Excitotoxicity and Nitric Oxide in Parkinson's Disease Pathogenesis

M. Flint Beal, MD

A potential role for excitotoxic processes in Parkinson's disease (PD) has been strengthened by the recent observations that there appears to be a mitochondrially encoded defect in complex I activity of the electron transport chain. An impairment of oxidative phosphorylation will enhance vulnerability to excitotoxicity. Substantia nigra neurons possess TV-methyl-D-aspartate receptors and there are glutamatergic inputs into the substantia nigra from both the cerebral cortex and the subthalamic nucleus. After activation of excitatory amino acid receptors, there is an influx of calcium followed by activation of neuronal nitric oxide (NO) synthase, which can then lead to the generation of peroxynitrite. Consistent with such a mechanism, studies of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine neurotoxicity in both mice and primates have shown that inhibition of neuronal NO synthase exerts neuroprotective effects. Studies utilizing excitatory amino acid receptor antagonists have been inconsistent in mice but show significant neuroprotective effects in primates. These results raise the prospect that excitatory amino acid antagonists for neuronal NO synthase inhibitors might be useful in the treatment of PD.

Beal MF. Excitotoxicity and nitric oxide in Parkinson's disease pathogenesis. Ann Neurol 1998;44(Suppl 1):S110-S114

A potential role for excitotoxicity in Parkinson's disease (PD) is an intriguing possibility. This possibility is sup­ported by the recent observation that there appear to be systemic defects in complex I activity of the electron transport chain. This has been most clearly demon­strated by recent studies using cybrid cell lines. These are discussed elsewhere in this supplement by Schapira et al. The initial studies demonstrating that complex I defects could be transferred from PD platelets into mitochondrial-deficient cell lines were carried out by Swerdlow and colleagues. These authors demonstrated that the complex I defect that had been detected in platelets of PD patients could be transferred into mitochondrial-deficient cell lines. Furthermore, after transfer of these defects there was increased free radical production. There was also an increase in susceptibility of these cell lines to toxicity mediated by 1-methyl-phenylpyridinium ions (MPP ). This provides further evidence for an interaction between a potential envi­ronmental factor and a genetic defect. The cell death induced by MPP was apoptotic in nature. These cell lines also show markedly impaired calcium buffering to a variety of stimuli. This is consistent with other ob­servations that mitochondrial defects are associated with impaired intracellular calcium buffering.

The observation that there is impaired energy me­tabolism that may be due to a mitochondrial defect in PD raises the possibility that slow or weak excitotoxic­

ity may contribute to the ensuing neuronal degenera­tion. The possibility that impaired energy metabolism could result in excitotoxicity was originally demon­strated by the work of Novelli and coworkers. They showed that inhibitors of either oxidative phosphoryla­tion or of the sodium-potassium ATPase allow gluta-mate to become neurotoxic at concentrations that or­dinarily exert no neurotoxicity. The mechanism for this was presumed to be a reduction in ATP, which is cru­cial for maintaining the normal resting potential of the cell membrane. When the cell membrane is depolarized from its usual —90 mV to between —60 and —30 mV, the voltage-dependent magnesium block of the iV-methyl-D-aspartate (NMDA) receptor is relieved, leading to persistent receptor activation. Consistent with this notion, Zeevalk and Nicklas3 carried out el­egant experiments in the cultured chick retina. They showed that partial neuronal depolarization induced by inhibitors of either glycolysis or oxidative phosphoryla­tion led to N M D A receptor activation and cell death in the absence of any increase in extracellular glutamate concentrations. In follow-up experiments they showed that graded titration of the membrane potential with potassium mimicked the toxicity produced by graded metabolic inhibition. It is therefore possible that sim­ilar mechanisms could occur in the pathogenesis of PD. The substantia nigra neurons in humans contain N M D A receptors that might be activated by such a

From the Neurochemistry Laboratory, Neurology Service, Massa­chusetts General Hospital and Harvard Medical School, Boston, MA.

Address correspondence to Dr Beal, Neurology Service/Warren 408, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114.

SI 10 Copyright © 1998 by the American Neurological Association

mechanism, and these are depleted in PD.5 NMDA receptors in the substantia nigra are more sensitive than AMPA receptors to endogenous glutamate.6

After activation of NMDA receptors there is an in­flux in intracellular calcium, which has been closely linked to the ensuing cell death. This was originally demonstrated by the work of Choi.7 Measurement of ionized calcium with low-affinity fluorescent calcium indicators confirmed that levels predict excitotoxic neu­ronal death.8 After activation of NMDA receptors, the increases in intracellular calcium are buffered by mito­chondria. It has been demonstrated that accumulation of calcium in mitochondria followed by mitochondrial depolarization are critical features of excitotoxic cell death.9,10 The increases in accumulation of calcium in mitochondria are associated with increased free radical production. Increases in cellular calcium are also as­sociated with activation of nitric oxide synthase (NOS). The increases in generation of Superoxide and nitric oxide (NO) can lead to the production of per-oxynitrite.12 Peroxynitrite is produced by the direct chemical interaction of Superoxide radical with NO. Peroxynitrite appears to be a critical mediator of cell death both in vitro and in vivo. Peroxynitrite can me­diate one or two electron oxidations. These can lead to oxidation of proteins, lipids, or DNA. Peroxynitrite can also mediate tyrosine nitration. Tyrosine nitration can impair the activity of a variety of enzymes and can also inactivate tyrosine kinases, which are utilized by a variety of growth factors.

The role of NO in excitotoxicity was originally re­ported by Dawson and colleagues.13 They showed that after brief exposure of cultured cortical neurons to NMDA, inhibitors of NOS, calmodulin antagonists, and reduced hemoglobin, which scavenges NO, mark­edly attenuated neurotoxicity. Removal of l.-arginine from the medium also blocked toxicity. NOS inhibi­tors also block glutamate neurotoxicity in cultured stri-atal and hippocampal neurons. Pretreatment of the cul­tures with quisqualate, which preferentially kills NOS neurons, blocked glutamate neurotoxicity in cultured cortical and striatal neurons.14 Interestingly, there was no effect of NOS inhibitors on kainate and AMPA neurotoxicity in vitro. In more recent studies, Dawson and colleagues15 demonstrated that cultured neurons from mice with a knockout of the neuronal NOS gene were markedly resistant to NMDA but not to AMPA or kainate neurotoxicity. Our own studies are consis­tent with these results. We studied the effects of a se­lective inhibitor of neuronal NOS in vivo. We found that 7-nitroindazole protected against striatal lesions produced by NMDA but had no effect on lesions pro­duced by either kainate or AMPA.'' Furthermore, we recently demonstrated that mice deficient in the neu­ronal NOS gene are resistant to NMDA striatal le­sions.17 This resistance correlated with a reduction in

3-nitrotyrosine levels induced by the toxin compared to wild-type animals. These observations strongly im­plicate peroxynitrite in the pathogenesis of the cell damage.

The best-established animal model of PD is that pro­duced by l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). In humans and in nonhuman primates, MPTP produces clinical, biochemical, and neuropatho-logic changes that model those in idiopathic PD. The pathogenesis of the lesions involves the conversion of MPTP to an active metabolite, MPP + , by mono-amine oxidase (MAO) B.19 MPP+ is selectively taken up by the dopamine transporter, which is subsequently accumulated within mitochondria. There it disrupts oxidative phosphorylation by binding to or near the rotenone-binding site on complex I. This can result in increased intracellular calcium, followed by increased free radical production by mitochondria and activa­tion of neuronal NOS. This can, in turn, lead to in­creased production of NO, which can then react with Superoxide to produce peroxynitrite.

Direct evidence for excitotoxicity and MPTP toxic­ity has been controversial. MK-801 does not protect mesencephalic dopaminergic neurons from MPP in vitro. However, these cells may be less dependent on oxidative phosphorylation than those in vivo. ' ' Tur-ski and colleagues22 initially reported that the neuro-toxic effects of intranigral MPP+ in vivo can be almost completely prevented by either systemic or intranigral administration of a variety of NMDA antagonists, in­cluding CPP and MK-801. Subsequent studies by Son-salla et al,23 using a similar dosing regimen, failed to confirm these results. Other studies found no protec­tion with MK-801 against MPTP-induced dopamine depletion in mice.24 However, Chan et al25 found that MK-801 produced partial but significant attenuation of MPTP-induced dopamine depletions in mice. Sim­ilarly, we found that administration of MK-801 or ei­ther of two competitive NMDA antagonists for 24 hours partially but significantly attenuated striatal do­pamine depletions at both 24 hours and 1 week.2f' The glutamate release inhibitor lamotrigine also produces dose-dependent neuroprotection against MPTP-induced dopamine depletion in mice. 7 Riluzole, an­other putative glutamate release inhibitor, attenuates 6-hydroxydopamine neurotoxicity in rats and MPTP neurotoxicity in mice.28'29 We found that intrastriatal administration of MPP+ leads to dopamine depletion that was partially attenuated by prior decortication at 1 week, and that it was blocked at 24 hours after sys­temic administration of MK-801 for 24 hours.30 Prior decortication or administration of MK-801 for 48 hours by Alzet pump protected against MPP -induced loss of neurons in the substantia nigra.31

Two studies examined the effects of NMDA antag­onists against MPTP-induced dopaminergic neurotox-

Beal: Excitotoxicity in Parkinsons Disease S i l l

icity in primates. Zuddas et al32 showed that repetitive low doses of MK-801 partially attenuated dopamine depletions and protected against neuronal loss in the substantia nigra. Similarly, Lange et al"" showed that CPP, a competitive NMDA antagonist, can signifi­cantly protect against depletion of substantia nigra do-paminergic neurons and striatal dopamine depletions in primates. Systemic administration of riluzole, a pu­tative glutamate release inhibitor, also showed some neuroprotection against MPTP toxicity in primates."

We investigated the effects of 7-nitroindazole, a rel­atively selective inhibitor of neuronal NOS, in MPTP toxicity in mice.35 We found that 7-nitroindazole dose-dependently protected against MPTP-induced dopa­mine depletion with two different dosing regimens of MPTP that produce various degrees of dopamine depletion. At a dose of 50 mg/kg, 7-nitroindazole produced almost complete protection in both dosing regimens. We also demonstrated that after administra­tion of MPTP there was a significant increase in 3-nitrotyrosine levels. After administration of 7-nitro­indazole these increases in 3-nitrotyrosine levels were blocked. Our observations were confirmed and ex­tended by the work of Przedborski and colleagues," ' who confirmed that 7-nitroindazole showed marked neuroprotection against MPTP-induced dopamine de­pletions. Furthermore, these authors demonstrated that it also protected against depletion of tyrosine hydroxy-lase neurons or increase in silver-stained neurons in the substantia nigra. They found that mice that were defi­cient in neuronal NOS were resistant to MPTP neu-rotoxicity. In their studies there was no effect of 7-nitroindazole on MPP levels after administration of MPTP. We subsequently utilized another relatively selec­tive neuronal NOS inhibitor, S-methyl-thiocitrulline.37

This compound works at a different site on the neuronal NOS enzyme, and it also produced significant neuro­protection against MPTP-induced dopamine depletion. The protection, however, was not as marked as that observed with 7-nitroindazole. L-Nitroarginine, which is a nonspecific NOS inhibitor, protected against MPP "-induced increases in hydroxyl radical generation and partially protected against MPTP-induced dopa­mine depletion."

We carried out studies on the effects of 7-nitroinda-zole on MPTP-induced neurotoxicity in baboons." We utilized an acute dosing regimen of MPTP. This resulted in a 94 to 98% depletion of dopamine in the putamen and caudate nucleus, respectively. 7-Nitro-indazole administered alone had no effect on dopamine levels. When 7-nitroindazole was co-administered with MPTP at a dose of 25 rng/kg bid, there was essentially complete protection against MPTP-induced dopamine depletion. There was also significant protection against the loss of tyrosine hydroxylase-positive neurons in the substantia nigra. Furthermore, administration of 7-nitro­

indazole protected against motor deficits, as assessed by total distance traveled and tangential velocity of move­ment. 7-Nitroindazole also produced protection against cognitive deficits as assessed by the object detour re­trieval test.

These results were recently called into question by a study suggesting that 7-nitroindazole also inhibits MAO-B and that this plays a major role in its neuro­protection against MPTP neurotoxicity. ° These au­thors demonstrated that 7-nitroindazole almost com­pletely blocked dopamine depletion produced by a single subcutaneous injection of 20 mg/kg MPTP. However, they found that there was a significant re­duction in striatal levels of MPP+. The MPP+ levels produced by a dose of 40 mg/kg MPTP in the pres­ence of 7-nitroindazole were similar to those produced by 20 mg/kg MPTP alone. The authors noted that, with comparable MPP levels, 7-nitroindazole produced a 20% protection against dopamine deple­tion. One potential confounding difficulty with these results, however, is that the authors administered the 7-nitroindazole intraperitoneally rather than subcutane-ously, which will result in rapid absorption. 7-Nitro­indazole administered at a dose of 50 mg/kg can sig­nificantly reduce cerebral blood flow. It is therefore possible that some of the effect on MPP+ levels ob­served may have been due to impaired uptake of MPTP into the brain.

We carried out studies to examine the effects of neu­ronal NOS (nNOS) inhibition on MPP+ induced sub­stantia nigra degeneration. ' After injection of MPP+

into the striatum it is retrogradely transported to the substantia nigra, resulting in ipsilateral degeneration of substantia nigra neurons. We examined the effects of MPP -induced striatal lesions in substantia nigra de­generation in mutant mice lacking either the nNOS or the endothelial NOS (eNOS) gene. The substantia nigra degeneration is caused by retrograde transport of MPP to the substantia nigra, because comparably sized excitotoxic lesions of the striatum do not result in depletion of substantia nigra neurons. We found that MPP -induced neuronal degeneration was significantly attenuated in the nNOS-deficient mice but not in the eNOS-deficient mice. This parallels findings in exper­imental stroke models and our findings with malonate-induced neuronal degeneration in the striatum. " Fur­thermore, we found that MPP causes increased striatal 3-nitrotyrosine concentrations in controls and in eNOS-deficient mice, which are significantly atten­uated in the nNOS-deficient mice. 3-Nitrotyrosine is believed to be a relatively specific neurochemical marker for peroxynitrite-mediated nitration. 3 We also found significant increases in DHBA to salicylate, an index of free radical generation, in control and in eNOS-deficient mice but not in nNOS-deficient mice. This, again, suggests an effect of peroxynitrite, which

S112 Annals of Neurology Vol 44(Suppl 1) September 1998

can generate hydroxyl radicals directly through an ac­tivated transition state. The attenuation of increases in DHBA to salicylate may therefore reflect a reduction in the hydroxyl radical-like effects of peroxynitrite.

We examined the ability of 7-nitroindazole to block MPP+-induced substantia nigra degeneration in rats. 7-Nitroindazole was dissolved in peanut oil and ad­ministered at a dose of 50 mg/kg. It was administered both before MPP+ injections and then at approxi­mately 12-hour intervals for up to 48 hours after the MPP+. The administration of 7-nitroindazole signifi­cantly attenuated MPP+-induced degeneration of the substantia nigra of rats. There was almost complete protection against the ensuing cell loss. This observa­tion provides further evidence that the effects of 7-nitroindazole on MPTP neurotoxicity are unlikely to be the result of a nonspecific effect on MPTP metab­olism or MPP uptake. These results are also consis­tent with those observed in the nNOS-deficient mice.

The source of NO in the substantia nigra is unclear. One potential source could be inducible NOS in either microglia or astrocytes. ' Prior studies showed that neuronal injury leads to the expression of inducible NOS in astrocytes.46 Neuronal injury can also lead to expression of nNOS in neurons. There are therefore several potential sources for NO in the substantia nigra. We examined the distribution of nNOS-positive neurons in the substantia nigra compared to tyrosine hydroxylase neurons. ' We found immunohistochemi-cal evidence for a small population of nNOS-positive interneurons distributed throughout the substantia nigra. These nNOS-positive neurons showed direct ap­position of nNOS processes with substantia nigra ty­rosine hydroxylase-positive neurons. This is therefore a plausible source for NO that might be involved in the pathogenesis of nigral cell injury. These observations taken together provide strong evidence implicating NO and peroxynitrite in the pathogenesis of MPP -induced degeneration of substantia nigra neurons.

In summary, there is evidence implicating both ex-citotoxicity and peroxynitrite in the pathogenesis of MPTP neurotoxicity. If similar mechanisms occur in PD, this suggests that excitatory amino acid antagonists or neuronal NOS inhibitors might be useful therapeu­tic strategies.

The secretarial assistance of Sharon Melanson is gratefully acknowl­edged. Supported by National Institutes of Health grant NS31579 and NS10828.

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