The role of mitochondria in brain aging and the effects of melatonin.

Abstract

Melatonin is an endogenous indoleamine present in different tissues, cellular compartments and organelles including mitochondria. When melatonin is administered orally, it is readily available to the brain where it counteracts different processes that occur during aging and age-related neurodegenerative disorders. These aging processes include oxidative stress and oxidative damage, chronic and acute inflammation, mitochondrial dysfunction and loss of neural regeneration. This review summarizes age related changes in the brain and the importance of oxidative/nitrosative stress and mitochondrial dysfunction in brain aging. The data and mechanisms of action of melatonin in relation to aging of the brain are reviewed as well.

Full text

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182 Current Neuropharmacology, 2010, 8, 182-193

1570-159X/10 $55.00+.00 ©2010 Bentham Science Publishers Ltd.
The Role of Mitochondria in Brain Aging and the Effects of Melatonin
Germaine Escames1,2, Ana López1,2, José Antonio García1,2, Laura García1,2,
Darío Acuña-Castroviejo1,2,3, José Joaquín García4 and Luis Carlos López1,2,*
1Centro de Investigación Biomédica, Instituto de Biotecnología, Universidad de Granada, Granada, Spain;
2Departamento de Fisiología, Facultad de Medicina, Universidad de Granada, Granada, Spain; 3Servicio de Análisis
Clínicos, Hospital Universitario San Cecilio, Granada, Spain; 4Departamento de Farmacología y Fisiología, Facultad
de Medicina, Zaragoza, Spain
Abstract: Melatonin is an endogenous indoleamine present in different tissues, cellular compartments and organelles
including mitochondria. When melatonin is administered orally, it is readily available to the brain where it counteracts
different processes that occur during aging and age-related neurodegenerative disorders. These aging processes include
oxidative stress and oxidative damage, chronic and acute inflammation, mitochondrial dysfunction and loss of neural
regeneration. This review summarizes age related changes in the brain and the importance of oxidative/nitrosative stress
and mitochondrial dysfunction in brain aging. The data and mechanisms of action of melatonin in relation to aging of the
brain are reviewed as well.
Keywords: Melatonin, mitochondria, oxidative stress, brain, aging, neurodegenerative diseases, neural stem cells.
INTRODUCTION: MELATONIN SYNTHESIS AND
ITS ACTION´S MECHANISMS

doleamine derived from tryptophan. It is present in bacteria,
eukaryotes, plants and all phyla of muticellular animals.
Because of its ancient origin, it is thought that melatonin
posseses numerous functions acquired throughout evolution
[59].
Melatonin is an ancient and highly conserved in-

steps. First, tryptophan hydroxylase catalyzes the conversion
of tryptophan to 5-hydroxytryptophan, which is then decar-
boxylated by aromatic amino acid decarboxylase to produce
serotonin. Therefore, the enzyme arylalkylamine N-acetyl-
transferase (AANAT) converts serotonin to N-acetylserotonin,
which is then O-methylated by the action of the hydroxyindole-
O-methyltransferase (HIOMT) to produce melatonin [47].
In mammals, melatonin is synthesized by the pineal gland
in a circadian manner and it is released into blood and
into the cerebrospinal fluid (CSF). In the blood melatonin
can concentrate up to 0.5 nM [47, 132].
The biosynthesis of melatonin requires four enzymatic

(10–15-fold increase) during the night than during the
daytime. This circadian rhythm is present in all living
organisms, from unicellular to multicellular organisms in-
cluding humans. In vertebrates, the rhythm is generated by a
biological clock situated in the suprachiasmatic nucleus
(SCN) of the hypothalamus, and synchronized to 24 h pri-
marily by the light–dark cycle acting via the SCN [79]. In
humans, these rhythms are developed during the first months

Blood melatonin concentrations exhibit higher values
*Address correspondence to this author at the Centro de Investigación
Biomédica, Parque Tecnológico de Ciencias de la Salud, Universidad de
Granada, Avenida del Conocimiento, s/n, E-18100 Armilla, Granada, Spain;
Tel: +34-958-241000 ext; 20198; Fax: +34-958-819132;
E-mail: luisca@ugr.es
of life and reach the greatest magnitude between the 4 and
7th year of age. At puberty, there is a drop in melatonin con-
centrations, and thereafter plasma concentrations diminish
gradually. After that, melatonin production in the pineal
gland declines progressively with age [79]. As a consequence,
in many elderly individuals the day–night differences in
melatonin secretion are almost absent. Therefore, in old
animals and elderly humans the levels of melatonin available
to the organism are a small proportion of that of young
individuals [133].

gland, melatonin is also generated in many tissues and
organs of the body, and this extrapineal production of
melatonin is much more greater than that produced by the
pineal [152]. The expression of genes for the key enzymes
for melatonin synthesis, AANAT and HIOMT has been
found in many organs [146]. Interestingly, melatonin is con-
centrated by subcellular compartments including nucleus and
mitochondria, the latter showing 100-200 times more mela-
tonin than cytosol [5, 95].
In addition to the melatonin produced by the pineal

different indoleamine levels in different tissues, cell types
and subcellular compartments and presumably related to the
specific actions of the indoleamine in these tissues. Some
of these actions are especially remarkable for the potential
benefits in the brain, especially during the physiological
aging and in pathophysiological age-related disorders: 1) In
contrast to numerous synthetic and natural drugs, melatonin
is readily administrated orally and it is readily available
to the brain [88], 2) numerous in vitro and in vivo studies
have demonstrated the capacity of melatonin to counteract
oxidative stress and oxidative damage [7, 134, 135, 136], 3)
melatonin reduces chronic and acute inflammation in the
brain [6], and 4) melatonin plays an important role in
the mitochondrial homeostasis, especially in response to mi-
tochondrial damage [7].
The multiple sources of melatonin are associated to

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The Role of Mitochondria in Brain Aging and the Effects of Melatonin Current Neuropharmacology, 2010, Vol. 8, No. 3 183
MITOCHONDRIA,
BRAIN
MELATONIN AND AGING

demand and it possess highly active mitochondria metabolism
with high oxygen utilization (20% of the total oxygen
inspired). Consequently, reactive oxygen species (ROS)
generation in the brain is intense. In addition, the brain is
very susceptible to free radical damage because of its high
concentrations of polyunsaturated fatty acids [48] and transi-
tion metals such as iron, which is involved in the generation
of the hydroxyl radical [65] , moreover, the brain contains
low concentrations of cytosolic antioxidants [40, 134].
Remarkably, it has been shown that the cognitive functions,
motor ability, exploratory capacity and neuromuscular
coordination in mice are decreased upon aging in parallel
with an increase of protein oxidation and a reduction in
mitochondrial complex activities in the brain of these
animals [49, 114].
The mammalian brain is a tissue with high energy

between aging, increased oxidative damage and mitochondrial
dysfunction. Oxidative damage to mitochondria influences
different structural and functional components of mitochondrial
DNA (mtDNA), proteins and membrane lipids. Three factors
make mtDNA particularly vulnerable to reactive species:
mtDNA is located close to the mitochondrial inner mem-
brane, near the generation of ROS; mtDNA is not
extensively condensed and protected by histones; mtDNA
repair is limited [26], the expression of the entire mtDNA is
essential for the maintenance of mitochondrial bioenergetic
function, while only about 7% of the nuclear genome is
expresses during cell differentiation [109].
Several lines of evidences have shown the relation

humans in a wide variety of aged tissues including the
brain, and the correlation of the rise in mtDNA deletions and
mitochondrial respiratory chain malfunction during aging
has been amply demonstrated [14, 29, 86, 162]. The global
deletion levels rarely exceed 1% and, therefore, the contribu-
tion of mtDNA deletions to the aging process appear to be
unlikely. However, the deletions levels in particular areas
and cell types are still unknown. On the other hand, the
mechanisms of mtDNA deletions during aging are still
controversial but oxidative damage to DNA associated with
single or double-stranded breaks has been proposed. This
idea has been supported for several studies: the relative
amount of mtDNA deletions correlates with the levels of 8-
hydroxy-2'-deoxyguanosine (8-OHdG) [62], and treatment of
human skin fibroblasts with sub-lethal dose of oxidative sub-
stances and environmental insult inducers of ROS results in
the formation and accumulation of the 4977 bp deletion in
mtDNA [16, 41].
Age-related mtDNA deletions have been detected in

protein-coding genes and D-Loop have been also found
to accumulate in some post-mitotic tissues during human
aging [36, 108, 160]. However, it seems that the proportion
of mutant mtDNAs is too low to cause a significant impact
on mitochondrial function in aging tissues. On the other
hand, the distribution of the mutant mtDNA in cells and
tissues is still unknown and clarification of this matter
could resolve important questions regarding the importance of
mtDNA point mutations and duplications in tRNA,
mtDNA mutations in aging [99]. Additionally, mitochondria
polymerase ? (POLG) deficient mice accumulate high
levels of mtDNA mutations and deletions resulting in a
premature aging phenotype with COX deficient cells in the
brain and heart but without an increase of ROS generation
and oxidative damage [87, 156, 159]. Thus, these results
support the idea of a direct involvement of mtDNA
mutations in aging but cast doubt on the vicious cycle
theories of aging and oxidative stress [93].

account the lack of oxidative stress in POLG deficient mice:
aging is the result of alterations in many pathways and,
therefore, the aging phenotype in the mutator mice should
not be compared to normal aging; alterations in POLG
may be downstream from mechanisms that generate ROS;
and extensive and equally distributed mtDNA mutations
(in contrast to the mosaic distribution of mtDNA mutations
observed in normal aging) could prevent the generation of
ROS [93]. In contrast to POLG mutant mice, skin fibroblasts
harboring mtDNA point mutations associated with aging
show an alteration in the expression profile of antioxidant
enzymes [161].
Some explanations have been proposed, however, to

mitochondrial protein oxidation has been also reported in a
variety of organisms during aging [99]. The proteins containing
Fe-S clusters seem to be the most susceptible to oxidation
[99]. Several reports have revealed that oxidation of
aconitase, adenine nucleotide translocase and mitochondrial
respiratory chain complexes may occur during aging and
consequently the activities of these enzymes diminish with
increasing age [9, 168, 169]. Oxidative injury is not limited
to mtDNA or proteins but also to mitochondrial membranes.
This may lead to a progressive lipid peroxidation (LPO)
and cross linking damage, with simultaneous changes in
the respiration rate, ATP synthesis, membrane fluidity and
permeability, Ca2+ homeostasis and apoptosis [3].
In addition to the research related to mtDNA alterations,

dant enzymes in response to the oxidative environment in the
aging cells has been found in human blood [10, 33, 57] and
muscle [102, 121], and in a variety of tissues from rats and
mice, including skeletal muscles, brain and heart, which are
tissues with high energy demand. Additional information
about the physiological changes in mammalian aging has
been uncovered by studies performed in the senescence-
accelerated mouse (SAM) [150]. SAM includes two strains,
one prone to accelerated senescence (SAMP) and one resis-
tant to accelerated senescence (SAMR). SAMP8, a sub-strain
of SAMP, shows relatively strain specific age-associated
phenotypic pathologies such as a shortened life span
and early manifestation of senescence (including loss of
activity, alopecia, lack of hair glossiness, skin coarseness,
periphthalmic lesions, increased lordokyphosis and systemic
senile amyloidosis), similar to several geriatric disorders
observed in humans [148, 150]. SAMP8 mice show a general
hyperoxidative status manifested by increased mitochondrial
electron leakage and ROS production, elevated LPO and
protein carbonyl content, changes in the antioxidant enzymes
activities and increase of GSSG:GSH ratio [23, 68, 92, 105,
117, 149, 171]. The results are a loss in the mitochondrial
respiratory chain activity, ATP synthesis and energy status of
Alterations in the expression and activities of the antioxi-

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184 Current Neuropharmacology, 2010, Vol. 8, No. 3
Escames et al.
the organism, suggesting that the mechanism of senescence
acceleration in SAMP8 mice is related to free radical damage
[138,139]. SAMP8 mice also show an age-dependent
increase in IFN-? and TNF-?, a reduction in IL-2 levels
and an rise in nitric oxide (NO•) levels [140], suggesting
the existence of an inflammatory process during aging.
The increase of NO• levels is particularly relevant since
this molecule reacts with (O2
peroxynitrite anion [126], which irreversible impairs the
mitochondrial respiratory chain and decreases the efficiency
of oxidative phosphorylation, leading to energy depletion
and cell death [20, 25].
._) in mitochondria yielding

scavenges free radicals, stimulates other antioxidant systems
of the cells and inhibits the expression of iNOS with the
subsequent reduction in the levels of both ROS and RNS [3,
6, 8]. In recent studies, Jou and colleagues [73, 74] demon-
strated that cybrids with 80% common deletion in mtDNA
showed an increase in oxidative stress and an rise in suscep-
tibility to a secondary oxidative stress induced by H2O2 ex-
posure. Interestingly, melatonin reduced ROS generation in
both conditions preventing ROS-mediated depolarization of
mitochondrial membrane potential and subsequent opening
of the mitochondrial permeability transition pore (MPTP)
and cytochrome c release [74]. The protection provided by
melatonin was superior to other antioxidants such us vitamin
E and mitochondria targeted coenzyme Q (MitoQ) [74].
Numerous studies have shown that melatonin directly

damage and mitochondrial dysfunction have been also tested
in the brain of mice with accelerated aging, SAMP8 [24].
Chronic melatonin administration in the drinking water
for 9 months (10 mg/kg b.w.) completely prevented the
mitochondrial impairment maintaining or even increasing
ATP production. Likewise, melatonin prevented the rise in
mitochondrial LPO and increased GPx and GRd activities
normalizing the GSSG/GSH ratio [24]. Melatonin also
counteracted the oxidative damage to the mitochondrial
membranes of the brain during aging since it prevented rigidity
in the mitochondrial membrane and preserved the content
and structural integrity of cardiolipin molecules [54, 125].
Immune function in the brain is other important factor in
aging. Nitrite levels accurately reflect the nitrosative stress
status that is caused by inflammation. Importantly,
age-dependent nitrosative status in brain mitochondria was
prevented by melatonin administration [24]. The effect of
melatonin in the reduction of nitrosative stress has been
amply studied in animal models of sepsis. The administration
of pharmacological doses of melatonin in rodents with sepsis
induced by lipopolysacharide injection or cecal ligation and
puncture (CLP) produced a decrease in the expression and
activity of iNOS, and consequently nitrite levels, nitrosative/
oxidative stress and mitochondrial function were normalized
[42, 43, 45, 96, 97]. Interestingly, the increase of iNOS
expression was more pronounced in aged rats (18 m.o.) than
young rats (3 m.o.), but melatonin was able to reduce the
expression to the same levels in both groups [42, 44].
The effects of melatonin in counteracting oxidative
MITOCHONDRIA, MELATONIN AND NEURODE-
GENERATIVE DISORDERS

diseases of different etiologies that may share mitochondrial
Aged-related disorders include neurodegenerative
dysfunction, oxidative/nitrosative stress and apoptosis in
particular brain areas as a final common pathway. Conse-
quently, neuronal loss may be associated to mitochondrial
dysfunction in those disorders. Alzheimer disease (AD) is a
predominantly sporadic late-onset disorder characterized by
progressive dementia with a relatively long course. Progressive
neuronal loss, particularly in the cortex and the hippocampus,
is typically observed in the brain of Alzheimer patients. The
two main histopathological features of AD are the accumulation
of extracellular neuritic plaques, mainly represented by
?-amyloid (A?), and of neurofibrillary tangles, mainly
represented by the hyper-phosphorylated forms of the
microtubule-associated protein tau [56]. Additionally, some
evidence indicates that mitochondria are involved in the
pathology of AD, including a reduction in brain energy
metabolism shown by positron emission tomography [11],
defects of mitochondrial metabolic enzymes [104, 144],
mitochondrial respiratory chain complex deficiencies [19, 85]
and an increase of mtDNA deletion level in the substantia
nigra neurons [15]. On the contrary, a recent review found
little evidence in support a role of mtDNA mutations in the
development of AD [69].

in a metal-catalyzed reaction, which induces neuronal
cell death in a ROS-mediated process resulting in damage
to neuronal membrane lipids, proteins and nucleic acids.
This suggests that the use of antioxidants such as vitamin E,
melatonin or estrogens may be beneficial in AD [90, 110].
In AD patients, melatonin levels are reduced in blood
and CSF and that reduction seems to go in parallel to the
progression of AD neuropathology [165, 174]. Moreover,
CSF melatonin levels are already decreased in pre-clinical
AD individuals [165].
It has been shown that ?-amyloid peptide generates ROS

to reduce the neurodegenerative manifestations in AD [123].
When neuroblastoma cells were incubated with A?, more
than 80% of the neurons died due to apoptosis, but the
presence of melatonin reduced cellular death and DNA
damage in a dose-related manner [124]. In human platelets,
melatonin also protected against A?-induced damage [21,
122]. Recently, melatonin treatment has been tested in the
APP + PS1 double transgenic (Tg) mouse, which is considered
a mouse model with characteristics of the neuropathology
of AD [119]. Melatonin administred in the drinking water
(100 mg/L water) for four months protected AD mice from
cognitive impairment in a variety of tasks of working
memory, spatial reference learning/memory, and basic
mnemonic function. Immunoreactive A? deposition was
significantly reduced in hippocampus (43%) and entorhinal
cortex (37%) of melatonin-treated AD mice. The levels
of tumor necrosis factor (TNF)-alpha were reduced in the
hippocampus of AD mice treated with melatonin, as well as
the cortical mRNA expression of SOD-1, GPx and catalase.
Taken together, the results suggest that melatonin's cognitive
benefits could involve its anti-A? aggregation, anti-
inflammatory, and/or antioxidant properties [119]. In AD
patients, melatonin stabilizes cognitive function over a 2–
3 year period [21, 22]. An additional retrospective study
reported that individuals with mild cognitive impairment
given melatonin for sleep enhancement also showed
The administration of melatonin has been tested in order

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The Role of Mitochondria in Brain Aging and the Effects of Melatonin Current Neuropharmacology, 2010, Vol. 8, No. 3 185
significantly better cognitive performance in two widely
utilized cognitive assessment tests [52].

disorder characterized by bradykinesia, rigidity and tremor.
PD is accompanied by the loss of about 60% of dopaminergic
neurons of the substantia nigra pars compacta. Mitochondrial
involvement in PD is suggested by deficiencies of complex I
(C-I) in substantia nigra [142], with a parallel reduction
in GSH levels, suggesting the existence of oxidative stress.
In platelets of PD patients, C-I is also decreased and in
some cases is accompanied by complex II (C-II), complex III
(C-III) and complex IV (C-IV) deficiencies.
Parkinson disease (PD) is a mainly sporadic late-onset

genetically supported by the finding of POLG mutations in
early-onset Parkinsonism in different families [32, 98]. In
some cases, the POLG mutations were accompanied by mtDNA
deletions, ragged-red and cytochrome c oxidase-negative
fibers and low activities of mitochondrial complexes containing
mitochondrial DNA-encoded subunits [32, 98]. Additionally,
analysis of single substantia nigra neurons from individuals
with PD and age-mathed controls showed an age-related
accumulation of high levels of mtDNA deletions [14]. On
the contrary, a recent review of the evidence for primary
mtDNA mutations in PD led to the conclusion that there is
no convincing proof for a primary role of mtDNA mutations
in this neurodegenerative disorder [69].
Mitochondrial involvement in the pathology of PD has been

SNCA, LRRK2 and HTRA2) are recognized to be associated
with the familial form of PD and the proteins encoded by
these genes interact directly or indirectly with mitochondria
and seem to be involved in apoptosis [35]. Moreover, some
environmental toxins seem to interact with the products of
these genes, which provoke oxidative damage, mitochondrial
dysfunction and cell death [2]. These environmental toxins
influence PD, as shown by the C-I inhibitory effects of 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone
and paraquat. The C-I inhibition is prevented by free radical
scavengers indicating oxidative damage to C-I. Moreover,
MPTP also stimulates NMDA-dependent nNOS activity
thereby increasing NO• production [155], and decreasing
the content of mtDNA [111]. In mouse models of PD
induced by MPTP melatonin administration normalized
complex I activity and oxidative status in mitochondria from
substantia nigra and striatum.
A series of nuclear genes (PARK2, PARK7, PINK1,

recently shown that this indole reduced the activity of the
mitochondrial iNOS (i-mtNOS), thus decreasing mitochondrial
NO• levels and preventing the respiratory inhibition
produced by NO• at the level of complex IV [153]. Melatonin
also protects against excitotoxicity by reducing the autoxidation
of dopamine (DA) which occurs in PD [80]. These effects
were demonstrated in MPTP-induced PD in mice [4, 120]
and in PC12 cells incubated with 6-hydroxydopamine [106].
Melatonin also abrogated cell death induced by cysteamine
pretreatment of the PC12 cells; cystamine treatment involves
mitochondrial iron sequestration [50].
Looking for the targets of melatonin action, it was

in subcortical astrocytes may facilitate the bioactivation
of DA to neurotoxic free radical intermediates and thereby
The age-associated accumulation of redox-active iron
predispose the nervous system to PD and other neurode-
generative diseases. In rats injected with kainic acid to
produce excitotoxicity-induced
melatonin significantly attenuated apoptosis, an effect linked
to the reduction in oxidative damage and an increased
GSH content [27]. In a spontaneous, age-induced model of
apoptosis using cerebellar granule cells, it was shown that
melatonin and Ca2+-channel blockers such as amlodipine,
inhibited spontaneous apoptosis [103]. This antagonism
between melatonin and Ca2+-channels was also demonstrated
in electrophysiological and binding experiments [46]. Striatal
neurons growing in low density culture in serum-free
medium and in the absence of glia die within 3 days by
apoptosis. The presence of melatonin rescues striatal neurons
from impending cell death, which has important conse-
quences in neurodegenerative diseases involving nigrostriatal
pathway such as in PD [71].
apoptotic cell death,

disorder characterized by ataxia, chorea and dementia. It is
known to be caused by an alteration in a gene for nDNA
encoding huntingtin, a widely expressed protein of unknown
function but associated with inappropriate apoptosis. The
pathology of HD involves mainly the GABA-containing
neurons of the caudate nucleus and putamen [142]. Excito-
toxicity has been suggested to play an important role in this
disease. This includes activation of NMDA-dependent
neuronal nitric oxide synthase (nNOS) and NO• production.
NO• and particularly peroxynitrite mediate oxidative damage.
Increases in mtDNA copy number and multiple mtDNA
depletions have been found in HD patients, which are
especially common in the frontal and temporal lobes of the
cerebral cortex, although its significance is unclear [67, 130].
These mtDNA alterations could be a consequence of the
increase of the oxidative damage to DNA reflected by an
elevation of the levels of 8-hydroxydeoxyguanosine [130].
There are also deficiencies in the activities of C-II, C-III and
C-IV in caudate and in a lesser extend in putamen in HD.
Huntington’s disease (HD) is a neurodegenerative

in cells and animals models of HD, which include calcium
dyshomeostasis and anomalous mitochondrial dynamics
[130]. Melatonin treatment has been used in a rat model
of Huntington disease induced by 3-nitropropionic acid,
a mycotoxin that inhibits the mitochondrial succinate
dehydrogenase or complex II [157]. The inhibition of complex
II by 3-nitropropionic acid was accompanied by rises in LPO
and protein carbonyl content, and a decrease in SOD activity
in the brain cortex and striatum. Melatonin administered
intraperitoneally (1 mg/kg b.w./day) for 8 days prevented the
deleterious effects induced by the acid [157].
Others mitochondrial abnormalities have been found

sporadic disorder clinically characterized by progressive
muscle weakness, atrophy, spasticity widespread paralysis
and premature death. The disease is caused by the degeneration
and death of upper and lower motor neurons in the cortex,
brainstem, and spinal cord [64]. About 5%–10% of patients
have a familial form of ALS (FALS), and about 20% of
these harbor mutations in the SOD1 gene that encodes the
Cu,Zn-superoxide dismutase 1 (SOD1) [64]. Mouse models
overexpressing mutant SOD1 also develop motor neuron
degeneration. Most pathogenic mutations do not impair
Amyotrophic lateral sclerosis (ALS) is a late-onset

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186 Current Neuropharmacology, 2010, Vol. 8, No. 3
Escames et al.
SOD1 activity, and some studies have proved that a portion
of mutant SOD1 is localized in mitochondria, both in FALS
patients and in the animal models. As a result, it has been
hypothesized that mutant SOD1 may damage mitochondria
by some misunderstood mechanism [64].
A recent investigation has used melatonin in cellular and
mouse models of ALS, as well as 31 ALS patients [163].
First, NSC-34 cells, a widely used motoneuron-neuroblastoma
fusion line, were exposed to 2 or 10 mM of glutamate
for 3 days. A mortality of 29.2% and 52.1% of cells were
detected, respectively; treatment with 50 mM melatonin
recovered the survival by 17.2 % (2 mM glutamate) and
8.5 % (10 mM glutamate). Melatonin reduced the elevated
ROS production in this cellular model. Second, in
the SOD1G93A-transgenic mouse (an ALS mouse model),
melatonin was administrated in the drinking water (0.5
mg/ml water) at 28 days old, resulting in a delayed disease
progression and extended survival; third, daily doses of 300
mg melatonin were administrated in ALS patients as novel
suppositories at bedtime. Melatonin treatment decreased
the serum protein carbonyls compared with the elevated
levels presented in the serum of the matched untreated
ALS patients [163].
NEURAL STEM CELLS IN AGING AND AGED-
RELATED DISORDERS: THE IMPORTANCE OF
MITOCHONDRIA AND OXIDATIVE STRESS

embryonic stem cells (ESC) that have the potential to
differentiate into any cell type in the organism; multipotent
cells derived from adult tissue including umbilical cord
blood and amniotic fluid, which can differentiate into a
limited number of cells types of their own lineage, e.g.,
mesoderm; and precursor cells, which are adult stem cells
committed to differentiation. In the brain, both neural
stem cells and neural progenitor cells are responsible for
neurogenesis. Neural stem cells produce additional stem
cells as well as offspring that go on to differentiate into
oligodendrocytes, other glial cells and neurons. Neural
progenitor cells have a limited replicative potential that
are committed to the neuronal lineage [175]. The vast
majority of cells in the adult central nervous system (CNS)
are generated during the embryonic and early postnatal
period, but some proportion of neurogenesis is also occurs
during adulthood. The functional significance of the adult
neurogenesis is not fully understood but it has been shown
to be involved in several brain function and pathologies
[38, 175]. Neurogenesis in the adult brain from mammals
is concentrated in the subventricular zone (SVZ) of the lat-
eral ventricule wall and the subgranular zone (SGZ) of the
dentate gyrus of the hippocampus [175]. New cells generated
from these regions migrate toward their final destinations,
where they differentiate into mature cells and are integrated
into the CNS [76, 175].
Stem cells can be classified in three types: pluripotent

and SGZ, which are associated with a decline of neurogenesis
during aging [38, 175]. These changes may affect the
different steps on neurogenesis: 1) proliferation of new cells;
2) survival of newly born cells; 3) migration of these cells
Age-related changes have been observed in both SVZ
toward target areas; and 4) differentiation into mature
functional cells.

in rodents by use of cell proliferation markers such as
bromo-deoxyuridine (BrdU) and tritiated thymidine [76,
175]. Most results have shown that cell proliferation declines
during aging in both SVZ and SGZ [76, 175]. However, the
difference in the proliferation decline is not clear between
middle age and senescence since some studies have reported
significant changes in both ages groups [18, 129] but others
did not [39, 128].
Neural stem cell proliferation has been widely studied

the newborn cells seems to be unaffected by aging [38].
However, when some brain areas are damaged, the survivals
of newborn cells show clear differences between young
and old animals. In one study of stroke simulation, rats
subjected to ischemia showed an induction of neural stem
cell proliferation in the SGZ. One day after the ischemia
induction young adults exhibited a 5.7 fold increase in BrdU
labeled cells compared to a 10.6-fold increase in old adults.
Remarkably, 65.5% of the labeled cells survived 28 days
after ischemia in young adults, whereas in old adult brains
only 15.3% remained [167].
On the contrary, the short-term survival pattern in

that when hippocampi was first damaged by the action
of kianic acid, a specific agonist for kainite receptors with
neuroexcitotoxic and epileptogenic properties, the survival of
rat fetal hippocampal cells injected into the ventricles of rats
was 30% in middle-aged and old brains compared with the
72 % survival in young brains. Both studies suggested that
changes in the environment of the brain areas during aging
may be critical for the survival of new cells [167, 173]. Thus,
the increases of glucocorticoids and the decreases of the
levels of Insulin-like growth factor 1 (IGF-1) in aged brain
have been associated to a decrease in neurogenesis [1, 91,
115].
In other study, Zaman and Shetty [173] observed

increased in the rat hippocampus during aging [116], also
may play an important role in neural and progenitor stem
cell survival. It has been shown that curcumin, an antioxidant
and anti-inflammatory agent, modulates the proliferation of
embryonic neural progenitor cells with a biphasic effect in
cultured cells. In mouse brain, curcumin administration
resulted in a significant rise in the number of newly-generated
cells in the SGZ of hippocampus [82]. Oxidative stress
disrupts the differentiation of oligodendrocyte precursors
and neural progenitor cells into mature oligodendrocyte and
neurons [51, 143]. On the contrary, the increase of the anti-
oxidant capacity protects neural progenitor cells in vitro and
potentiates the formation of cellular networks that
provide neuroprotection in vivo [100, 143].
Interestingly, oxidative stress, which is particularly

and an increase in the resistance of neurons to dysfunction
and apoptosis have been detected in rodents undergoing
dietary restriction. Both oxidative stress and dietary
restriction are interlinked and related to mitochondrial
function. However, few studies have focused in the role
of mitochondria in proliferation, survival and differentiation
An increase in newly generated neural cells in adult brain

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The Role of Mitochondria in Brain Aging and the Effects of Melatonin Current Neuropharmacology, 2010, Vol. 8, No. 3 187
of neural stem cells, and most of them are related with
the mitochondrial apoptotic pathway induced by some toxic
agent [77, 78,151, 154].

(ESC) have focused on the activity of mitochondrial genome
and of these data could be extrapolated to neural stem cells.
Transmitochondrial embryonic stem cells harboring patho-
genic mtDNA mutations have shown to be compromised in
neuronal differentiation when the mitochondrial respiratory
chain function is severely affected [84]. Thus, some authors
have hypothesized that stem cell competence may be verified
using functional mitochondrial characteristics [94]. Differen-
tiation of mouse and human embryonic stem cells (ESC)
results in changes in mitochondrial structure, morphology
and pattern of cytoplasmic localization. Mitochondria
in stem cells tend to localize perinuclearly [94]. Moreover,
ESC have relatively few mitochondria with poorly developed
cristae [28, 118], and restricted oxidative capacity. As cells
are allowed to differentiate, the number of mtDNA copies
increase and these differentiated cells contain elevated
numbers of mitochondria with distinct cristae, dense
matrices and high membrane potentials. These features
suggest the initiation of metabolic activity through oxidative
phosphorylation (OXPHOS) [145]. Because ESC display
low oxygen consumption and thus, poor OXPHOS, an
elevation in ATP content per cell may therefore reflect a
loss of stemness and the subsequent onset of differentiation
[28, 94]. Therefore, preservation of immature mitochondria
with a perinuclear arrangement, reduced expression of
OXPHOS enzymes and low metabolic activity in ESC has
led to the suggestion that these mitochondrial properties
might be important for the maintenance of pluripotency and
should be considered as another ESC marker. Departures
from this profile indicate that cells are differentiating or
perhaps becoming senescent.
Interestingly, some studies in embryonic stem cells

elevated ATP production and, thus, by a greater generation
of ROS. Undoubtedly, the intracellular levels of ROS are
higher in differentiated than in undifferentiated ESC, due to
the increase in OXPHOS metabolism in the former [141]. An
increase in ROS levels might have a role in cell signaling
and regulation of proliferation and differentiation. Exposure
to low levels of ROS has been reported to enhance ESC dif-
ferentiation whereas continuous exposure to high levels of
ROS results in inhibition of differentiation [141]. Therefore,
differentiating cells probably activate effective antioxidant
systems, including catalase, GPx and others. In summary,
successful differentiation of embryonic cells in vivo or
ESC in vitro involves initiation of mtDNA transcription and
replication, an increase in the number of mitochondria,
and regulation of the enzymes required for aerobic
metabolism in order to fulfill the elevated ATP requirements
of fully differentiated cells.
The increase in mitochondrial mass is accompanied by

stem cells may also be affected with aging. It has been
reported that the capacity of the newly born cells to migrate
from SGZ to the granule cell layer is decreased with aging
[63]. Several studies have also shown different grades
of reduction in the differentiation of neural stem cells into
The migration and differentiation of neural and progenitor
neurons [38] and the reduction in the dendritic maturation
during aging [38, 129].

physiological aging, some insults can also dramatically af-
fect this process. Neurodegenerative diseases are character-
ized by loss of neurons and newly-generated neurons should
appear in the damaged area to repair this injur. However,
recent studies have documented alterations in neurogenesis
in neurodegenerative diseases. Cell proliferation is signifi-
cantly repressed in SVZ of PD patients and animals models
of PD, resulting in a decrease in the numbers of neural stem
cells and neuroblasts. Importantly, this reduction is more
significant in PD patients with cognitive impairments than in
those without [66]. AD is characterized by the accumulation
of A? that suppresses the proliferation of neural stem and
progenitor cells and the neuronal differentiation in cell
culture [60]. Mouse models of AD with accumulation of A?
and mice given A? intravenously show a defect in neuronal
production, survival and differentiation in the SGZ, as well
as migration of neuroblasts [37, 61, 164]. Another proof
of the involvement of neural stem cell in the pathology of
AD is the fact that long term administration of cholinesterase
inhibitors, which improve cognitive function in AD patients,
promotes the survival of newly-generated neurons and
increases neurogenesis in adult mice [75]. The pathophysiology
of HD includes atrophy of the caudate nucleus and putamen,
which are adjacent to the SVZ. Related to that, Curtis and
colleagues [30, 31] observed an enhanced thickness of the
SVZ together with an increase of cell proliferation in the
brain of HD patients. Subsequently, Batista and colleagues
[13] reported that the ability of neural stem cells dissociated
from the SVZ of the R6/2 mice, a mouse model of HD, to
self-renew increases in parallel with the progression of the
disease. Likewise, they observed the presence of migrating
neuroblasts and newly-generated neurons in the striatum
of these mice. However, the migration of neuroblasts toward
the olfactory bulb was significantly suppressed [13].
Contrary to the SVZ, cell proliferation and neurogenesis of
the SGZ are decreased in the mouse models of HD [55, 89],
while the relation of these findings with the neuropathology
of DH has not been clarified.
In addition of the alterations in neurogenesis during

pathology of neurodegenerative diseases, stem cell research
focused in the cell replacement therapy is a promising treat-
ment for neurodegenerative disorders. Different strategies
are currently being examined for the treatment of neurode-
generative disorders using neural stem cells, including
approaches involving transplantation of exogenous cells or
promoting proliferation of endogenous cells. In both cases it
is believed that the increase in neural stem cells in the brain
attenuates anatomic and functional deficits associated with
diseases of the CNS via cell replacement, release of specific
neurotransmitters and production of neurotrophic factors that
protect injured neurons and promote neuronal growth.
Independently of the involvement of neurogenesis in the

fibroblast growth factor (bFGF) increased the SVZ cell
proliferation and increased migration of neuroblasts to the
striatum and the regeneration of the striatal projection
neurons [72]. The result was an amelioration of the motor
In mouse models of HD, the administration of basic

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188 Current Neuropharmacology, 2010, Vol. 8, No. 3
Escames et al.
dysfunction and the increase of the life-span of these mice.
The rise of hippocampal neurogenesis by the enrichment
of the mouse environment also delayed the progression of
HD symptoms in the mouse model [158]. Recently, mouse
neural stem cells were transplanted intraventricularly into
R6/2 HD mouse model combined with dietary trehalose,
which reduces cellular aggregate formation. The combined
treatment resulted in an improvement of motor function,
reduction in aggregate formation and increase of the
life-span of the animals [170]. However, the information
related to the migration and the survival of the grafted neural
stem cells was not provided in this study [83]. Previously, it
was shown that fetal cell transplantation ameliorated
neuronal dysfunction and improved motor function in both,
the HD mouse model and HD patients [12, 113].

models of ALS. Transplantation of neural stem cells isolated
from fetal spinal cord or neurons generated from the NT2
human teratrocarcinoma cell line in the spinal cord of ALS
mice were effective in the functional improvement and in
the delay of the progression of the disease [53, 166]. The
transgenic SODG93A mouse model of ALS was transplanted
with human neural stem cells overexpressing vascular
endothelial growth factor (VEGF) resulting in a functional
improvement and extended survival. The immunohisto-
chemical analysis demonstrated that the transplanted neural
stem cells migrated into anterior horn of the spinal cord and
differentiated into motoneurons [70]. Thus, a clinical trial of
mesenquimal stem cell transplantation in ALS patients has
recently finished phase I and is currently underway on phase
II [107].
Stem cell transplantation has been examined in animal

has been evaluated in mouse model of AD (3xTg-AD) which
express pathogenic forms of amyloid precursor protein, pre-
senilin, and tau [17]. The results showed that hippocampal
neural stem cell transplantation rescues the spatial learning
and memory deficits in aged 3xTg-AD mice. However, these
improvements were not associated with an alteration of Aß
or tau pathology. Interestingly, the mechanism underlying
the improved cognition involves an augmentation of
hippocampal synaptic density, mediated by brain-derived
neurotrophic factor (BDNF) [17]. To further confirm this
result, aged 3xTg-AD mice were treated with recombinant
BDNF, which mimicked the beneficial effects of neural stem
cell transplantation. On the contrary, depletion of neural
stem cell-derived BDNF failed to improve cognition or
restore hippocampal synaptic density [17].
Recently, the effect of neural stem cell transplantation

cells into the striatum of PD patients has been carried out
since early 1990s in patients with advanced disease [83].
However, the evidence of poor survival of the transplanted
cells in the brain together with the difficulties to obtain
enough fetal tissue for transplantation have led to a
redesigned stem cell therapeutical strategy in PD [58, 83].
Thus, dopaminergic neurons have been generated from
embryonic stem cells, mesenchymal stem cells and neural
stem cells following different experimental protocols [83].
Dopaminergic neurons generated from monkey embryonic
stem cells and human neural stem cells have been trans-
planted into striatum of monkeys with PD induced by MPTP.
Transplantation of human fetal ventral mesencephalic
The results showed a behavioral improvement of the PD
monkeys [131, 147]. Transplantation of immortalized neural
stem cells into the striatum of a rat model of PD also induced
functional improvement [172]. Other strategies include the
transplantation of neural stem cells transfected with specific
genes such as tyrosine hydroxylase (TH) and GTP cyclohy-
drolase I (GTPCH1) [83].

promising strategy but several issues must be clarified before
of general use in clinical medicine. The concerns include: 1)
it must be verified which type of stem cell is most suitable
for each purpose; 2) stem cells that escape differentiation and
selection processes might expand and produce tumor in the
graft site following transplantation; 3) highly purified popu-
lations of neural cell types derived from embryonic or neural
stem cells may contain other neuronal or glial cells types that
could generated unpredictable interactions among grafted
cells or host cells; and 4) earliest studies have demonstrated
that the long-term survival and phenotypic stability of stem
cell-derived neurons or glial cells in the graft following
transplantation are unsuccessful [83]. Hence, it is necessary
to establish which factors are involved in the poor survival
and stability of the transplanted cell. Among them, the highly
toxic oxidative and nitrosative environment in the aged brain
and its interaction with mitochondrial function should be
taken into account.
The use of stem cells for therapeutical purposes is a
NEURAL STEM CELLS AND MELATONIN

and/or differentiation begins to have experimental support,
the role of melatonin remains unclear. One can presume that,
in view of the specific and significant effects of melatonin on
mitochondrial physiology, the indoleamine may also affect
mitochondrial physiology in stem cells. It was recently
reported that melatonin modulates the proliferative and
differentiative ability of the neural stem cells from fetal
mouse brain in a concentration and exposure-timing dependent
manner [112]. When pharmacological concentrations of
melatonin (1-100 ?M) were applied during the proliferation
period, the proliferation was diminished. Interesting, neural
differentiation of these cells increased without affecting
astroglial differentiation. Other data point towards a net
hippocampal neurogenesis in adult mice by melatonin [127].
Interestingly, it was shown that pinealectomy causes loss of
pyramidal neurons in rat CA1/3 hippocampal layers, an ef-
fect reversed by melatonin administration [34]. Melatonin
also promotes neurogenesis and motor recovery after mild
focal ischemia or cranial irradiation in mice [81, 101]. New
experimental data suggest that melatonin induces neurogenesis
in dentate gyrus of adult pinealectomized rats [137]. The
effects of melatonin on neural proliferation and differentiation
might be partly a result of melatonin´s activity in mitochondria.
Clearly, additional studies are required to uncover the
underlying actions of melatonin on neural stem cells.
Whereas a role of mitochondria in stem cell proliferation
ACKNOWLEDGEMENTS

Marie Curie International Reintegration Grant Programme
(COQMITMEL-266691) within the 7th European Community
Framework Programme, from Ministerio de Ciencia e
Innovación, Spain (SAF2009-08315, SAF2009-14037), from
This study was partially supported by grants from the

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The Role of Mitochondria in Brain Aging and the Effects of Melatonin Current Neuropharmacology, 2010, Vol. 8, No. 3 189
Instituto de Salud Carlos III, Spain (RD06/0013/0008,
RD06/0013/1017, PI08-1664), and from the Consejería
de Innovación, Ciencia y Empresa, Junta de Andalucía
(P07-CTS-03135 and CTS-101).
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Received: March 18, 2010 Revised: April 24, 2010 Accepted: May 05, 2010