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: firstname.lastname@example.org 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. S44 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. References 1. 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Age-associated mitochondrial oxidative decay: improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-L-carnitine and/or R-alpha-lipoic acid. Proc Natl Acad Sci USA 2002;99:1876 –1881. 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- S48 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.