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Bioenergetic approaches for neuroprotection in Parkinson's disease.

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Bioenergetic Approaches for Neuroprotection
in Parkinson’s Disease
M. Flint Beal, MD
There is considerable evidence suggesting that mitochondrial dysfunction and oxidative damage may play a role in the
pathogenesis of Parkinson’s disease (PD). This possibility has been strengthened by recent studies in animal models,
which have shown that a selective inhibitor of complex I of the electron transport gene can produce an animal model
that closely mimics both the biochemical and histopathological findings of PD. Several agents are available that can
modulate cellular energy metabolism and that may exert antioxidative effects. There is substantial evidence that mitochondria are a major source of free radicals within the cell. These appear to be produced at both the iron-sulfur clusters
of complex I as well as the ubiquinone site. Agents that have shown to be beneficial in animal models of PD include
creatine, coenzyme Q10, Ginkgo biloba, nicotinamide, and acetyl-L-carnitine. Creatine has been shown to be effective in
several animal models of neurodegenerative diseases and currently is being evaluated in early stage trials in PD. Similarly,
coenzyme Q10 is also effective in animal models and has shown promising effects both in clinical trials of PD as well as
in clinical trials in Huntington’s disease and Friedreich’s ataxia. Many other agents show good human tolerability. These
agents therefore are promising candidates for further study as neuroprotective agents in PD.
Ann Neurol 2003;53 (suppl 3):S39 –S48
Parkinson’s disease (PD) is the second most common
neurodegenerative disease, affecting approximately 1%
of the population older than age 65 years. It affects
more than one million people in the United States.
The cardinal clinical manifestations include bradykinesia, rest tremor, rigidity, and postural instability. The
cause of the illness is a selective degeneration of dopaminergic neurons in the substantia nigra compacta.
Much evidence has accumulated implicating mitochondrial defects in the pathogenesis of Parkinson’s
disease (PD). Investigations of 1-methyl-4-phenyl1,2,3,6-tetrahydrodropyridine (MPTP) toxicity, which
produces parkinsonism in humans and laboratory animals, showed that it is mediated by inhibition of respiratory complex I. MPTP first came to light as a contaminant of synthetic opiates, which had led to an
outbreak of parkinsonism in young individuals in
southern California. MPTP is metabolized to MPP⫹,
which is preferentially taken up by dopamine neurons
and selectively inhibits complex I of the electron transport chain.1 In idiopathic PD, there is a 30 to 40%
decrease in complex I activity in the substantia nigra,2,3
as well as reduced staining for complex I subunits, although preserved staining for other subunits of the
electron transport complexes.4 Reduced complex I activity in PD platelets also has been reported in several
studies.5,6 There have been two studies, which demonstrated that cybrids made from individuals with PD
show selective reductions in complex I activity, as
well as increased free radical production, and an increased susceptibility to the MPTP metabolite
MPP⫹.7,8 However, one recent study of cybrids in
PD failed to show significant and specific reductions
in complex I activity.9 As one might predict, cybrids
made from patients with autosomal dominant PD associated with ␣-synuclein mutations do not show
complex I defects.10
There has been some genetic evidence suggesting
that complex I defects play a role in parkinsonism. A
family with multisystem degeneration with parkinsonism has been reported with an 11778 mitochondrial
DNA mutation that produces a complex I defect.11
Another family recently has been described that had a
novel mitochondrial 12sRNA point mutation associated with parkinsonism, deafness, and neuropathy.12
Cybrid studies have shown that a complex I defect is
associated with PD in one large family.13 In a study of
monozygotic twins who were discordant for PD, several novel homeoplasmic sequence variants, including
two missense mutations in complex I subunits, were
detected in four of the pairs.14 Furthermore, a total of
20 known polymorphisms effecting both complex I
From the Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York Presbyterian Hospital,
New York, NY.
Address correspondence to Dr Beal, Neurology Department, New
York Hospital–Cornell Medical Center, 525 East 68th Street, New
York, NY 10021. E-mail: fbeal@mail.med.cornell.edu
Published online Mar 24, 2003, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.10479.
© 2003 Wiley-Liss, Inc.
S39
and transfer RNA mutations were found. Mitochondrial DNA sequences, however, tended to be identical,
and the disease did not affect siblings of each pair. The
pathogenic relevance of several of these mutations
therefore is questionable. In addition, an out-of-frame
cytochrome b gene deletion has been detected in a patient with parkinsonism that was associated with impaired complex III assembly and an increase in free
radical production.15
In a direct sequencing study of complex I in transfer
RNA mutations, we recently observed no homoplasmic
mutations, suggesting either that the observed complex
I defects are caused by heteroplasmic mutations or that
they may involve interactions between the nuclear genome and the environment.16 We also recently directly
sequenced mitochondrial DNA from postmortem
brain tissue of neuropathologically confirmed PD patients.17 Once again, we did not detect any homoplasmic mitochondrial DNA mutations associated with
PD. This suggests that if mitochondrial DNA mutations play a role in PD, the pathogenetic effects may be
very complicated. It recently has been demonstrated
that nuclear background determines the biochemical
phenotype of the deafness-associated mitochondrial 12s
RNA mutation.18 A nuclear mitochondrial DNA mutation affecting hearing impairment also has been demonstrated in mice.19 Furthermore, mitochondrial DNA
variant susceptibility to dilated cardiomyopathy is different in two different human populations.20 These
findings suggest that there are complex interactions between the nuclear and mitochondrial DNA, and that
expression of a mitochondrial disease may occur only
in selective nuclear DNA backgrounds. This may make
the study of mitochondrial DNA defects in parkinsonism extremely complex.
A major finding suggesting that a complex I defect
may play a critical role in the pathogenesis of PD
comes from recent studies with the environmental
toxin rotenone. The possibility that pesticides and
other environmental toxins are involved in the pathogenesis of PD is suggested by several epidemiological
studies.21,22 Patients with certain glutathione transferase polymorphisms and exposure to pesticides seem
to have an increased incidence of PD.23 Furthermore,
an atypical PD syndrome has been described in association with the consumption of fruits and herbal tea
containing insecticides in the French West Indies.24
Rotenone is a natural occurring compound derived
from the roots of certain plant species, which has been
used as an insecticide for vegetables and to kill fish
populations in lakes or reservoirs. Rotenone is known
to be a high-affinity–specific inhibitor of complex I of
the electron transport chain.
A recent study examined the effects of rotenone
when infused intravenously into rats.25 The rats developed progressive degeneration of nigrostriatal neurons
S40
Annals of Neurology
Vol 53 (suppl 3)
2003
Table. Bioenergetic Agents Effective in Parkinson’s Disease
Models
Agent
Coenzyme Q10
Creatine
Ginkgo biloba
Carnitine
Nicotinamide
Lipoic acid
Proposed Mechanism of Action
Cofactor of complex I, II, III and antioxidant
Increases PCr, inhibits the MPT
Antioxidant and preserves mitochondrial function
Facilitates fatty acid transport, increases
repiration
Precursor of NADH, inhibitor of polyADP-ribose polymerase
Coenzyme for ␣-ketoglutarate dehydrogenase, antioxidant
PCr ⫽ creatine/phosphocreatine; MPT ⫽ mitochondrial permeability transition pore; NADH ⫽ nicotinamide adenine dinucleotide.
with a loss of immunoreactivity for tyrosine hydroxylase, dopamine transporter, and vesicular monoamine
transporter. Furthermore, the nigral neurons showed
cytoplasmic inclusions that were highly suggestive of
Lewy bodies in that they stained with antibodies to
ubiquitin and ␣-synuclein, and electron microscopy
showed a dense core surrounded by fibrillar elements
similar to Lewy bodies. The rats showed bradykinesia,
postural instability, unsteady gait, and some evidence
of tremor that improved after treatment with the dopamine agonist, apomorphine. These findings suggest
that rotenone can produce a selective degeneration of
nigrostriatal neurons consistent with the neuropathological and clinical manifestations of PD. They are remarkable because they show that an inhibitor of complex I of the electron transport chain, which acts
uniformly throughout the brain, produces a selective
degeneration of nigrostriatal neurons. They therefore
indicate the substantia nigra neurons are particularly
susceptible to complex I inhibitors. This is consistent
with the findings of decreased complex I activity in PD
postmortem tissue and platelets. It has been suggested
that the selective effects of rotenone may be mediated
by oxidative damage. This is also consistent with prior
studies showing extensive oxidative damage in the substantia nigra of PD patients.
If mitochondrial defects and oxidative damage play a
role in the pathogenesis of PD, then one would suspect
that agents that may improve mitochondrial function or
exert antioxidative effects could be neuroprotective.
There are several agents that currently are under investigation for their potential neuroprotective effects based
on their capacity to modify mitochondrial dysfunction.
These include creatine, coenzyme Q10 (CoQ10), Ginkgo
biloba, nicotinamide, riboflavin, acetyl-carnitine, and lipoic acid (Table). Of these creatine, CoQ10, G. biloba
and nicotinamide have all been assessed in the MPTP
model of PD. As noted above, MPTP toxicity in primates replicates all the clinical signs of PD, including
tremor, rigidity, akinesia, and postural instability (reviewed in Beal26).
Mitochondria and Reactive Oxygen Species
In addition to their critical role in ATP synthesis, mitochondria are also the major source of reactive oxygen
species (ROS) in most cell types. ROS include superoxide, hydrogen peroxide (H2O2), and hydroxyl free
radical (•OH). It has been suggested that as much as
2% of the oxygen consumed by mitochondria is converted to superoxide, which then is converted by manganese superoxide dismutase into H2O2. Recently,
CuZn superoxide dismutase has been localized in the
intermembrane space of mitochondria.27 This enzyme
may be important in preventing the exit of mitochondrially derived superoxide into the cytoplasm where it
could damage critical cellular components. Approximately 50% of superoxide derived from the electron
transport chain is directed toward the intermembrane
space.28
The principal sites of production of ROS are
thought to be ubiquinone and an as yet undetermined
site in complex I. A recent study of rat brain mitochondria showed that the highest rate of mitochondrial
ROS generation was observed in mitochondria respiring on the complex II substrate succinate.29 This production of ROS appeared to be dependent on reverse
electron transport through complex I, because it was
inhibited by rotenone. It was also very sensitive to
changes in mitochondrial membrane potential, being
inhibited by reductions in membrane potential such as
those associated with ATP generation. Mitochondria
respiring on the complex I substrates glutamate and
malate produce very little ROS unless complex I is inhibited by rotenone. It is noteworthy that although
ubiquinone produces ROS with both substrates, they
represent a relatively minor component of the overall
ROS generation.
Another recent study of isolated rat brain mitochondria also showed that most of ROS generation produced by succinate occurs at complex I through reverse
electron transfer rather than at the ubiquinone site.30
Similarly, complex I substrates produced very little
ROS unless rotenone or antimycin A were present. In
these studies, the authors used the flavoprotein inhibitor diphenyliodonium, which has been shown to block
succinate-induced H202 production, consistent with
flavin mononucleotide being the source of mitochondrial ROS rather than complex I iron-sulfur clusters.
Other data, however, favor some of the distal complex
I iron-sulfur clusters in generation of ROS.
Bioenergetics
Creatine is a guanidine compound found in meatcontaining products and produced endogenously by
the liver, kidneys, and pancreas.31 The production of
creatine requires the amino acids arginine and glycine
as well as methionine. L-Arginine:glycine amidinotransferase results in the production of guanidinoacetate,
which, in turn, is methylated by S-adenosylmethionine to produce creatine.32 Creatine is taken up
into brain and cardiac and skeletal muscle by a
sodium-dependent transporter that has been cloned
and sequenced.33 The creatine/phosphocreatine (PCr)
system functions as a spatial energy buffer between the
cytosol and mitochondria, using a unique mitochondrial creatine kinase (CK) isoform.34 The mitochondrial CK isoform exists in the intermembrane space of
the mitochondria35 where it can convert from an octameric to a dimeric form. The octameric form facilitates the functional coupling between the porin molecule on the outer mitochondrial membrane and the
adenine nucleotide translocase in the inner mitochondrial membrane. Together, they form components of
the mitochondrial permeability transition pore, whose
opening (which promotes apoptosis) is inhibited when
mitochondrial CK is in the octameric form.36 It has
been demonstrated that the octameric form is converted into the dimeric form in the presence of free
radicals such as peroxynitrite thereby promoting opening of the pore and apoptosis.37 Creatine administration can protect mitochondrial CK from being converted into the dimeric form. Both creatine and PCr
can attenuate peroxynitrite-mediated mitochondrial
CK inactivation with consequent dimerization and
opening of the PTP.38 Another potential neuroprotective effect of creatine administration is increasing glutamate uptake into synaptic vesicles, which has been
shown to be energy dependent and which can be fueled by PCr.39
The potential of creatine to be protective can be illustrated in numerous models of neurodegeneration.
Creatine administration protects against glutamate and
␤-amyloid toxicity in rat hippocampal neurons.40 Creatine is also beneficial in animal models of traumatic
brain injury and cerebral ischemia.41,42 In addition,
preincubation of anoxic rat hippocampal slices with
creatine attenuated the decrease in PCr and ATP content.43
We initially studied the effects of oral creatine supplementation on striatal lesions produced by malonate
and 3-nitropropionic acid, which are reversible and irreversible inhibitors of complex II, respectively, and
which model Huntington’s disease (HD).44 After administration of 3-nitropropionic acid there was attenuation of ATP and phosphocreatine depletion, reduced
lactate accumulation, and reduced oxidative stress. We
also examined the effects of creatine supplementation
on MPTP-induced parkinsonism.45 We found that creatine produced dose-dependent protection against dopamine loss, as well as an attenuation of neuron loss in
the substantia nigra of mice treated with MPTP. Sub-
Beal: Bioenergetics in Parkinson’s
S41
sequent work has shown that creatine significantly improves survival and neuronal survival in transgenic
mouse models of both amyotrophic lateral sclerosis
(ALS) and HD.46 – 48 In the transgenic mouse model of
ALS, there is also a delayed onset loss of neurons in the
substantia nigra of approximately 20 to 25%. This loss
of neurons is of particular interest because it is late in
onset and slowly progressive, similar to the cell loss
that occurs in human PD. This cell loss was completely
prevented by 1% creatine administration in mice studied at 110 days of age.
Another potential bioenergetic treatment for PD is
CoQ10, which recently has been studied in a small pilot clinical trial. CoQ10 is an important cofactor of the
electron transport chain where it accepts electrons from
complexes I and II.49,50 It consists of a quinone head
attached to a chain of isoprene units numbering 9 to
10 in various mammalian species. The quinone head
can alternately assume three different redox states,
namely, ubiquinone (Q) the fully oxidized form; the
free radical ubisemiquinone (•QH), which is the partially reduced form; and ubiquinol (QH2), the fully reduced form. Ubiquinone initially is reduced to the
semiquinone radical and then transfers electrons one at
a time to complex III of the electron transport chain.
CoQ10, which is also known as ubiquinone, serves as
an important antioxidant in both mitochondrial and
lipid membranes.51,52 It is a particularly important antioxidant in the inner mitochondrial membrane where
it can directly scavenge free radicals.53 Ubiquinol has
also recently been documented to directly interact with
nitric oxide.54 There is also substantial evidence that
ubiquinol also may act as an antioxidant in concert
with ␣-tocopherol,55 because it reduces ␣-tocopheroxyl
radical back to ␣-tocopherol.53,56,57 In rat liver subject
to oxidant stress, mitochondrial CoQ9 levels are oxidized before the onset of massive lipid peroxidation
and the subsequent depletion of ␣-tocopherol.58 In rat
mitochondria, supplementation with succinate results
in a reduction of CoQ to ubiquinol, thereby preserving
␣-tocopherol concentrations during oxidation.51 This
suggests that ␣-tocopherol is the direct radical scavenger, and ubiquinol primarily acts to regenerate
␣-tocopherol. Another interaction occurs between dihydrolipoic acid and CoQ.59 Dihydrolipoic acid reduces ubiquinone to ubiquinol by the transfer of a pair
of electrons, thereby increasing the antioxidant capacity
of ubiquinol in biomembranes. Lipoic acid has been
shown to maintain a normal ratio of reduced to oxidized ubiquinone after MPTP administration in
vivo.60
The effects of oral supplementation with CoQ or
␣-tocopherol on the rate of mitochondrial superoxide
radical generation have been examined in skeletal muscle, liver, and kidney of 24-month-old mice.51 In this
study, the administration of ␣-tocopherol produced a
S42
Annals of Neurology
Vol 53 (suppl 3)
2003
sevenfold increase in mitochondrial ␣-tocopherol content, whereas CoQ10 administration increased both total CoQ content and ␣-tocopherol by approximately
fivefold. In these mice, the rate of superoxide radical
generation from submitochondrial particles was inversely related to ␣-tocopherol content, but unrelated
to CoQ content. This study therefore provides in vivo
evidence that at least part of the antioxidant effects of
CoQ are mediated by its ability to reduce the
␣-tocopheroxyl radical.
A potentially very interesting effect of CoQ is its interaction with mitochondrial uncoupling proteins.
CoQ has been shown to be an obligatory cofactor for
uncoupling protein function.61,62 This has been demonstrated for uncoupling proteins 1, 2, and 3. The effect originally was examined in liposomes; it subsequently was demonstrated that CoQ increased proton
conductance in rat kidney mitochondria that are oxidizing succinate.62 This increase required fatty acids
and was prevented by guanosine diphosphate. CoQ activated proton conductance in these studies only when
it was likely to be reduced to CoQH2. Activation was
abolished by superoxide dismutase, indicating that
CoQ might mediate uncoupling through the production of superoxide. This subsequently was shown to be
the case when CoQ was replaced by an exogenous system that generates superoxide using xanthine plus xanthine oxidase.
This effect is important because uncoupling proteins
may reduce the generation of free radicals,63 important
mediators of oxidative damage. Through an interaction
with CoQ, uncoupling proteins (UCPs) may adjust
electron transfer by regulating the quinone pool according to cellular context and needs.62 This may be
an adjustment in response to the formation of ROS
and biological parameters such as the need for ATP
production.64
CoQ10 has been shown to exert neuroprotective effects in the central nervous system in several in vivo
models. It produces significant protection against experimental ischemia,65 attenuating ATP and glutathione depletion as well as neuronal injury in the hippocampus. We found that oral administration of
CoQ10 significantly attenuated ATP depletion and
produced dose-dependent neuroprotective effects
against striatal lesions produced by the mitochondrial
toxin malonate.66 CoQ10 administration also significantly attenuated striatal lesions produced by aminoxyacetic acid.67 The role of CoQ10 has also been studied
in MPTP toxicity. We demonstrated significant protection against dopamine depletion and loss of tyrosine
hydroxylase immunostained neurons in 24-month-old
mice treated with MPTP.68 We also found that CoQ10
produces marked neuroprotective effects against the
systemic administration of the mitochondrial toxin
3-nitroproprionic acid.69 This is an irreversible inhibi-
tor of succinate dehydrogenase that produces selective
striatal lesions in both rats and primates, closely resembling those found in HD. Administration of CoQ10
for 1 week before coadministration of 3-nitropropionic
acid resulted in a 90% neuroprotection against the striatal lesions and significantly attenuated the reductions
in reduced CoQ9 and reduced CoQ10. More recently,
we have demonstrated that CoQ10 produces neuroprotective effects in transgenic mouse models of both ALS
and HD.69,70
On the basis of these results, we, and others, have
examined the effects of CoQ10 in patients with neurodegenerative diseases. We initially tested the oral administration of 360mg daily of CoQ10 on elevated occipital cortex lactate concentrations in patients with
HD.71 In this study, we obtained lactate concentrations before, during, and after the discontinuation of
CoQ therapy. CoQ10 treatment produced a 37% reduction in occipital cortex lactate concentrations,
which was reversed after discontinuation of therapy.
Recently, a clinical trial was performed by the Huntington’s Study Group, which examined the effects of
CoQ10 with or without the N-methyl-D-aspartate receptor antagonist remacemide.72 The trial encompassed
340 patients who were treated for 30 months. Patients
were randomized to CoQ10 600mg daily, remacemide,
or a combination of the two in a 2 ⫻ 2 factorial design. In this study, remacemide demonstrated no efficacy. Administration of CoQ10 resulted in a 14% slowing of disease progression as assessed by a total
functional capacity rating scale, but the effect did not
reach significance because the study was not powered
to detect an effect of this magnitude. Nevertheless,
there was significant improvement on several secondary
outcome measurements.
Studies of PD patients have shown that the ratio of
reduced to oxidized CoQ10 is significantly reduced in
platelets,73 although in another study serum levels were
unaltered.74 We measured CoQ10 levels in mitochondria isolated from platelets of PD patients and found
significant reductions that directly correlated with decreases in complex I activity.75 Oral administration of
CoQ10 to PD patients was well tolerated and resulted
in significant, dose-dependent increases in plasma
CoQ10 levels.
We recently completed a phase II clinical study of
CoQ10 in de novo PD patients (Parkinson Study
Group, unpublished findings). Patients were treated
with placebo or 300, 600, or 1,200mg of CoQ10 for
10 months. The primary outcome measure was the
change in the Unified Parkinson’s Disease Rating Scale
(UPDRS) between baseline and final visits. Secondary
outcome measures were changes in complex I activity
of the mitochondrial electron transport chain in platelets and serum CoQ10 levels. This study demonstrated
a dose-dependent reduction in disease progression of
44% as assessed by the UPDRS. A larger phase III
study is required to determine whether these results
can be replicated. Interestingly, there was a dosedependent increase in plasma CoQ10 levels, with the
largest increase occurring between the 600 and
1,200mg doses, consistent with the magnitude of
changes in clinical efficacy. These findings indicate that
CoQ10 is an extremely promising agent for study as a
neuroprotectant for PD.
CoQ10 and its analog, idebenone, also have been
studied in patients with Friedreich’s ataxia where it has
been reported to significantly reduce cardiac mass76,77
and to significantly improve cardiac and skeletal muscle bioenergetics.78 The latter study examined the effects of 6 months of treatment with 400mg daily of
CoQ10 and vitamin E 2,100IU/day in 10 Friedreich’s
ataxia patients using in vivo phosphorous magnetic resonance spectroscopy. After 3 months of treatment, the
cardiac PCr to ATP ratio showed a mean increase of
178%, and the maximum rate of skeletal muscle mitochondrial ATP production was increased by 139% in
comparison with their respective baseline values. These
improvements were sustained after 6 months of therapy. There were, however, no significant improvements
on neurological or echocardiographic evaluation. These
findings also warrant a larger trial of Friedreich’s ataxia
patients who can be studied over a longer time frame.
Several other agents that modulate cerebral energy
metabolism or that exert antioxidant effects are also
potential neuroprotective treatments for PD. G. biloba
is a plant extract composed of a complex chemical mixture that exerts neuroprotective effect against models of
mitochondrial damage and oxidative stress. It has been
shown to significantly reduce the generation of lipid
peroxides in brain homogenates and in rat brain synaptosomes,79 and to protect primary cultures of cerebellar neurons against oxidative damage80 and hippocampal neurons from toxicity produced by either
hydrogen peroxide or nitric oxide.81 G. biloba has been
reported to protect dopamine neurons from MPTPinduced neurotoxicity82 and to be effective in models
of focal and global ischemia. Finally, we found that G.
biloba extract has beneficial effects on survival in transgenic mice that model ALS.83
Nicotinamide is a precursor of nicotinamide adenine
dinucleotide (NADH), which is a substrate for complex I of the electron transport chain. It is also an inhibitor of polyADP-ribose polymerase, an enzyme that
is activated by DNA damage and that, in turn, depletes
both NADH and ATP. Several studies have shown
that nicotinamide, like other polyADP-ribose polymerase inhibitors, protects against MPTP neurotoxicity.84
Similar results have been observed in mice with a
knockout of polyADP-ribose polymerase.85 Our studies further demonstrate that nicotinamide attenuates
Beal: Bioenergetics in Parkinson’s
S43
neuronal injury and ATP depletion produced by focal
ischemia, malonate, and MPTP.66,86,87
Carnitine and acetyl-L-carnitine are agents that facilitate the entry and exit of fatty acids from mitochondria. Carnitine facilitates the entry of long chain fatty
acids into mitochondria for subsequent ␤-oxidation
and the removal of short chain and medium chain fatty
acids that accumulate during normal and abnormal
metabolism. Short and medium chain fatty acids are
esterified to carnitine by the action of carnitine acetyltransferase. The acetylcarnitine esters are then transported out of mitochondria by carnitine acetylcarnitine
translocase. Acetyl- L-carnitine may have better brain
penetration and may be useful as an agent for elevating
brain carnitine levels.
Carnitine delays mitochondrial depolarization in response to a variety of stressors including oxidative
damage.88 Acetyl- L-carnitine increases cellular respiration, mitochondrial membrane potential, and cardiolipin levels in hepatocytes of 24-month-old rats.89
These biochemical effects are paralleled by increases in
ambulatory activity of aged rats. Carnitine and acetylL-carnitine attenuate neuronal damage produced by
3-nitroproprionic acid, rotenone, and MPTP in
vitro.90,91 After ischemia reperfusion in rats, acetyl-Lcarnitine resulted in a more rapid recovery of ATP and
PCr and lactate levels.92
Lipoic acid is a disulfide compound that is found
naturally in mitochondria as a coenzyme for pyruvate
dehydrogenase and ␣-ketoglutarate dehydrogenase and
also has antioxidant effects. It has been shown to protect against peroxynitrite-induced nitration and
␣-antiproteinase inactivation and is neuroprotective in
rodent models of both focal and global cerebral ischemia.93–96 We found that ␣-lipoic acid exerts significant neuroprotective effects in a transgenic mouse
model of HD.97 In humans, a dose of 600mg/day decreased plasma indices of oxidative stress, low-density
lipoprotein oxidation, and urinary isoprostanes.98
Supplementation with ␣-lipoic acid in old rats improved ambulatory activity, decreased oxidative damage,
and improved mitochondrial function.99,100 Recent
studies of lipoic acid in combination with acetyl-Lcarnitine have demonstrated significant improvements in
mitochondrial function in old rats.101 This was shown
to occur in the absence of any increase in oxidative damage, which is observed when acetyl-L-carnitine is administered alone. Furthermore, examination of aged rats
treated with acetyl-L-carnitine and lipoic acid showed
significant improvements on cognitive tasks,100 including the Morris water maze test. These findings suggest
that this combination of agents could be beneficial for
treating age-related cognitive deficits.
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Annals of Neurology
Vol 53 (suppl 3)
2003
Conclusions
There is substantial evidence based on postmortem
studies of PD tissue as well as experimental animal
models indicating that mitochondrial dysfunction and
oxidative damage play a role in the pathogenesis of PD.
In the laboratory, experimental animal models of PD
have been produced with both MPTP and rotenone,
which are known to inhibit complex I of the electron
transport chain and to increase oxidative damage. Several agents are now available that can modulate cellular
energy metabolism and that thereby may exert antioxidative and protective effects. Several of these agents
have been shown to produce significant neuroprotective effects in the MPTP model of PD, including creatine, CoQ10, G. biloba, nicotinamide, and acetyl-Lcarnitine. Creatine has been shown to produce
significant neuroprotective effects in several animal
models of neurodegenerative diseases and is well tolerated in man. Similarly, CoQ10 is effective in several
animal models of neurodegenerative diseases and recently has shown very promising results in a phase II
study in PD patients. Many of the other agents described above also show good human tolerability.
These observations raise the possibility that these
agents, either alone or in combination, are worthy of
further study as possible neuroprotective agents in PD.
This work was supported by grants from National Institute of Neurological Disorders and Stroke, the Department of Defense, and the
Parkinson’s Disease Foundation.
The secretarial assistance of S. Melanson is gratefully acknowledged.
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Discussion
Rascol: Do you have any experiments combining multiple possible neuroprotective agents that are thought
to act via different mechanisms? Do they act in an additive or synergistic way?
Beal: Yes. In the Huntington’s mice we have been
able to show that there are additive effects of remacemide and CoQ. You can go from a 15 to 20% effect
on survival to a 33% effect. You also can demonstrate
additive effects for behavior and weight loss. We also
have tested a combination of four different agents in
this model: a transglutamenase inhibitor, a nitric oxide
synthase inhibitor, remacemide, and CoQ . When we
use the four agents, we can get even better protective
effects with increases in survival up to 46% in the
Huntington’s transgenics. Therefore, it appears that we
can get increased benefits with multiple agents just as
they have found with cancer chemotherapy.
Marek: In the study that was performed in Huntington patients, remacemide and CoQ were ineffective.
So, how reliable are these models in predicting the response in humans?
Beal: The problem may have been dosing. We chose
a dose in the mice that was based on what we previously had found to be protective against acute excitotoxic lesions. In the humans, the dose was limited by
tolerability. Patients became drowsy and developed hallucinations as has been found with other N-methyl-Daspartate receptor antagonists. The problem therefore
may be that in humans you cannot get up to those
dose levels that are neuroprotective in rodents.
Olanow: Have you tried any specific N-methyl-Daspartate receptor subunit blockers that might avoid
the side effects that occur when the entire receptor is
blocked?
Beal: I think that is a promising strategy that might
work. Some have been tested in animals and they do
have neuroprotective effects, but none have yet been
tested in humans.
Kordower: In the Huntington’s mouse model that
you use, I was very impressed by the loss of cells and
the loss of striatal volume, and yet my understanding is
that there is very little striatal degeneration in the R6/2
mice. Could you expand upon that?
Beal: Well, they do in fact have profound striatal
atrophy, but initial reports did suggest there was no
cell loss. Now, the reason you have striatal atrophy is
twofold. One is the overall cell bodies shrink and the
other is cell loss. There is good evidence for cell shrinkage in this model, and this is probably the major factor
Beal: Bioenergetics in Parkinson’s
S47
leading to striatal atrophy. However, electronmicroscopy studies also indicate that there is some degree of
cell loss, but you cannot pick it up by routine light
microscopy.
Kordower: In your neuroprotection studies in Huntington’s disease, do you use cell size as the primary
outcome measure?
Beal: We have shown that we can protect against the
loss of cell size in these studies with some neuroprotective agents.
Kordower: Does creatine treatment lead to hypertrophy in addition to preventing cell shrinkage?
Beal: We protected against shrinkage, and the cells
were not larger than normal.
Isacson: In the MPTP-treated mice that you study,
you presented data indicating that many different
agents can block degeneration. However, if you do
nothing, most of the dopaminergic neurons will recover. So, it is an appealing model because you can
demonstrate that some agents have powerful neuroprotective effects, but as Ken Marek pointed out, I am not
sure that the same conditions apply in PD patients or
that you can assume that you will obtain comparable
results.
Stocchi: Does oral creatine gain access to the central
nervous system and what do you think is the mechanism of action for neuroprotection? There was one
study in Italy of athletes that failed to demonstrate any
increase in power, although they felt less fatigue.
Beal: I think the data are relatively solid that the
drug has no effect on long-term athletic performance.
On the other hand, with very high output short-term
athletic performance there are data indicating improved
performance and an enhanced rate of regeneration of
phosphocreatine as demonstrated by phosphorus nuclear magnetic resonance studies. As a result, most high
output athletes in the United States, such as sprinters
and baseball, football, and hockey players, are taking it.
As to whether it get into the central nervous system?
We have demonstrated that it does based on direct biochemical measurements and phosphorus nuclear magnetic resonance. We have performed these studies in
both the mouse and patients and have shown that we
get an approximately 10 to 15% increase in creatine
and phophocreatine levels in the brain.
Schapira: In what brain area does this occur?
Beal: We have primarily found increases in the cere-
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Annals of Neurology
Vol 53 (suppl 3)
2003
bral cortex and specific increases in the motor cortex in
ALS patients. As to the mechanism responsible for
neuroprotection? One possibility is that it simply increases levels of phosphocreatine. It is also possible that
it could have direct effects on the mitochondrial transition pore working through the mitochondrial CK.
However, we have mice now that have a knockout of
mitochondrial CK in whom we still see protection with
creatine. This would argue that creatine is not acting
by way of a direct effect on mitochondria.
Tatton: We examined the capacity of creatine to
block apoptosis in four different types of cells in tissue
culture. We found that maximal protection was obtained with concentrations of approximately 10⫺6 molar. However, antiapoptotic effects were largely blocked
by protein synthesis inhibitors, suggesting that the drug
acts through a transcriptional mechanism. We did find
that creatine upregulated CK, but I believe that the
effects are caused by a transcriptional action of creatine
and not by an energetic action.
Beal: That is very possible, I agree.
Olanow: Have you tested creatine as well as Coenzyme Q in clinical trials of PD?
Beal: Schults and colleagues have tested CoQ in a
prospective double-blind clinical trial in PD. The study
is now completed but not yet published. Creatine, I
am told, is in a trial for PD in Munich but I have no
data to provide. There are also two trials of creatine in
ALS and a pilot trial in Huntington’s disease that is
combined with imaging. None of these results are currently available.
Olanow: In the CoQ study, which type of patient
was studied and what was the primary endpoint?
Beal: We studied patients with early PD who were
untreated and remained untreated throughout the
study. Change from baseline in UPDRS is the primary
end point. Secondary end point is complex I activity in
platelets.
Olanow: Are there renal complications with highdose creatine and are there any problems with CoQ?
Beal: There have been reports of renal problems at
doses of 20gm/day, probably because such a large load
was being placed on the kidneys. In the trial, we used
a dose of 5gm/day, which is very well tolerated. CoQ
can be administered in doses of up to 1,200mg/day
without tolerability problems.
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