on 195 proteins in mitochondria from
HeLa cells and mouse liver, representing 20% of mito-
chondrial proteins such as proteins involved in the TCA
cycle, fatty acid b-oxidation, amino acid and carbohydrate
metabolism, membrane transport, and ETC. [199]. Along
with this line of evidence, long-chain acyl-CoA synthetase
1 was found to be acetylated at the lysine residue 633 in rat
liver mitochondria [199]. Lysine acetylation neutralizes the
positive charge and increases the hydrophobicity of the
lysine side chain. While the functional relevance of this
postranslational modification is not completely defined, it
is conceivable that these modifications can modify protein
conformation and, consequently, function. This is the case
for mitochondrial matrix acetyl-CoA synthetase, which is
reversibly acetylated at a lysine residue in the active site of
the enzyme; thus, SIRT3-induced deacetylation activates
the enzyme. Another mitochondrial target for both SIRT3
and SIRT4, is glutamate dehydrogenase which is inhibited
upon deacetylation. Remarkably, SIRT3 by reversibly
binding to mitochondrial complex I and by decreasing the
acetylation status, leads to an increase of its activity [97].
As a result, cytochrome b content increases, leading to
increased activity of electron transport chain (ETC.) com-
plexes, associated with stimulation of oxidative phos-
phorylation (OXPHOS). Thus, reversible acetylation of the
mitochondrial proteome, which represents an efficient
mechanism to regulate the activity of mitochondrial pro-
teins, may be relevant to aging process, where this
sequence of events, conceivably, can revert the mitochon-
drial dysfunction associated to age-related pathology [97].
In addition to regulation of mitochondrial enzyme activi-
ties, acetylation possibly modulates de novo synthesis of
mitochondrial acetyl-CoA. Since acetyl-CoA is the acety-
lation donor for all known acetyltransferases and the Km
for acetyl-CoA is high (330 lM), reasonably, in conditions
of carnitine deficiency the concentration of mitochondrial
acetyl-CoA could be a limiting factor in the acetylation
reactions, such as during aging. In fact, carnitine acetyl-
transferase is decreased in aging and, in these conditions,
exogenous acetylcarnitine transported into the mitochon-
dria by generating the necessary acetyl group for building
up of acetyl-CoA, modulates the acetylation status of
mitochondrial proteins. Likewise, following acetylcarnitine
supplementation it is plausible that a nuclear CAT might
contribute to nuclear acetyl-CoA. Consistent to this, global
transcription in yeast, has been recently evidenced to be
regulated by the steady-state level of acetyl-CoA supplied
by the nuclear acetyl-CoA synthetase [198], which is under
the control of the histone acetylase activity rather than the
histone deacetylase activity. Notably, recent evidence
indicates that lysine acetylation is a prevalent modification
in enzymes that catalyze intermediate metabolism [196].
1907
Virtually every enzyme in glycolysis, gluconeogenesis, the
tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid
metabolism, and glycogen metabolism was found to be
acetylated in human liver tissue [196]. The importance of
lysine acetylation in the regulation of chromatin dynamics
and gene expression is well recognized as an emerging
areas of interest. Recent finding extends the scope of cell
regulation by lysine acetylation to an extent comparable
to that of other major posttranslational modifications such
as phosphorylation and ubiquitination. Most intermediate
metabolic enzymes in fact are acetylated and it has been
demonstrated that acetylation can directly affect the
enzyme activity or stability. Acetylation of metabolic
enzymes was found to change in response to the alterations
of extracellular nutrient availability, providing evidence for
a physiological role of dynamic acetylation in metabolic
regulation. Lysine acetylation is an evolutionarily con-
served mechanism involved in regulation of metabolism in
response to nutrient availability and cellular metabolic
status. Acetylation may play a key role in the coordination
of different metabolic pathways in response to extracellular
conditions [196]. The carnitine system has a key role in in
the deacylation–reacylation processes of membrane phos-
pholipids. Accordingly evidence exist which indicates that
carnitine and acetylcarnitine increase fluidity in rat brain
microsomes and liposomes [183]. Due to their amphiphilic
structure, these molecules may directly interact with the
surface charges on cell membranes. The carboxylic group,
in particular can interact with charges on membrane
phospholipids, glycolipids, and proteins. Recent evidence
demonstrate that acetylcarnitine is a critical factor for the
elongation–desaturation of the n-3 polyunsaturated fatty
acids to form 22:6n-3, DHA, in mitochondria [200]. ALC
is thought to provide an intra-mitochondrial source of
acetyl groups and donate them in the elongation pathway.
Changes in DHA content of membranes can influence
synaptic plasticity, inflammatory response, gene expres-
sion, ion channels, membrane-bound proteins and neuro-
transmission. Likewise, in mitochondria phospholipids
may be important for assembly of the respiratory chain
complexes. Consequently, acetylcarnitine prevents damage
associated with inhibition of these complexes, whereby
preserving mitochondrial membrane integrity or improving
overall metabolic functions through promoting acetylation
processes of critical protein and lipid targets.
Recent evidence suggests that L-carnitine and acetyl-
carnitine supplementation is important to attenuate oxida-
tive stress, and mitochondrial dysfunction associayed to
various physiopathological conditions such as aging,
stress-related disease states of brain, and in metabolic
diseases where one common denominator is a reduction
in tissue lactic acidosis and consequent formation of
ROS. One major mechanism of action of ALC is the
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improvement of mitochondrial bioenergetics which allows
the neuron to produce ATP necessary to maintain the
normal membrane potential and restore altered redox bal-
ance (i.e., synthesis of glutathione) [41, 201], through a
shift in both the mitochondrial and cytosolic redox state
and/or through the induction of antioxidant genes [35, 142,
202]. As a consequence, intracellular reducing power
increases being available for detoxification through the
glutathione system. Thus, due to their capacity in increas-
ing mitochondrial energetics and function, both carnitine
and acetylcarnitine molecules are critical for the prevention
of neurodegeneration and the regulation of neurotrans-
mission [97, 182, 183].
Acetylcarnitine supplementation can be of therapeutic
benefit for chronic degenerative diseases, Alzheimer’s
disease (AD) slowing the progression of mental deterio-
ration in this disease, multiple sclerosis, chronic fatigue
syndrome, depression in the elderly, HIV infection, dia-
betic neuropathies, it provides to protection during meta-
bolic stress such as hypoxia, ischemia and reperfusion of
the brain and of the heart, cognitive impairment resulting
from alcoholism, and ageing, brain injury, autism, Par-
kinson disease, Down syndrome, Huntington’s disease,
cerebellar ataxia, age-associated mental decline, hepatic
encephalopathy and ammonia neurotoxicity [183]. By
restoring tissue carnitine in the elderly, acetylcarnitine
supplementation facilitates the elimination of potentially
toxic acyl-CoA metabolites derived from fatty acid oxi-
dation. Besides the afore mentioned role on mitochondrial
bioenergetics, which allows brain cells to restore altered
redox balance [3, 45], ALC is neuroprotective through a
variety of other effects such as the increase in PKC activity
[41], and modulation of synaptic plasticity via NMDA
receptor expression and increase in the production of
neurotrophins [202]. Importantly, treatment with ALC
induces up-regulation of the gene coding for Hsp72
explaining its protective effects in inflammation, neurode-
generative disorders, and aging [203–205]. Another gene
identified that is positively modulated by acetylcarnitine in
the brain is the mitochondrial voltage-dependent anion
channel (VDAC) protein [206]. VDAC, also known as
porin, is a small pore-forming protein of the mitochondrial
outer membrane. It is the primary pathway for diffusion of
ions and metabolites across the outer membrane and plays
a key role in apoptosis by forming the outer pore compo-
nent of the mitochondrial permeability transition (MPT)
complex. Defects in the function of complexes II and III
have been observed in Huntington’s disease patients [207],
while complex IV is involved in the mitochondrial dys-
function underlying the pathogenesis of AD. Acetylcarni-
tine has been shown to reverse age-related decreases in the
activity of complex III and oxidative phosphorylation and
the inhibition of complex IV, resulting in restoration of
123
Neurochem Res (2010) 35:1880–1915
normal GSH content and increase in the amount of cyto-
chrome b and aa3 hemes mitochondria isolated from old
rats [97, 208], enhancing mtDNA transcription, the stabil-
ity of mitochondrial mRNA, and mitochondrial protein
synthesis. Further to this, in rodents ALC up-regulates the
expression of metabotropic glutamate receptor 2 (mGlu2)
in dorsal root ganglia (DRG) of the spinal cord with a
mechanism demonstrated to be dependent on activation of
the NF-kappaB pathway through acetylation of p65/RelA.
On the basis of this action ALC is suggested for clinical
management of neuropathic pain [209–211].
Due to its central role in fatty acid utilization, the car-
nitine system is a determinant in insulin regulation of fat
and glucose metabolic rate. Insulin resistance has been
described as a ‘marker of early mitochondrial carnitine-
dependent dysfunction’ [212]. For example, increased lipid
accumulation in muscle leads to prolonged inhibition of
muscle isoform of carnitine palmitoyltransferase I enzyme
(M-CPT I). This insulin-regulated enzyme facilitates bidi-
rectional glucose and fatty-acid carbon flux, which conse-
quently promotes insulin resistance [212]. Abnormalities in
M-CPT I expression have also been reported in metabolic
disorders (e.g., metabolic syndrome, diabetes mellitus
[186, 213]. In healthy subjects, L-carnitine infusion in the
presence of hyperinsulinemia increases OCTN2 mRNA
expression and total skeletal muscle carnitine content
[214]. In streptozotocin (STZ)-induced diabetic rats, car-
nitines show to modulate insulin-like growth factor axis
[215]. Importantly, tissue export of acetylcarnitine lower
local concentration of acetylCoA, which in turn, stimulates
PDH activity and this basic mechanism contributes to
enhance glucose oxidation [186]. Consistently, carnitine
administration to obese rats not only restores tissue carni-
tine content but also enhances glucose disposal and mito-
chondrial performance [186]. This highlights the crucial
role of carnitine system in regulating mitochondrial
emergetics, in particular CAT enzyme whose overexpres-
sion has been proven to promote glucose uptake and mit-
igate lipid-induced suppression of glucose oxidation,
counteracting the Randle effects ‘‘in vivo’’ (i.e., lipid-
induced lowering of glucose oxidation) [216].
Carnitine, exogenously administered, attenuates organic
aciduria induced by alcohol given chronically to rats as
well s prevents neuronal injury in the hippocampus by
inhibiting the increase in oxidized glutathione and mito-
chondrial dysfunction [184, 185]. Studies in our laboratory
have shown that ALC reduces Ab toxicity in primary
cortical neuronal cultures by increasing both HO-1 and
Hsp70 expression [217]. In rats chronic ALC treatment
increases life-span, improves cognitive behaviour and
long-term memory performance [202]. Furthermore,
chronic ALC treatment has been proven to prevent age-
related changes in mitochondrial respiration and decrease
Neurochem Res (2010) 35:1880–1915
oxidative stress biomarkers through the up-regulation of
vitagene products [2, 3, 35, 41, 201, 204]. Consistently, we
have demonstrated that acetyl-L-carnitine induces HO-1 in
a dose and time dependent manner, an effect associated
with up-regulation of other Hsps and high expression of the
redox-sensitive transcription factor Nrf2, a mechanism
following the hormetic law-dose response [3, 201]. These
evidence, intriguingly related with modulation of mito-
chondrial biogenesis via the transcriptional control of the
nuclear respiratory factor-1 (NRF-1) [97, 99], highlight the
crucial role of the carnitine system and the clinical rele-
vance of its insufficiency in mitochondrial energetics and
metabolic disruptions associated with aging, overnutrition
and neurodegenerative disorders.
Conclusions and Future Directions
While the anatomical paradigm of medicine and the
Mendelian paradigm of genetics have been powerful pre-
dictors of medical relationships for the past century, they
are failing to direct us toward solutions for the common
age-related diseases. Continually increasing resources are
being expended to combat the age-related diseases that
include diabetes and metabolic syndrome, Alzheimer’s
disease, Parkinson’s disease, cardiovascular disease, and
cancer. Yet the causes of these diseases remain an open
question, while their incidence and morbidity either remain
constant or increase [218]. Huge investments in biomedical
research in the recent past have resulted in some important
accomplishments, such as sequencing of the human gen-
ome, identification of thousands of human chromosomal
single nucleotide polymorphisms (SNPs), or regional
clusters of chromosomal SNPs. However, these accom-
plishments have failed to reveal the genetic causes for the
common age-related diseases [67]. When a current para-
digm fails to make productive predictions, then hypothesis-
based research begins to fail. To resolve the crisis and
return to productive ‘‘normal science,’’ a new paradigm
must be generated that comprise the potential of the pre-
vious founding paradigm whereby adding new elements
that address the current problems being compared and
possibly overcome [219].
Life involves the dynamic interplay between structure
and energy. For the eukaryotic cell, this duality was
solidified approximatelyt 2 billion years ago by the sym-
biosis of what appears to have been a glycolytic motile cell,
which gave rise to the nucleus–cytosol, and an oxidative a-
proteobacterium, which evolved into the mitochondrion
[67]. Initially, each organism was free living and contained
all of the genes for an independent life form. However,
over the subsequent 1.2 billion years, the single-cell
descendants of the initial symbiosis went through
1909
subsequent alternative arrangements of genomic reorgani-
zation and biochemical traduction. An ultimative arrange-
ment was then reached in which mitochondria became
specialized in energy production and the nucleus–cytosol
became specialized in structure. This final design prompted
the development of multicellularity and the evolution of
higher plants and animals, including humans [220]. In this
reorganization, the transfer of virtually all of the genes of
the mitochondrial genome into the chromosomal nDNA
was finalized. In spite of this, mtDNA persisted, today still
retaining 13 polypeptide-encoding genes plus a small and
large rRNA gene and 22 tRNA genes. All of the mtD-
NAencoded polypeptides are core subunits of the enzyme
complexes of the mitochondrial energy-generating appa-
ratus, oxidative phosphorylation (OXPHOS). The 13
polypeptides of the mtDNA include 7 of the 45 polypep-
tides of complex I (ND1, -2, -3, -4L, -4, -5, -6), 1 of the
11 polypeptides of complex III (cytochrome b), 3 of the 13
polypeptides of complex IV (COI, -II, -III), and 2 of the 15
polypeptides of complex V (ATP6 and -8). All of the other
genes of the mitochondrial genome are dispersed across the
chromosomes and include the mitochondrial DNA poly-
merase g (POLG), RNA polymerase, ribosomal proteins or
metabolic enzymes. Why it was beneficial for the first
1,500 mitochondrial genes to be transferred to the nucleus,
and not for the last 13, still remains an intriguing unraveled
question [67, 220, 221].
Mitochondria are major cellular sources (and targets) of
free radicals, play key roles in the regulation of calcium
homeostasis, and are effectors of the intrinsic apoptotic
pathway due to the release of signaling molecules that
activate and trigger specific caspase cascades. Mitochon-
drial dysfunction is inherent in a variety of human disor-
ders from the classical mitochondrial diseases arising from
mitochondrial DNA mutations (encephalomyopathies) to
those involving mitochondrial signaling pathways to the
rest of the cell, modulated by organellar dynamics and
culminating in programmed cell death. The role of mito-
chondria in normal aging has been the focus of extensive
research in the last decades and being complementary to
and maturing from the free radical theory of aging, to the
oxidative stress theory of aging, to the mitochondrial oxi-
dative stress theory of aging, and today addressing the tight
co-regulation of mitochondrial energy and redox signaling.
In this scenario, it appears conceivably the function of the
carnitine system as a prototypical vitagene operating at the
functional interface of energy distribution between ances-
tral mechanisms of cell proliferation and differentiation
and homeostatic mitochondrial dependent processes of cell
survival which require energy for cellular stress response
and redox homeostasis [41]. Very importantly, this new
envisioned role exploited for the carnitine system, partic-
ularly, acetylcarnitine as a molecule endowed with the
123
1910
capability of potentiating the cellular stress response
pathways appear to be a promising alternative therapeutic
approach for those pathophysiological conditions, such as
neurodegeneration or cancer, where hormetic stimulation
of the vitagene pathway is strongly warranted.
Acknowledgments Work from the authors’ laboratories was sup-
ported by grants from MIUR, FIRB RBRN07BMCT, I.N.B.B., and by
‘‘Fondi Ateneo’’ 2008 and 2009. We are very much honoured to
contribute to this Special Issue in honour of Abel Lajtha. Everybody
in the field of Neurochemistry knows Abel Lajtha and his great
contribution to the advancement of research in Neurochemistry. He
has been the Editor in Chief of the Journal ‘‘Neurochemical
Research’’ for so many years holding very stimulating and interesting
Meetings of the Editorial Board to which we are very proud to have
participated. It is difficult to imagine future Editorial Board Meetings
without him. We would like to thank him very much for his great
contribution to the advancement and success of Neurochemical
Research and ask him to continue helping us with its precious efforts
and advices.
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