Bioenergetic Approaches for Neuroprotection           in Parkinson’s Disease                                              ...
and transfer RNA mutations were found. Mitochon-            Table. Bioenergetic Agents Effective in Parkinson’s Diseasedri...
tremor, rigidity, akinesia, and postural instability (re-   creatine requires the amino acids arginine and glycineviewed i...
sequent work has shown that creatine significantly im-       sevenfold increase in mitochondrial ␣-tocopherol con-proves s...
tor of succinate dehydrogenase that produces selective       44% as assessed by the UPDRS. A larger phase IIIstriatal lesi...
neuronal injury and ATP depletion produced by focal          Conclusionsischemia, malonate, and MPTP.66,86,87             ...
9. Aomi Y, Chen CS, Nakada K, et al. Cytoplasmic transfer of         28. Han D, Antunes F, Daneri F, Cadenas E. Mitochondr...
48. Klivenyi P, Ferrante RJ, Matthews RT, et al. Neuroprotective      67. Brouillet E, Henshaw DR, Schulz JB, Beal MF. Ami...
84. Cosi C, Marien M. Decreases in mouse brain NADϩ and ATP            101. Liu J, Killilea DW, Ames BN. Age-associated mi...
leading to striatal atrophy. However, electronmicros-        bral cortex and specific increases in the motor cortex incopy...
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Approcci bioenergetici per la neuroprotezione nella malattia parkinsoniana


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Approcci bioenergetici per la neuroprotezione nella malattia parkinsoniana

  1. 1. Bioenergetic Approaches for Neuroprotection in Parkinson’s Disease M. Flint Beal, MDThere is considerable evidence suggesting that mitochondrial dysfunction and oxidative damage may play a role in thepathogenesis 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 modelthat closely mimics both the biochemical and histopathological findings of PD. Several agents are available that canmodulate cellular energy metabolism and that may exert antioxidative effects. There is substantial evidence that mito-chondria are a major source of free radicals within the cell. These appear to be produced at both the iron-sulfur clustersof complex I as well as the ubiquinone site. Agents that have shown to be beneficial in animal models of PD includecreatine, coenzyme Q10, Ginkgo biloba, nicotinamide, and acetyl-L-carnitine. Creatine has been shown to be effective inseveral 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 asin clinical trials in Huntington’s disease and Friedreich’s ataxia. Many other agents show good human tolerability. Theseagents therefore are promising candidates for further study as neuroprotective agents in PD. Ann Neurol 2003;53 (suppl 3):S39 –S48Parkinson’s disease (PD) is the second most common studies.5,6 There have been two studies, which demon-neurodegenerative disease, affecting approximately 1% strated that cybrids made from individuals with PDof the population older than age 65 years. It affects show selective reductions in complex I activity, asmore than one million people in the United States. well as increased free radical production, and an in-The cardinal clinical manifestations include bradykine- creased susceptibility to the MPTP metabolitesia, rest tremor, rigidity, and postural instability. The MPPϩ.7,8 However, one recent study of cybrids incause of the illness is a selective degeneration of dopa- PD failed to show significant and specific reductionsminergic neurons in the substantia nigra compacta. in complex I activity.9 As one might predict, cybrids Much evidence has accumulated implicating mito- made from patients with autosomal dominant PD as-chondrial defects in the pathogenesis of Parkinson’s sociated with ␣-synuclein mutations do not showdisease (PD). Investigations of 1-methyl-4-phenyl- complex I defects.101,2,3,6-tetrahydrodropyridine (MPTP) toxicity, which There has been some genetic evidence suggestingproduces parkinsonism in humans and laboratory ani- that complex I defects play a role in parkinsonism. Amals, showed that it is mediated by inhibition of re- family with multisystem degeneration with parkinson-spiratory complex I. MPTP first came to light as a con- ism has been reported with an 11778 mitochondrialtaminant of synthetic opiates, which had led to an DNA mutation that produces a complex I defect.11outbreak of parkinsonism in young individuals in Another family recently has been described that had asouthern California. MPTP is metabolized to MPPϩ, novel mitochondrial 12sRNA point mutation associ-which is preferentially taken up by dopamine neurons ated with parkinsonism, deafness, and neuropathy.12and selectively inhibits complex I of the electron trans- Cybrid studies have shown that a complex I defect isport chain.1 In idiopathic PD, there is a 30 to 40% associated with PD in one large family.13 In a study ofdecrease in complex I activity in the substantia nigra,2,3 monozygotic twins who were discordant for PD, sev-as well as reduced staining for complex I subunits, al- eral novel homeoplasmic sequence variants, includingthough preserved staining for other subunits of the two missense mutations in complex I subunits, wereelectron transport complexes.4 Reduced complex I ac- detected in four of the pairs.14 Furthermore, a total oftivity in PD platelets also has been reported in several 20 known polymorphisms effecting both complex IFrom the Department of Neurology and Neuroscience, Weill Med- Address correspondence to Dr Beal, Neurology Department, Newical College of Cornell University, New York Presbyterian Hospital, York Hospital–Cornell Medical Center, 525 East 68th Street, NewNew York, NY. York, NY 10021. E-mail: online Mar 24, 2003, in Wiley InterScience( DOI: 10.1002/ana.10479. © 2003 Wiley-Liss, Inc. S39
  2. 2. and transfer RNA mutations were found. Mitochon- Table. Bioenergetic Agents Effective in Parkinson’s Diseasedrial DNA sequences, however, tended to be identical, Modelsand the disease did not affect siblings of each pair. The Agent Proposed Mechanism of Actionpathogenic relevance of several of these mutationstherefore is questionable. In addition, an out-of-frame Coenzyme Q10 Cofactor of complex I, II, III and anti-cytochrome b gene deletion has been detected in a pa- oxidanttient with parkinsonism that was associated with im- Creatine Increases PCr, inhibits the MPTpaired complex III assembly and an increase in free Ginkgo biloba Antioxidant and preserves mitochon- drial functionradical production.15 Carnitine Facilitates fatty acid transport, increases In a direct sequencing study of complex I in transfer repirationRNA mutations, we recently observed no homoplasmic Nicotinamide Precursor of NADH, inhibitor of poly-mutations, suggesting either that the observed complex ADP-ribose polymeraseI defects are caused by heteroplasmic mutations or that Lipoic acid Coenzyme for ␣-ketoglutarate dehydro- genase, antioxidantthey may involve interactions between the nuclear ge-nome and the environment.16 We also recently directly PCr ϭ creatine/phosphocreatine; MPT ϭ mitochondrial permeabil-sequenced mitochondrial DNA from postmortem ity transition pore; NADH ϭ nicotinamide adenine dinucleotide.brain tissue of neuropathologically confirmed PD pa-tients.17 Once again, we did not detect any homoplas- with a loss of immunoreactivity for tyrosine hydroxy-mic mitochondrial DNA mutations associated with lase, dopamine transporter, and vesicular monoaminePD. This suggests that if mitochondrial DNA muta- transporter. Furthermore, the nigral neurons showedtions play a role in PD, the pathogenetic effects may be cytoplasmic inclusions that were highly suggestive ofvery complicated. It recently has been demonstrated Lewy bodies in that they stained with antibodies tothat nuclear background determines the biochemical ubiquitin and ␣-synuclein, and electron microscopyphenotype of the deafness-associated mitochondrial 12s showed a dense core surrounded by fibrillar elementsRNA mutation.18 A nuclear mitochondrial DNA mu- similar to Lewy bodies. The rats showed bradykinesia,tation affecting hearing impairment also has been dem- postural instability, unsteady gait, and some evidenceonstrated in mice.19 Furthermore, mitochondrial DNA of tremor that improved after treatment with the do-variant susceptibility to dilated cardiomyopathy is dif- pamine agonist, apomorphine. These findings suggestferent in two different human populations.20 These that rotenone can produce a selective degeneration offindings suggest that there are complex interactions be- nigrostriatal neurons consistent with the neuropatho-tween the nuclear and mitochondrial DNA, and that logical and clinical manifestations of PD. They are re-expression of a mitochondrial disease may occur only markable because they show that an inhibitor of com-in selective nuclear DNA backgrounds. This may make plex I of the electron transport chain, which actsthe study of mitochondrial DNA defects in parkinson- uniformly throughout the brain, produces a selectiveism extremely complex. degeneration of nigrostriatal neurons. They therefore A major finding suggesting that a complex I defect indicate the substantia nigra neurons are particularlymay play a critical role in the pathogenesis of PD susceptible to complex I inhibitors. This is consistentcomes from recent studies with the environmental with the findings of decreased complex I activity in PDtoxin rotenone. The possibility that pesticides and postmortem tissue and platelets. It has been suggestedother environmental toxins are involved in the patho- that the selective effects of rotenone may be mediatedgenesis of PD is suggested by several epidemiological by oxidative damage. This is also consistent with priorstudies.21,22 Patients with certain glutathione trans- studies showing extensive oxidative damage in the sub-ferase polymorphisms and exposure to pesticides seem stantia nigra of PD have an increased incidence of PD.23 Furthermore, If mitochondrial defects and oxidative damage play aan atypical PD syndrome has been described in associ- role in the pathogenesis of PD, then one would suspectation with the consumption of fruits and herbal tea that agents that may improve mitochondrial function orcontaining insecticides in the French West Indies.24 exert antioxidative effects could be neuroprotective.Rotenone is a natural occurring compound derived There are several agents that currently are under inves-from the roots of certain plant species, which has been tigation for their potential neuroprotective effects basedused as an insecticide for vegetables and to kill fish on their capacity to modify mitochondrial dysfunction.populations in lakes or reservoirs. Rotenone is known These include creatine, coenzyme Q10 (CoQ10), Ginkgoto be a high-affinity–specific inhibitor of complex I of biloba, nicotinamide, riboflavin, acetyl-carnitine, and li-the electron transport chain. poic acid (Table). Of these creatine, CoQ10, G. biloba A recent study examined the effects of rotenone and nicotinamide have all been assessed in the MPTPwhen infused intravenously into rats.25 The rats devel- model of PD. As noted above, MPTP toxicity in pri-oped progressive degeneration of nigrostriatal neurons mates replicates all the clinical signs of PD, includingS40 Annals of Neurology Vol 53 (suppl 3) 2003
  3. 3. tremor, rigidity, akinesia, and postural instability (re- creatine requires the amino acids arginine and glycineviewed in Beal26). as well as methionine. L-Arginine:glycine amidinotrans- ferase results in the production of guanidinoacetate,Mitochondria and Reactive Oxygen Species which, in turn, is methylated by S-adenosyl-In addition to their critical role in ATP synthesis, mi- methionine to produce creatine.32 Creatine is taken uptochondria are also the major source of reactive oxygen into brain and cardiac and skeletal muscle by aspecies (ROS) in most cell types. ROS include super- sodium-dependent transporter that has been clonedoxide, hydrogen peroxide (H2O2), and hydroxyl free and sequenced.33 The creatine/phosphocreatine (PCr)radical (•OH). It has been suggested that as much as system functions as a spatial energy buffer between the2% of the oxygen consumed by mitochondria is con- cytosol and mitochondria, using a unique mitochon-verted to superoxide, which then is converted by man- drial creatine kinase (CK) isoform.34 The mitochon-ganese superoxide dismutase into H2O2. Recently, drial CK isoform exists in the intermembrane space ofCuZn superoxide dismutase has been localized in the the mitochondria35 where it can convert from an oc-intermembrane space of mitochondria.27 This enzyme tameric to a dimeric form. The octameric form facili-may be important in preventing the exit of mitochon- tates the functional coupling between the porin mole-drially derived superoxide into the cytoplasm where it cule on the outer mitochondrial membrane and thecould damage critical cellular components. Approxi- adenine nucleotide translocase in the inner mitochon-mately 50% of superoxide derived from the electron drial membrane. Together, they form components oftransport chain is directed toward the intermembrane the mitochondrial permeability transition pore, whosespace.28 opening (which promotes apoptosis) is inhibited when The principal sites of production of ROS are mitochondrial CK is in the octameric form.36 It hasthought to be ubiquinone and an as yet undetermined been demonstrated that the octameric form is con-site in complex I. A recent study of rat brain mito- verted into the dimeric form in the presence of freechondria showed that the highest rate of mitochondrial radicals such as peroxynitrite thereby promoting open-ROS generation was observed in mitochondria respir- ing of the pore and apoptosis.37 Creatine administra-ing on the complex II substrate succinate.29 This pro- tion can protect mitochondrial CK from being con-duction of ROS appeared to be dependent on reverse verted into the dimeric form. Both creatine and PCrelectron transport through complex I, because it was can attenuate peroxynitrite-mediated mitochondrialinhibited by rotenone. It was also very sensitive to CK inactivation with consequent dimerization andchanges in mitochondrial membrane potential, being opening of the PTP.38 Another potential neuroprotec-inhibited by reductions in membrane potential such as tive effect of creatine administration is increasing glu-those associated with ATP generation. Mitochondria tamate uptake into synaptic vesicles, which has beenrespiring on the complex I substrates glutamate and shown to be energy dependent and which can be fu-malate produce very little ROS unless complex I is in- eled by PCr.39hibited by rotenone. It is noteworthy that although The potential of creatine to be protective can be il-ubiquinone produces ROS with both substrates, they lustrated in numerous models of neurodegeneration.represent a relatively minor component of the overall Creatine administration protects against glutamate andROS generation. ␤-amyloid toxicity in rat hippocampal neurons.40 Cre- Another recent study of isolated rat brain mitochon- atine is also beneficial in animal models of traumaticdria also showed that most of ROS generation pro- brain injury and cerebral ischemia.41,42 In addition,duced by succinate occurs at complex I through reverse preincubation of anoxic rat hippocampal slices withelectron transfer rather than at the ubiquinone site.30 creatine attenuated the decrease in PCr and ATP con-Similarly, complex I substrates produced very little tent.43ROS unless rotenone or antimycin A were present. In We initially studied the effects of oral creatine sup-these studies, the authors used the flavoprotein inhibi- plementation on striatal lesions produced by malonatetor diphenyliodonium, which has been shown to block and 3-nitropropionic acid, which are reversible and ir-succinate-induced H202 production, consistent with reversible inhibitors of complex II, respectively, andflavin mononucleotide being the source of mitochon- which model Huntington’s disease (HD).44 After ad-drial ROS rather than complex I iron-sulfur clusters. ministration of 3-nitropropionic acid there was attenu-Other data, however, favor some of the distal complex ation of ATP and phosphocreatine depletion, reducedI iron-sulfur clusters in generation of ROS. lactate accumulation, and reduced oxidative stress. We also examined the effects of creatine supplementationBioenergetics on MPTP-induced parkinsonism.45 We found that cre-Creatine is a guanidine compound found in meat- atine produced dose-dependent protection against do-containing products and produced endogenously by pamine loss, as well as an attenuation of neuron loss inthe liver, kidneys, and pancreas.31 The production of the substantia nigra of mice treated with MPTP. Sub- Beal: Bioenergetics in Parkinson’s S41
  4. 4. sequent work has shown that creatine significantly im- sevenfold increase in mitochondrial ␣-tocopherol con-proves survival and neuronal survival in transgenic tent, whereas CoQ10 administration increased both to-mouse models of both amyotrophic lateral sclerosis tal CoQ content and ␣-tocopherol by approximately(ALS) and HD.46 – 48 In the transgenic mouse model of fivefold. In these mice, the rate of superoxide radicalALS, there is also a delayed onset loss of neurons in the generation from submitochondrial particles was in-substantia nigra of approximately 20 to 25%. This loss versely related to ␣-tocopherol content, but unrelatedof neurons is of particular interest because it is late in to CoQ content. This study therefore provides in vivoonset and slowly progressive, similar to the cell loss evidence that at least part of the antioxidant effects ofthat occurs in human PD. This cell loss was completely CoQ are mediated by its ability to reduce theprevented by 1% creatine administration in mice stud- ␣-tocopheroxyl radical.ied at 110 days of age. A potentially very interesting effect of CoQ is its in- Another potential bioenergetic treatment for PD is teraction with mitochondrial uncoupling proteins.CoQ10, which recently has been studied in a small pi- CoQ has been shown to be an obligatory cofactor forlot clinical trial. CoQ10 is an important cofactor of the uncoupling protein function.61,62 This has been dem-electron transport chain where it accepts electrons from onstrated for uncoupling proteins 1, 2, and 3. The ef-complexes I and II.49,50 It consists of a quinone head fect originally was examined in liposomes; it subse-attached to a chain of isoprene units numbering 9 to quently was demonstrated that CoQ increased proton10 in various mammalian species. The quinone head conductance in rat kidney mitochondria that are oxi-can alternately assume three different redox states, dizing succinate.62 This increase required fatty acidsnamely, ubiquinone (Q) the fully oxidized form; the and was prevented by guanosine diphosphate. CoQ ac-free radical ubisemiquinone (•QH), which is the par- tivated proton conductance in these studies only whentially reduced form; and ubiquinol (QH2), the fully re- it was likely to be reduced to CoQH2. Activation wasduced form. Ubiquinone initially is reduced to the abolished by superoxide dismutase, indicating thatsemiquinone radical and then transfers electrons one at CoQ might mediate uncoupling through the produc-a time to complex III of the electron transport chain. tion of superoxide. This subsequently was shown to beCoQ10, which is also known as ubiquinone, serves as the case when CoQ was replaced by an exogenous sys-an important antioxidant in both mitochondrial and tem that generates superoxide using xanthine plus xan-lipid membranes.51,52 It is a particularly important an- thine oxidase.tioxidant in the inner mitochondrial membrane where This effect is important because uncoupling proteinsit can directly scavenge free radicals.53 Ubiquinol has may reduce the generation of free radicals,63 importantalso recently been documented to directly interact with mediators of oxidative damage. Through an interactionnitric oxide.54 There is also substantial evidence that with CoQ, uncoupling proteins (UCPs) may adjustubiquinol also may act as an antioxidant in concert electron transfer by regulating the quinone pool ac-with ␣-tocopherol,55 because it reduces ␣-tocopheroxyl cording to cellular context and needs.62 This may beradical back to ␣-tocopherol.53,56,57 In rat liver subject an adjustment in response to the formation of ROSto oxidant stress, mitochondrial CoQ9 levels are oxi- and biological parameters such as the need for ATPdized before the onset of massive lipid peroxidation production.64and the subsequent depletion of ␣-tocopherol.58 In rat CoQ10 has been shown to exert neuroprotective ef-mitochondria, supplementation with succinate results fects in the central nervous system in several in vivoin a reduction of CoQ to ubiquinol, thereby preserving models. It produces significant protection against ex-␣-tocopherol concentrations during oxidation.51 This perimental ischemia,65 attenuating ATP and glutathi-suggests that ␣-tocopherol is the direct radical scaven- one depletion as well as neuronal injury in the hip-ger, and ubiquinol primarily acts to regenerate pocampus. We found that oral administration of␣-tocopherol. Another interaction occurs between di- CoQ10 significantly attenuated ATP depletion andhydrolipoic acid and CoQ.59 Dihydrolipoic acid re- produced dose-dependent neuroprotective effectsduces ubiquinone to ubiquinol by the transfer of a pair against striatal lesions produced by the mitochondrialof electrons, thereby increasing the antioxidant capacity toxin malonate.66 CoQ10 administration also signifi-of ubiquinol in biomembranes. Lipoic acid has been cantly attenuated striatal lesions produced by aminoxy-shown to maintain a normal ratio of reduced to oxi- acetic acid.67 The role of CoQ10 has also been studieddized ubiquinone after MPTP administration in in MPTP toxicity. We demonstrated significant protec-vivo.60 tion against dopamine depletion and loss of tyrosine The effects of oral supplementation with CoQ or hydroxylase immunostained neurons in 24-month-old␣-tocopherol on the rate of mitochondrial superoxide mice treated with MPTP.68 We also found that CoQ10radical generation have been examined in skeletal mus- produces marked neuroprotective effects against thecle, liver, and kidney of 24-month-old mice.51 In this systemic administration of the mitochondrial toxinstudy, the administration of ␣-tocopherol produced a 3-nitroproprionic acid.69 This is an irreversible inhibi-S42 Annals of Neurology Vol 53 (suppl 3) 2003
  5. 5. tor of succinate dehydrogenase that produces selective 44% as assessed by the UPDRS. A larger phase IIIstriatal lesions in both rats and primates, closely resem- study is required to determine whether these resultsbling those found in HD. Administration of CoQ10 can be replicated. Interestingly, there was a dose-for 1 week before coadministration of 3-nitropropionic dependent increase in plasma CoQ10 levels, with theacid resulted in a 90% neuroprotection against the stri- largest increase occurring between the 600 andatal lesions and significantly attenuated the reductions 1,200mg doses, consistent with the magnitude ofin reduced CoQ9 and reduced CoQ10. More recently, changes in clinical efficacy. These findings indicate thatwe have demonstrated that CoQ10 produces neuropro- CoQ10 is an extremely promising agent for study as atective effects in transgenic mouse models of both ALS neuroprotectant for PD.and HD.69,70 CoQ10 and its analog, idebenone, also have been On the basis of these results, we, and others, have studied in patients with Friedreich’s ataxia where it hasexamined the effects of CoQ10 in patients with neuro- been reported to significantly reduce cardiac mass76,77degenerative diseases. We initially tested the oral ad- and to significantly improve cardiac and skeletal mus-ministration of 360mg daily of CoQ10 on elevated oc- cle bioenergetics.78 The latter study examined the ef-cipital cortex lactate concentrations in patients with fects of 6 months of treatment with 400mg daily ofHD.71 In this study, we obtained lactate concentra- CoQ10 and vitamin E 2,100IU/day in 10 Friedreich’stions before, during, and after the discontinuation of ataxia patients using in vivo phosphorous magnetic res-CoQ therapy. CoQ10 treatment produced a 37% re- onance spectroscopy. After 3 months of treatment, theduction in occipital cortex lactate concentrations, cardiac PCr to ATP ratio showed a mean increase ofwhich was reversed after discontinuation of therapy. 178%, and the maximum rate of skeletal muscle mito-Recently, a clinical trial was performed by the Hun- chondrial ATP production was increased by 139% intington’s Study Group, which examined the effects of comparison with their respective baseline values. TheseCoQ10 with or without the N-methyl-D-aspartate re- improvements were sustained after 6 months of ther-ceptor antagonist remacemide.72 The trial encompassed apy. There were, however, no significant improvements340 patients who were treated for 30 months. Patients on neurological or echocardiographic evaluation. Thesewere randomized to CoQ10 600mg daily, remacemide, findings also warrant a larger trial of Friedreich’s ataxiaor a combination of the two in a 2 ϫ 2 factorial de- patients who can be studied over a longer time frame.sign. In this study, remacemide demonstrated no effi- Several other agents that modulate cerebral energycacy. Administration of CoQ10 resulted in a 14% slow- metabolism or that exert antioxidant effects are alsoing of disease progression as assessed by a total potential neuroprotective treatments for PD. G. bilobafunctional capacity rating scale, but the effect did notreach significance because the study was not powered is a plant extract composed of a complex chemical mix-to detect an effect of this magnitude. Nevertheless, ture that exerts neuroprotective effect against models ofthere was significant improvement on several secondary mitochondrial damage and oxidative stress. It has beenoutcome measurements. shown to significantly reduce the generation of lipid Studies of PD patients have shown that the ratio of peroxides in brain homogenates and in rat brain syn-reduced to oxidized CoQ10 is significantly reduced in aptosomes,79 and to protect primary cultures of cere-platelets,73 although in another study serum levels were bellar neurons against oxidative damage80 and hip-unaltered.74 We measured CoQ10 levels in mitochon- pocampal neurons from toxicity produced by eitherdria isolated from platelets of PD patients and found hydrogen peroxide or nitric oxide.81 G. biloba has beensignificant reductions that directly correlated with de- reported to protect dopamine neurons from MPTP-creases in complex I activity.75 Oral administration of induced neurotoxicity82 and to be effective in modelsCoQ10 to PD patients was well tolerated and resulted of focal and global ischemia. Finally, we found that significant, dose-dependent increases in plasma biloba extract has beneficial effects on survival in trans-CoQ10 levels. genic mice that model ALS.83 We recently completed a phase II clinical study of Nicotinamide is a precursor of nicotinamide adenineCoQ10 in de novo PD patients (Parkinson Study dinucleotide (NADH), which is a substrate for com-Group, unpublished findings). Patients were treated plex I of the electron transport chain. It is also an in-with placebo or 300, 600, or 1,200mg of CoQ10 for hibitor of polyADP-ribose polymerase, an enzyme that10 months. The primary outcome measure was the is activated by DNA damage and that, in turn, depleteschange in the Unified Parkinson’s Disease Rating Scale both NADH and ATP. Several studies have shown(UPDRS) between baseline and final visits. Secondary that nicotinamide, like other polyADP-ribose polymer-outcome measures were changes in complex I activity ase inhibitors, protects against MPTP neurotoxicity.84of the mitochondrial electron transport chain in plate- Similar results have been observed in mice with alets and serum CoQ10 levels. This study demonstrated knockout of polyADP-ribose polymerase.85 Our stud-a dose-dependent reduction in disease progression of ies further demonstrate that nicotinamide attenuates Beal: Bioenergetics in Parkinson’s S43
  6. 6. neuronal injury and ATP depletion produced by focal Conclusionsischemia, malonate, and MPTP.66,86,87 There is substantial evidence based on postmortem Carnitine and acetyl-L-carnitine are agents that facil- studies of PD tissue as well as experimental animalitate the entry and exit of fatty acids from mitochon- models indicating that mitochondrial dysfunction anddria. Carnitine facilitates the entry of long chain fatty oxidative damage play a role in the pathogenesis of PD.acids into mitochondria for subsequent ␤-oxidation In the laboratory, experimental animal models of PDand the removal of short chain and medium chain fatty have been produced with both MPTP and rotenone,acids that accumulate during normal and abnormal which are known to inhibit complex I of the electronmetabolism. Short and medium chain fatty acids are transport chain and to increase oxidative damage. Sev- eral agents are now available that can modulate cellularesterified to carnitine by the action of carnitine acetyl- energy metabolism and that thereby may exert antioxi-transferase. The acetylcarnitine esters are then trans- dative and protective effects. Several of these agentsported out of mitochondria by carnitine acetylcarnitine have been shown to produce significant neuroprotec-translocase. Acetyl- L-carnitine may have better brain tive effects in the MPTP model of PD, including cre-penetration and may be useful as an agent for elevating atine, CoQ10, G. biloba, nicotinamide, and acetyl-L-brain carnitine levels. carnitine. Creatine has been shown to produce Carnitine delays mitochondrial depolarization in re- significant neuroprotective effects in several animalsponse to a variety of stressors including oxidative models of neurodegenerative diseases and is well toler-damage.88 Acetyl- L-carnitine increases cellular respira- ated in man. Similarly, CoQ10 is effective in severaltion, mitochondrial membrane potential, and cardio- animal models of neurodegenerative diseases and re-lipin levels in hepatocytes of 24-month-old rats.89 cently has shown very promising results in a phase IIThese biochemical effects are paralleled by increases in study in PD patients. Many of the other agents de-ambulatory activity of aged rats. Carnitine and acetyl- scribed above also show good human tolerability.L-carnitine attenuate neuronal damage produced by These observations raise the possibility that these3-nitroproprionic acid, rotenone, and MPTP in agents, either alone or in combination, are worthy ofvitro.90,91 After ischemia reperfusion in rats, acetyl-L- further study as possible neuroprotective agents in PD.carnitine resulted in a more rapid recovery of ATP andPCr and lactate levels.92 Lipoic acid is a disulfide compound that is found This work was supported by grants from National Institute of Neu-naturally in mitochondria as a coenzyme for pyruvate rological Disorders and Stroke, the Department of Defense, and the Parkinson’s Disease Foundation.dehydrogenase and ␣-ketoglutarate dehydrogenase andalso has antioxidant effects. It has been shown to pro- The secretarial assistance of S. Melanson is gratefully acknowledged.tect against peroxynitrite-induced nitration and␣-antiproteinase inactivation and is neuroprotective inrodent models of both focal and global cerebral isch- Referencesemia.93–96 We found that ␣-lipoic acid exerts signifi- 1. Nicklas WJ, Vyas I, Heikkila RE. Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-cant neuroprotective effects in a transgenic mouse pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-model of HD.97 In humans, a dose of 600mg/day de- 1,2,3,6-tetrahydropyridine. Life Sci 1985;36:2503–2508.creased plasma indices of oxidative stress, low-density 2. Bindoff LA, Birch-Martin M, Cartlidge NEF, et al. Mitochon-lipoprotein oxidation, and urinary isoprostanes.98 drial function in Parkinson’s disease. Lancet 1989;1:49. 3. Schapira AHV, Cooper JM, Dexter D, et al. Mitochondrial Supplementation with ␣-lipoic acid in old rats im- complex I deficiency in Parkinson’s disease. J Neurochemproved ambulatory activity, decreased oxidative damage, 1990;54:823– 827.and improved mitochondrial function.99,100 Recent 4. Hattori N, Tanaka M, Ozawa T, Mizuno Y. Immunohisto-studies of lipoic acid in combination with acetyl-L- chemical studies on complexes I, II, III and IV of mitochon- dria in Parkinson’s disease. Ann Neurol 1991;30:563–571.carnitine have demonstrated significant improvements in 5. Haas RH, Nasirian F, Nakano K, et al. Low platelet mito-mitochondrial function in old rats.101 This was shown chondrial complex I and complex II/III activity in early un-to occur in the absence of any increase in oxidative dam- treated Parkinson’s disease. Ann Neurol 1995;37:714 –722.age, which is observed when acetyl-L-carnitine is admin- 6. Parker WD Jr, Boyson SJ, Parks JK. Abnormalities of the elec- tron transport chain in idiopathic Parkinson’s disease. Annistered alone. 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  9. 9. 84. Cosi C, Marien M. Decreases in mouse brain NADϩ and ATP 101. Liu J, Killilea DW, Ames BN. Age-associated mitochondrial induced by 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine oxidative decay: improvement of carnitine acetyltransferase (MPTP): prevention by the poly(ADP-ribose) polymerase in- substrate-binding affinity and activity in brain by feeding old hibitor, benzamide. Brain Res 1998;809:58 – 67. rats acetyl-L-carnitine and/or R-alpha-lipoic acid. Proc Natl 85. Mandir AS, Przedborski S, Jackson-Lewis V, et al. Poly(ADP- Acad Sci USA 2002;99:1876 –1881. ribose) polymerase activation mediates 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Proc Natl Acad Sci USA 1999;96:5774 –5779. Discussion 86. Ayoub IA, Lee EJ, Ogilvy CS, et al. Nicotinamide reduces Rascol: Do you have any experiments combining mul- infarction up to two hours after the onset of permanent focal tiple possible neuroprotective agents that are thought cerebral ischemia in Wistar rats. Neurosci Lett 1999;259: to act via different mechanisms? Do they act in an ad- 21–24. 87. Schulz JB, Henshaw DR, Matthews RT, Beal MF. Coenzyme ditive or synergistic way? Q10 and nicotinamide and a free radical spin trap protect Beal: Yes. In the Huntington’s mice we have been against MPTP neurotoxicity. Exp Neurol 1995;132:279 –283. able to show that there are additive effects of remace- 88. Di Lisa F, Bobyleva-Guarriero V, Jocelyn P, et al. Stabilising mide and CoQ. You can go from a 15 to 20% effect action of carnitine on energy linked processes in rat liver mi- on survival to a 33% effect. You also can demonstrate tochondria. Biochem Biophys Res Commun 1985;131: additive effects for behavior and weight loss. We also 968 –973. have tested a combination of four different agents in 89. Hagen TM, Ingersoll RT, Wehr CM, et al. Acetyl-L-carnitine fed to old rats partially restores mitochondrial function and this model: a transglutamenase inhibitor, a nitric oxide ambulatory activity. Proc Natl Acad Sci USA 1998;95: synthase inhibitor, remacemide, and CoQ . When we 9562–9566. use the four agents, we can get even better protective 90. Snyder JW, Kyle ME, Ferraro TN. L-carnitine delays the kill- effects with increases in survival up to 46% in the ing of cultured hepatocytes by 1-methyl-4-phenyl-1,2,3,6- Huntington’s transgenics. Therefore, it appears that we tetrahydropyridine. Arch Biochem Biophys 1990;276: can get increased benefits with multiple agents just as 132–138. they have found with cancer chemotherapy. 91. Virmani MA, Biselli R, Spadoni A, et al. Protective actions of L-carnitine and acetyl-L-carnitine on the neurotoxicity evoked Marek: In the study that was performed in Hunting- by mitochondrial uncoupling or inhibitors. Pharmacol Res ton patients, remacemide and CoQ were ineffective. 1995;32:383–389. So, how reliable are these models in predicting the re- 92. Aureli T, Miccheli A, Di Cocco ME, et al. Effect of acetyl-L- sponse in humans? carnitine on recovery of brain phosphorus metabolites and lac- Beal: The problem may have been dosing. We chose tic acid level during reperfusion after cerebral ischemia in the a dose in the mice that was based on what we previ- rat—study by 13P- and 1H-NMR spectroscopy. Brain Res ously had found to be protective against acute excito- 1994;643:92–99. 93. Muller U, Krieglstein J. Prolonged pretreatment with alpha- toxic lesions. In the humans, the dose was limited by lipoic acid protects cultured neurons against hypoxic, tolerability. Patients became drowsy and developed hal- glutamate-, or iron-induced injury. J Cereb Blood Flow Metab lucinations as has been found with other N-methyl-D- 1995;15:624 – 630. aspartate receptor antagonists. The problem therefore 94. Panigrahi M, Sadguna Y, Shivakumar BR, et al. ␣-Lipoic acid may be that in humans you cannot get up to those protects against reperfusion injury following cerebral ischemia dose levels that are neuroprotective in rodents. in rats. Brain Res 1996;717:184 –188. Olanow: Have you tried any specific N-methyl-D- 95. Whiteman M, Tritschler H, Halliwell B. Protection against peroxynitrite-dependent tyrosine nitration and ␣1-antiproteinase aspartate receptor subunit blockers that might avoid inactivation by oxidized and reduced lipoic acid. FEBS Lett the side effects that occur when the entire receptor is 1996;379:74 –76. blocked? 96. Wolz P, Krieglstein J. Neuroprotective effects of alpha-lipoic Beal: I think that is a promising strategy that might acid and its enantiomers demonstrated in rodent models of focal work. Some have been tested in animals and they do cerebral ischemia. Neuropharmacology 1996;35:369 –375. have neuroprotective effects, but none have yet been 97. Andreassen OA, Ferrante RJ, Dedeoglu A, Beal MF. Lipoic tested in humans. acid improves survival in transgenic mouse models of Hun- tington’s disease. Neuroreport 2001;12:3371–3373. Kordower: In the Huntington’s mouse model that 98. Marangon K, Devaraj S, Tirosh O, et al. Comparison of the you use, I was very impressed by the loss of cells and effect of alpha-lipoic acid and alpha-tocopherol supplementa- the loss of striatal volume, and yet my understanding is tion on measures of oxidative stress. Free Radic Biol Med that there is very little striatal degeneration in the R6/2 1999;27:1114 –1121. mice. Could you expand upon that? 99. Kriegstein AR. Cortical neurogenesis and its disorders. Curr Beal: Well, they do in fact have profound striatal Opin Neurol 1996;9:113–117. atrophy, but initial reports did suggest there was no100. Liu J, Head E, Gharib AM, et al. Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA cell loss. Now, the reason you have striatal atrophy is oxidation: partial reversal by feeding acetyl-L-carnitine and/or twofold. One is the overall cell bodies shrink and the R-alpha -lipoic acid. Proc Natl Acad Sci USA 2002;99: other is cell loss. There is good evidence for cell shrink- 2356 –2361. age in this model, and this is probably the major factor Beal: Bioenergetics in Parkinson’s S47
  10. 10. leading to striatal atrophy. However, electronmicros- bral cortex and specific increases in the motor cortex incopy studies also indicate that there is some degree of ALS patients. As to the mechanism responsible forcell loss, but you cannot pick it up by routine light neuroprotection? One possibility is that it simply in-microscopy. creases levels of phosphocreatine. It is also possible that Kordower: In your neuroprotection studies in Hun- it could have direct effects on the mitochondrial tran-tington’s disease, do you use cell size as the primary sition pore working through the mitochondrial CK.outcome measure? However, we have mice now that have a knockout of Beal: We have shown that we can protect against the mitochondrial CK in whom we still see protection withloss of cell size in these studies with some neuroprotec- creatine. This would argue that creatine is not actingtive agents. by way of a direct effect on mitochondria. Kordower: Does creatine treatment lead to hypertro- Tatton: We examined the capacity of creatine tophy in addition to preventing cell shrinkage? block apoptosis in four different types of cells in tissue Beal: We protected against shrinkage, and the cells culture. We found that maximal protection was ob-were not larger than normal. tained with concentrations of approximately 10Ϫ6 mo- Isacson: In the MPTP-treated mice that you study, lar. However, antiapoptotic effects were largely blockedyou presented data indicating that many different by protein synthesis inhibitors, suggesting that the drugagents can block degeneration. However, if you do acts through a transcriptional mechanism. We did findnothing, most of the dopaminergic neurons will re- that creatine upregulated CK, but I believe that thecover. So, it is an appealing model because you can effects are caused by a transcriptional action of creatinedemonstrate that some agents have powerful neuropro- and not by an energetic action.tective effects, but as Ken Marek pointed out, I am not Beal: That is very possible, I agree.sure that the same conditions apply in PD patients or Olanow: Have you tested creatine as well as Co-that you can assume that you will obtain comparable enzyme Q in clinical trials of PD?results. Beal: Schults and colleagues have tested CoQ in a Stocchi: Does oral creatine gain access to the central prospective double-blind clinical trial in PD. The studynervous system and what do you think is the mecha- is now completed but not yet published. Creatine, Inism of action for neuroprotection? There was onestudy in Italy of athletes that failed to demonstrate any am told, is in a trial for PD in Munich but I have noincrease in power, although they felt less fatigue. data to provide. There are also two trials of creatine in Beal: I think the data are relatively solid that the ALS and a pilot trial in Huntington’s disease that isdrug has no effect on long-term athletic performance. combined with imaging. None of these results are cur-On the other hand, with very high output short-term rently available.athletic performance there are data indicating improved Olanow: In the CoQ study, which type of patientperformance and an enhanced rate of regeneration of was studied and what was the primary endpoint?phosphocreatine as demonstrated by phosphorus nu- Beal: We studied patients with early PD who wereclear magnetic resonance studies. As a result, most high untreated and remained untreated throughout theoutput athletes in the United States, such as sprinters study. Change from baseline in UPDRS is the primaryand baseball, football, and hockey players, are taking it. end point. Secondary end point is complex I activity inAs to whether it get into the central nervous system? platelets.We have demonstrated that it does based on direct bio- Olanow: Are there renal complications with high-chemical measurements and phosphorus nuclear mag- dose creatine and are there any problems with CoQ?netic resonance. We have performed these studies in Beal: There have been reports of renal problems atboth the mouse and patients and have shown that we doses of 20gm/day, probably because such a large loadget an approximately 10 to 15% increase in creatine was being placed on the kidneys. In the trial, we usedand phophocreatine levels in the brain. a dose of 5gm/day, which is very well tolerated. CoQ Schapira: In what brain area does this occur? can be administered in doses of up to 1,200mg/day Beal: We have primarily found increases in the cere- without tolerability problems.S48 Annals of Neurology Vol 53 (suppl 3) 2003