TOXICOLOGY AND APPLIED PHARMACOLOGY 134, I - 17 ( 1995)

CONTEMPORARY ISSUES IN TOXICOLOGY Excitotoxins, Aging, and Environmental Neurotoxins: Implications for Understanding Human Neurodegenerative Diseases

RALPH DAWSON, JR.,* M. FLINT BEAL,t STEPHEN C. BONDY,:j: DONATO A. DI MONTE,§ AND GARY E. ISOM"

"'Depar!men/ o(Pharmacodynamics. l-"niiwsity o!Florida. (jainesville. Florida 32610: tNeurochemistry Laboratory. Massachusetts General Hospital. Bo.won ..\fassachu.1e11s 02114: tDepartment o(Communi1.1· and Environmental Medicine. l.Jniversily o(Cafifi1rnia. Irvine. Irvine. Califi>rnia 92717: §The Parkinson's lnstitute. Sunnyvale. Califi1rnia 94086: and' Departmelll o/Phar111acolog1· and Toxicologr. Purdue Unil'ersi1y. West Lafayette. Indiana 47907

Received October 24. 1994: accepted April I 0. 1995 group. By 2020, 16% of the total population is projected to Excitotoxins, Aging, and Environmental Neurotoxins: Im­ be elderly. Reports by the Congressional Office of Technol­ plications for Understanding Human Neurodegenerative Dis­ ogy Assessment ( 1990) and the National Research Council eases. DAWSON, R.,JR., BEAL, M. F., BONDY,S.C., DI MONTE, D. A., AND IsoM, G. E. (1995). Toxicol. Appl. Pharmacol. 134, ( 1992) have both concluded that environmental and indus­ 1-17. trial neurotoxicants pose a risk to the general population and highlighted the elderly as a special "at risk" population. We are an aging society and current demographic trends point Research in the area of neurotoxicology needs additional to a likely increase in age-related neurodegenerative diseases. focus in exploring the special risk factors to neurotoxic in­ The aged population may have a number of unique risk factors that result in a predisposition to neuronal damage from environ­ sult exhibited by our elderly population. Research in the mental neurotoxins. This symposium addressed the involve­ fields of aging and neurodegenerative diseases is in the fore­ ment of excitatory amino acids as final common mediators of front of basic and clinical neuroscience. The special ex­ neuronal death associated with various types of neurotoxic in­ pertise of the toxicologist needs to be focused on the toxi­ sult. The roles of oxidative stress, mitochondrial energy metab­ cokinetic and pharmacodynamic actions of chemicals, olism, and disruption of calcium homeostasis were discussed in drugs, and environmental agents and the problems they relation to excitoxicity and several experimental models of hu­ pose to the elderly population. This symposium focused on man neurodegenerative diseases. The neurotoxic actions of kai­ the role of excitatory amino acids as common mediators of nic acid, 3-nitropropionic acid, cyanide, l-methyl-4-phenyl- neuronal cell death in neurological disorders and the aging 1,2,3,6-tetrahydropyridine, and methamphetamine were exam­ CNS. The unique susceptibility and vulnerability of the ined for their relevance as models of human neurodegenerative disorders. The mechanisms of action of excitotoxins in experi­ aged CNS to direct- or indirect-acting excitotoxins were mental models ofHuntingtons's disease and Parkinson's disease highlighted by the speakers and the model systems that they were explored in light of the enhanced susceptibility and poten­ use to study these problems. tial vulnerability of the aP,e'i nervous system to neurotoxins that The term "excitotoxin" was coined by John Olney in the perturb cellular metabolism and homeostatic processes. Bioen­ 1970s to describe the neurotoxic properties of the acidic ergetic defects and oxidative stress were found to be critical links amino acids, glutamate (GLU) and aspartate (ASP), and in a neurotoxic cascade of events that trigger the sustained re­ their analogues (Olney, 1980). These endogenous amino lease of excitotoxic amino acids. The interrelationships among acids were known since the early 1960s to produce mem­ the aging process, the pathophysiology of neurodegenerative brane depolarization of neurons when ionophoretically ap­ diseases, and the mechanism of action of various neurotoxins were addressed from the unifying perspective of the excitotoxic plied (Curtis et al., 1960). Since then, other endogenous substances have been found to be potential excitatory neu­ hypothesis of neuronal death. 1c 1995 Academic Press, Inc. rotransmitters (Table I). The neurotoxic actions of GLU on the retina were first recognized by Lucas and Newhouse We are an aging society in both absolute numbers and ( 1957), and Olney ( 1980) conducted his pioneering charac­ proportionately. Dementia and neurodegenerative diseases terization of excitotoxic amino acids in the CNS in the in the aging population are major and growing medical and 1970s. Currently, the study of the pharmacology, neuro­ social problems. Dementia has its highest incidence in the chemistry, and neurotoxicology of excitatory amino acids population over age 75, which is the fastest growing age is one of the most active areas in neuroscience, including

004 l-008X/95 $12.00 Copyright 1 ~ 1 1995 b) Academic Press. Inc. All rights of reproduction in any form resened. 2 DAWSON ET AL.

TABLE I TABLE J Some Endogenous and Exogenous Excitotoxins The Major Classes of Glutamate Receptor Subtypes

Endogenous cxcitotoxins Exogenous cxcitotoxins Subtypes Effector systems

21 Glutamate Kainic acid NMDA" Cation channel (Ca • Na+. K +)

1 Aspartate Domoic acid AMPA ' Cation channel (Na·. K +)

1 Quinolinate 11-/1/-0xalamino-1-alanine (BOAA) Kainate (KA) Cation channel (Na', K ) lV-Acctyl-aspartylglutamate 11-N-Mcthvlamino-1 -alanine (BMAA) Metahotropic G proteins (G,, Gr) (NAAG) " N-Methyl-D-aspartic acid. 1-Homocysteate lhotcnic acid "Amino-3-hydroxy-5-methylisoxazolc-4 propionic acid. L-Cysteine sultinate Quisqualic acid

1-Cysteine Willardiinc characterized. Excitotoxins have been powerful tools with great experimental and heuristic value to neurotoxicolo­ gists and neuroscientists. the study of their clinical significance in neurological disor­ A key purpose of this symposium was to provide infor­ ders (Choi. 1988: Lipton and Rosenberg, l 994; Table 2). mation and stimulate discussion about the links between GLU and ASP are considered to be the predominant excit­ neurotoxicants. the aging processes, and human neurode­ atory neurotransmitters in the CNS, although other candi­ generative diseases. This introduction will be a starting date neurotransmitters exist (Table t ). There are a number point for the discussion of some of these potential links and of naturally occurring excitotoxic amino acids. which in­ will also briefly describe the factors that contribute to turn­ clude kainic acid, domoic acid. and BMAA (Table 1). Ex­ ing excitatory amino acids from neurotransmitters to neu­ citatory amino acids (EAAs) are involved in learning and rotoxins. GLU is unique among other neurotransmitters in memory, sensory processing, cardiovascular and neuroen­ requiring a relationship with glia for both normal biosyn­ docrine control, the motor system, and numerous other vi­ thesis and inactivation (Fig. I). GLU is a two-edged sword tal functions (Daw et al.. 1993, van den Pol and Trombley. as a neurotransmitter since "too little or too much" results 1993). The anatomy of the major pathways. which utilize in functional and/or pathologic consequences. EAA as neurotransmitters, is well known (Storm-Mathisen Some interactions that merit further study in regard to and Ottersen, 1988) and there has been an explosive growth toxic agents, aging, and excitotoxicity are listed in Table 4. in the knowledge of the receptor pharmacology of EAAs These processes relate to both the pre- and the postsynaptic based on molecular cloning studies of receptor subtypes events involving GLU efflux and inactivation. receptor and subunits (Nakanishi, 1992, Gasic and Hollmann. 1992). Four receptor subtypes have been extensively char­ acterized by both molecular and functional studies (Table GLUl'AMATE 3). Other subtypes exist and are currently being more fully NEURON GLIALCELL

TABLE 2 gin ..+---1---1-- gin Neurodegenerative Diseases and Neurological Disorders pag I 1gin synthetase Linked to Abnormal Excitatory Amino Acid Metabolism glu+

Olivopontoccrehcllar atrophy glu

Amyotrophic lateral sclerosis glu Huntington's disease '-;:::======:. I POSTSYNAPTIC NEUIWN Alzheimer's disease FIG. I. Overview of the neuronal-glial glutamate cycle. GLU is syn­ Parkinson's disease thesiled from GLN hy the mitochondrial enzyme. PAG. GLU is stored in Temporal Johe epilepsy synaptic vesicles and is also present in high concentrations of the cyto­ plasm. GLlJ is released and taken up hy hoth glia and neurons hy a high­ AIDS-related dementia affinity. sodium-dependent transport system. Glial cells convert GLU to GLN via the enzyme glutaminc synthetase and GLN can he taken up by Stroke/ischemia neurons. EXCITOTOXINS. AGING. AND ENVIRONMENT AL NEUROTOXINS 3

TABLE 4 crease in the brain as a function of age, whereas GLU Potential Links between the Aging Process, Excitotoxins, content tends to decrease in cortical areas (Rajeswari and and Environmental Neurotoxicants Radha, 1984; Wallace and Dawson, 1990). Recently, Seiler ( 1993) has proposed that abnormal ammonia detoxifica­ Energy metabolism tion may contribute to the pathophysiology of neurodegen­ Antioxidant capacity erative diseases. Data from the laboratory of Dawson and Protein modification co-workers would support an age-related deficit in ammo­ nia handling, which would also alter GLU synthesis and Calcium homeostasis release. Calcium-dependent GLU release appears normal Ammonia detoxification or elevated in aged rats depending on the brain region and Growth factor regulation intensity of depolarization conditions (Dawson el al., 1989; Meldrum et al., I 992a; Cobo et al., 1993; Palmer et al.. 1994). If the aged brain is less responsive to feedback inhi­

bition from NH4 as shown by Wallace and Dawson ( 1992), stimulation, and postreceptor events. The exact processes then agents that perturb release may be more effective in that lead to neuronal death are multifaceted and complex. sustaining prolonged periods of excessive GLU release or A trigger event must occur that results in a loss of control of delayed G LU inactivation. A number of neurotoxicants are the compartmentation of EAAs. GLU efflux can be known to influence GLU (Table 5). By contrast, metabolic calcium-dependent or -independent and involves release extremes like hypoxia and ischemia produce severe but from synaptic vesicles or direct cytosolic release (Nicholls, acute elevations of GLU efflux and neuronal death (Ben­ 1993; Bernath, 1991 ). There is an approximately I 0,000- veniste et al., 1989). Neurodegenerative processes are more fold difference between the I 0 mM intracellular concentra­ insidious in nature and are more likely related to modest tion ofGLU and the= I µM concentration normally found but sustained elevation of extracellular EAA concentra­ in the brain extracellular space. The dissipation of the nor­ tions. Candidate mechanisms for neurotoxic agents acting mal ionic gradients due to excessive depolarization or en­ as indirect excitotoxins would include altered synthesis, re­ ergy collapse results in the massive release of nonneuro­ lease, or reuptake ofGLU or the stimulation of nonspecific transmitter GLU and this has been shown to be the mecha­ efflux ofGLU from the cytosolic compartment of neurons nism of both ischemic and hypoxia neuronal death (Choi, or glia. Trimethyltin (TMT) produces substantial damage 1992). A number of postreceptor events occur that result in to the hippocampus and related limbic and cortical areas the amplification and expression of neuronal death (Choi, (Brown et al.. 1979). TMT has been shown to stimulate 1992). These involve ionic events, energy depletion, calcium-mediated enzymatic events, and free radical gener­ ation. These mechanisms are further explored and elabo­ rated on in the following sections. TABLE 5 A substantial body of research has focused on the role of Some Environmental Neurotoxins and Their Effects GLU in aging and neurological diseases. The release mech­ on Glutamate anisms and biosynthesis ofGLU will be briefly reviewed as Toxin Effects on glutamate a starting point for the discussion of the role of GLU in neurodegenerative processes and neuronal death. Phos­ Trimethyltin Increased glutamate release phate-activated glutaminase (PAG) is the major enzymatic Glutamate uptake blockade pathway in forebrain structures for neurotransmitter bio­ Ethanol Decreased glutamate release synthesis (Erecinska and Silver, 1990), whereas in the brain­ Blockade of NM DA receptor function stem ASP aminotransferase is important (Kihara and Aluminum Decreased glutamate release Kubo, 1989). PAG generates both GLU and NH4, which are products that are potentially toxic (Fig. I), and both act Methylmercury Increased glutamate release as powerful end product inhibitors of the enzyme. The Cyanide Increased glutamate release aging nervous system has been found to be significantly less Manganese Inhibition of uncoupled mitochrondrial respiration responsive to ammonia feedback inhibition of PAG (Wal­ with glutamate as substrate lace and Dawson, 1992). This age-related deficit is specific Toluene Increases in glutamate receptor binding to certain brain regions and end product inhibition by GLU and GLU analogues appears intact (Dawson and Wallace, Lead Decreased glutamate content in the motor cortex 1993). Calcium activation and phosphate activation of Permethrin Increased glutamate content in the brainstem PAG are also diminished in the aged nervous system (Wal­ Diazinon Decreased cortical content of ASP and GLU lace and Dawson, 1993). In general, ammonia levels in- 4 DAWSON FT AL

GLU ctllux from hrain (Naalsund and Fonnum. 1986): 1990). limited functional reserve capacity. aberrant however. TMT-stimulatcd GLU release docs not differ be­ calcium homeostasis (Gihson and Peterson. 1987: Land­ tween adult and aged F344 rats (Dawson et al.. 1995). ficld et al .. 1992). and reduced protein repair mechanisms Differences may also exist in the responsiveness of the aged (Stadtman. 1988). Ncurotoxins that inhibit electron nervous system to cxcitotoxins hascd on age-related alter­ transport in mitochondria or inhibit the production of high ations in receptor binding or coupling. In general. /'1/­ energy phosphate compounds lead to energy failure and mcthyl-D-aspartatc (NMDA) receptor numhcr decreases in both enhanced GLU release and increased vulnerability to the aged nervous system (Tamaru et al., 1991: Wenk et al.. extracellular GLU (Lipton and Rosenberg. 1994). Further­ 1991: Magnusson and Cotman. 1993). hut it is unknown more. GLU activation ofNMDA receptors enhances nitric whether this is due to a loss of vulncrahlc neurons with oxide formation which can contribute to free radical-medi­ NMDA receptors or an adaptive down regulation to protect ated ncurotoxicity (Lipton and Rosenberg. 1994 ). Free rad­ aged neurons. It was reported that elderly individuals that ical-mediated oxidative damage can result in a loss ofgluta­ consumed domoic acid-contaminated mussels had more minc synthctasc activity (Oliver et al .. 1990) and can also severe neurological deficits than younger exposed individu­ lead to increased levels ofhrain GLU. Ncurotoxicants with als (Teitelbaum ct al .. 1990: Auer. 1991 ). Domoic acid acts diverse specific mechanisms of action may ultimately trig­ at the kainic acid (KA) receptor subtype and KA receptor ger an excitotoxic cascade in the aged nervous system. The numhcr docs not decrease with age (Tamaru et al .. 1991 ). subsequent sections will explore the links among mitochon­ Dawson and Wallace ( 1992) examined the sensitivity of drial function. oxidative stress. and EAAs. aged female rats to kainic acid-induced seizures and found aged animals to he approximately fourfold more sensitive THE RELATION BETWEEN EXCITOTOXICITY AND than adult rats. These aged animals also showed a greater OXIDATIVE STRESS fall in hrain GLU and ASP content 2 hr after kainic acid This out Ii nc is not intended to constitute a thorough re­ treatment (Dawson and Wallace. 1992). Dawson and Wal­ view hut is intended to illustrate that a complex relation lace ( 1992) also found KA to induce a significantly greater exists between excitatory and oxidative events in the ner­ increase in ASP release from hrain slices of aged rats. These vous system. The modeling of neurological diseases using studies suggest that aged animals arc compromised in re­ selected toxic chemicals described in this article reveals that sponse to excessive activation of KA receptors. Olney and interactions between these two modalities arc likely to play co-workers ( 1991) have reported similar findings and these a role in hoth age-related and environmentally caused data tend to substantiate the human data on domoic acid changes in nerve function. This subject is described in toxicity. greater detail in a recent review (Bondy. 1995). All of the The aged nervous system is also known to have compro­ other sections constituting the current overview also de­ mised hrain glucose utilization and energy metabolism scribe agents that involve hoth oxidant and excitatory (Hoyer. 1990). It is also known that conditions that com­ events acting in concert. Thus. such a "double hazard" promise neuronal energy metabolism greatly exacerbate the combination is suspected to account for the neurotoxicity toxicity of exogenous EAA (Novelli ct al.. 1988). Hypogly­ of glutamate. 3-nitropropionic acid. cyanide. and 1-methyl- cemia is known to increase the stimulated release of ASP 4-phenyl-1.2.3.6-tctrahydropyridine (MPTP). and GLU (Burke and Nadler. 1989). The effects of hypogly­ Many toxic chemicals disrupt the production of cellular cemia on potassium-stimulated GLU and ASP release in energy reserves and therefore impair anabolic processes. aged rats have previously been examined and found to he This may he by inhibition of mitochondrial oxidative phos­ augmented to the same level as that of adult controls (Mel­ phorylation or hy reduction of the supply of adequate nu­ drum et al., l 992h ). Subsequent authors will also address trients to the cell. Another large class of toxic chemicals the pivotal role of energy metabolism and mitochondrial causes an excess energy demand by interfering with the sta­ function in age-related neurodegenerativc processes. bilization of energy-requiring events such as maintenance The ability of GLU receptor activation to induce of ionic gradients. for example. by compromising mem­ calcium-dependent processes related to free radical produc­ brane integrity. Thus, one of the most common events that tion and the resulting protein and lipid modifications that may occur as a result of exposure to a toxicant is excess compromise mitochondrial and cell membrane function all demand on, or deficient supply of. the energy requirements interrelate to potential mechanisms for sustained and pro­ of the cell. A hroad range of unrelated chemicals may have gressive neuronal damage and compromise. These phe­ in common the ability to disrupt the balance between cellu­ nomena arc important given the research literature that lar ATP supply and demand. This general feature may lead points to the aged nervous system having diminished anti­ to the emergence of pathological changes that reflect the oxidant defense mechanisms (Bondy and LeBel. 1993). de­ susceptibilities of the cell rather than being related more to ficient neuronal glucose and energy metabolism (Hoyer. a specific feature of the toxic agent. EXCITOTOXINS. AGING. AND ENVIRONMENT AL NEUROTOXINS 5

In the case of the central nervous system, several distinc­ ever, under normal conditions, appropriate protective sys­ tive biochemical and anatomical features lead to com­ tems minimize the intensity ofprooxidant activity. For ex­ monly recurring characteristics of expression of neurotox­ ample, mitochondrial cytochrome oxidase has superoxide icity. These features may be considered "weak points" in dismutase activity and the rate of leakage of free radicals that they are the first to become deranged both by neuro­ out of healthy mitochondria is very low. Another means by toxic agents and in neurological disorders. One of these vul­ which exposure to a toxicant can lead to abnormal rates of nerable features involves the uniquely fluctuating demand production of ROS is by excessive utilization ofglutathione made by nervous tissue upon intracellular concentrations in detoxifying conjugation or reduction reactions. of cations. Maintenance of cationic gradients is required by A variety of changes resulting from an impaired balance all tissues. Yet in order to allow nervous tissue function, between the supply and the demand of intracellular energy these gradients must be allowed to dissipate and then to be can also lead to an imbalance between the rates of produc­ rapidly restored. These fluxes are needed for the passage of tion and inactivation of ROS. Excess demand for ATP pro­ action potentials, the modulation of postsynaptic poten­ duction or reduced availability of oxygen, as in ischemia, tials, and the calcium-effected release of neurotransmitters. can compromise mitochondrial efficiency and lead to the This continuously changing ion gating causes entropy-rais­ appearance of excess ROS. ing reductions in the steepness of ionic gradients. The gra­ Both hyperexcitability and excess ROS generation are dients need to be rapidly restored by entropy-requiring ion relatively nonspecific consequences of neural insult and pumping or sequestering events. This demand largely ac­ may be associated with an extensive and varied range of counts for the high energy requirement of the CNS. neurotoxic exposures. There is clearly a relation between Failure to restore homeostasis after a depolarizing event these two symptoms of cellular malfunction. It has been very commonly leads to neuronal hyperexcitability. This is proposed that during ischemia, ROS and excitatory amino due both to the attenuation of the axonal resting potential acids may cooperate in effecting neuronal damage (Bose ct due to excess intracellular sodium and to increased levels of al.. 1992). However, the two phenomena are distinct enti­ cytosolic calcium leading to abnormal presynaptic neuro­ ties and, while they are often incurred simultaneously, the transmitter release. In the absence of adequate supplies of causal relation between them is not always clear. There is energy, both of these events may be increasingly acceler­ evidence ofa reciprocal activation of these processes. Thus, ated. The collapse of ion gradients may account for the ap­ the influx of and prolonged elevation of calcium that may pearance of hyperactivity, tremor, and convulsions that occur under conditions of energy deficiency may activate commonly accompany many neurotoxic and neurological ROS generation by: insults. While each deleterious chemical has its own specific (i) Activation of the arachidonic acid cascade. The en­ means of cellular disruption, the commonality of hyperex­ zymic conversion of this compound to prostaglandins, leu­ citation is superimposed on any distinctive features. kotrienes, and thromboxanes by cyclooxygenases and li­ Another recurring characteristic weak point of the sub­ poxygenases which directly utilize molecular oxygen leads optimally functioning cell is its tendency to generate exces­ to considerable ROS generation and this may be a means by sively high levels of reactive oxygen species (ROS) (free rad­ which excitatory events promote excess generation of ROS icals). ROS may be oxygen-centered radicals possessing un­ (Pellerin and Wolfe, 1991 ). paired electrons, such as superoxide anion and hydroxyl (ii) Stimulation of lipolysis and, thence, lipid peroxida­ radical, or covalent molecules such as hydrogen peroxide tion (Vanella ct al., 1992). (Halliwell and Gutteridge, 1989). Disruption of the electron (iii) Depression of oxidative phosphorylation and de­ transport chain can lead to the appearance of incompletely rangement of mitochondrial function (Zhang ct al.. 1990). reduced oxygen species. Some of these moieties, such as the (iv) Catalysis of conversion of xanthine dehydrogenase hydroxyl radical, have extremely short half-lives and they to xanthine oxidase (McCord, 1987). rapidly oxidize any organic molecule in their proximity. Appearance of excessive levels of ROS is generally symp­ Conversely, there is also evidence that: tomatic of inefficient mitochondrial combustion of sub­ (i) ROS can mobilize intramitochondrial stores of se­ strates. questered calcium (Richter and Kass. 1991 ). Antioxidant defense systems can be overwhelmed by ele­ (ii) ROS inhibits calmodulin-dependent processes, vated levels of ROS production. Mechanisms include in­ thereby elevating levels of cytosolic free calcium (Okabe ct duction of mixed function oxidases or conversion of xan­ al.. 1989). thine dehydrogenase to xanthine oxidase. Oxidases un­ (iii) ROS can lead to extracellular release of excitatory dertake an inherently hazardous task involving scission of amino acids (Pellegrino-Giampeitro ct al.. 1990). the relatively inert oxygen molecule. This invariably in­ volves the appearance of an active oxidizing species. How- The therapeutic possibilities resulting from recognition 6 DAWSON ET AL of these two overlapping "final common pathways" of neu­ If this theory of cell death is valid then one should be able rological insult include the use of antioxidant and calcium to produce selective neuronal degeneration in experimental channel-blocking drugs. These approaches are finding animals with mitochondrial toxins which mimics that seen increasing utility in the treatment of many neurological in HD and which occurs by excitotoxic mechanisms. Beal disorders, including Parkinson's disease. Alzheimer's, and co-workers therefore carried out studies with a number multiple sclerosis, epilepsy. amyotrophic lateral sclerosis, of compounds which are selective blockers of the electron stroke. and cerebral ischemia (Bondy and LeBeL 1993). transport chain. One of the most interesting of these is the Several common neurological diseases are suspected to in­ compound 3-nitropropionic acid. volve a combination of interacting excitatory and oxidative 3-Nitropropionic acid (3-NP) is a naturally occurring processes (Halliwell, 1989: Coyle and Puttfarcken. 1993: abundant plant and mycotoxin which has been associated Lipton and Rosenberg, 1994). More prolonged treatments with neurological illnesses in grazing animals and humans emphasize the use of antioxidant vitamins. while calcium (Ludolph et al.. 1991 ). 3-NP is an irreversible inhibitor of channel blockers and iron chelators find application in succinate dehydrogenase that inhibits both the Krebs cycle more acute situations or in intermittent therapies. The and complex II of the mitochondrial electron transport maintenance of low levels of iron and calcium within the chain (Alston el al.. 1977: Coles et al.. 1979). Ingestion by cell is essential to the establishment of an optimal milieu for livestock results in dyspnea. hindlimb weakness, knocking anabolic biochemical processes. A small increase in levels together of the hindlimbs while walking, and goose stepping of free iron within cells can dramatically accelerate rates of (Ludolph et al., 1991 ). The occurrence of illness in humans ROS production (Minnotti and Aust. 1989). and calcium has occurred in China after ingestion of mildewed sugar channel blockers can prevent cyanide-induced lipid perox­ cane, which contains the fungus Arthrinium. The illness oc­ idation and cell death (Maduh el al., 1988). curred mostly in children. with the oldest patient presenting While the treatment of a neurotoxic exposure obviously encephalopathy being 27 years old (Ludolph et al., 1991). needs to include procedures targeted toward mitigation of The illness is characterized by initial gastrointestinal distur­ the specific properties of the toxicant. the addition of more bance followed by encephalopathy with stupor and coma. general therapies, focused on protection against these recur­ Patients who recover have delayed onset of non progressive rent vulnerabilities of the CNS, is also of importance. This dystonia 7 -40 days after regaining consciousness. The pa­ approach is warranted in excitotoxin-mediated neuronal tients show facial grimacing, torticollis, dystonia, and jerk­ damage due to the prominent roles of calcium and ROS in like movements. CT scans show bilateral hypodensities in the mechanisms of cell death. the putamen and to a lesser extent in the globus pallid us. An earlier report of dystonia following ingestion of mildewed EXCITOTOXICITY IN HUNTINGTON'S DISEASE maize may have been the same illness (Woods and Pendle­ ton. 1925). The concept that excitotoxicity may play a role in the Studies of mice treated with a single or repeated doses of pathogenesis of Huntington's disease (HD) was suggested in 120 mg/kg of 3-NP showed depressed motor activity and 1976 after it was shown that kainic acid lesions in the stria­ bilateral symmetric lesions of the caudate-putamen (Gould tum of rats could reproduce many of the characteristic fea­ and Gustine. 1982). In rats repeated doses of 3-NP pro­ tures of HD (reviewed in Beal, 1992). Beal and co-workers duced progressive impairment of motor behavior with som­ later showed that NMDA agonists such as quinolinic acid nolence. wobbly gait. and finally recumbency with the ani­ provide an improved model since they spare striatal inter­ mals lying on their sides with the hindlimbs rigidly ex­ neurons, such as NADPH-diaphorase neurons, which are tended (Hamilton and Gould. 1987a). The most consistent spared in HD. However, it is difficult to envision how an lesions were found in the basal ganglia in all affected ani­ acute excitotoxic process could occur in HD. which evolves mals. with some animals showing damage in the CA 1 field over I(' - 15 years. One possibility is that a low-grade distur­ of the hippocampus and in the thalamus. Ultrastructural bance of energy metabolism may lead to slow excitotoxic studies in both mice and rats showed dendrosomatic swell­ neuronal degeneration in HD (Beal. 1992). An impairment ing, chromatin clumping. and marked mitochondrial swell­ of energy metabolism may result in neuronal depolariza­ ing, consistent with an excitotoxic injury. The pattern of tion, which can then lead to activation of voltage-depen­ neuronal injury could not be explained by the distribution dent NMDA receptors. This leads to an influx of calcium of inhibition ofsuccinate dehydrogenase activity, since bio­ and cell death by excitotoxic mechanisms. One of the chemical and histochemical studies showed it to be uniform downstream mechanisms is the generation of free radicals. throughout the cerebral cortex and basal ganglia (Gould The generation of free radicals by mitochondria is markedly and Gustine. 1982: Gould et al.. 1985). The lesions were augmented by increases in intracellular calcium in the hypothesized to be due to histotoxic hypoxia, since 3-NP range of those achieved following activation of NMDA re­ increased arterial blood pressure and arterial blood oxygen ceptors. compared with controls (Hamilton and Gould, 1987b). EXCITOTOXINS. AGING, AND ENVIRONMENTAL NEUROTOXINS 7

Recent in vitro studies showed that 3-NP has no direct significantly increased. Histologic studies confirmed a loss depolarizing effects on neurons in hippocampal slices of both Niss! and NADPH-diaphorase neurons, yet a (Riepe et al.. 1992), indicating that it does not act at excit­ sparing of tyrosine hydroxylase immunoreactivity in the atory amino acid receptors. Rather it leads to initial hyper­ striatum. polarization due to activation of ATP-sensitive potassium Systemic 3-NP lesions were also attenuated by prior de­ channels followed by depolarization due to loss of ATP. cortication and by the glutamate release inhibitor lamotrog­ Studies in cortical explants showed that 3-NP produces cel­ ins. Microdialysis studies showed that there was no signifi­ lular ATP depletion and neuronal damage by a secondary cant increase in extracellular glutamate concentrations. excitotoxic mechanism (Ludolph et al., I 992a). Pathologic These results are consistent with 3-NP causing excitotoxi­ changes were significantly attenuated by pretreatment with city by making neurons more vulnerable to endogenous lev­ excitatory amino acid antagonists. In cultured cerebellar els of glutamate. Furthermore, in vivo 3H-MK-801 receptor granule neurons 3-NP resulted in concentration and time­ autoradiography showed that systemic administration of 3- dependent neurotoxicity (Weller and Paul, 1993). Both NP was associated with activation of NMDA receptors MK-801 and the competitive NMDA antagonist 2-amino- (Wullnereta/., 1994). 5-phosphonovaleric acid delayed but did not prevent the 3- Beal et al. (I 993a) also examined the effects of chronic NP toxicity. low-dose administration of 3-NP subcutaneously for I Beal and co-workers studied 3-NP neurotoxicity in both month using Alzet pumps. We reasoned that a chronic low­ rats and nonhuman primates. In initial studies, the effects grade energy disturbance was much more likely to mimic of intrastriatal injections of 3-NP were examined. These in­ the situation which may occur in neurodegenerative dis­ jections produced dose-dependent lesions with neuronal eases, such as HD. Administration of 3-NP using this para­ loss and gliosis (Brouillet et al., I 993b). The injections pro­ digm resulted in subtle lesions confined to the dorsolateral duced ATP depletions at 3 hr, which persisted for up to 24 striatum, in which there was neuronal loss and gliosis (Beal hr after the injections (Beal et al.. 1993a). Using in vivo et al., 1993a). Furthermore, this mode of administration chemical shift magnetic resonance imaging we showed that was associated with the sparing of NADPH-diaphorase there were focal accumulations of lactate in the basal gan­ neurons, similar to findings in HD. A further characteristic glia. The lesions were strikingly age-dependent, which was feature of HD neuropathology is that there are proliferative subsequently confirmed (Bossi et al., 1993). This age depen­ changes in the dendrites of spiny neurons, with both recur­ dence correlated directly with the increases in lactate fol­ vatures and increased numbers of spines. Chronic low­ lowing administration of a uniform dose of 3-NP to animals grade administration of 3-NP resulted in identical changes of various ages. The lesions occurred by an excitotoxic in spiny neurons as assessed using the Golgi technique. mechanism since prior decortication, which removes the Wullner et al. (1994) also examined mRNA using in situ striatal glutamatergic input, significantly attenuated the le­ autoradiography. These studies showed a relative sparing sions. of mRNA for somatostatin compared with the amount of Beal and co-workers subsequently examined both sub­ mRNA for both substance P and enkephlin. Glial fibrillary acute and chronic administration of 3-NP in rats. Subacute acidic protein message was depleted in the center of the administration was performed by ip administration of 3-NP largest lesions, but increased at the periphery of the lesions. 3 over 5-7 days. This resulted in the development of motor [ H]Naloxone autoradiography showed a preservation of slowing and dystonic posturing in the rats. Similar to the the striosomal compartmentalization of the striatum. [3H]­ observations of Hamilton and Gould (I 987a), there were Mazindol autoradiography confirmed a preservation of do­ large symmetric lesions in the basal ganglia. With very large pamine terminals with lower doses of 3-NP. These studies lesions there was accompanying neuronal loss and gliosis in therefore show that chronic low-dose administration of 3- the CA 1 field of the hippocampus and gliosis in the cerebral NP can replicate most of the characteristic neurochemical cortex. Smaller lesions were confined to the basal ganglia. and neuropathologic features of HD. No lesions were observed in the spinal cord, suggesting that Beal and co-workers recently extended their studies to the movement disorder was due to the striatal lesions. Mag­ nonhuman primates. The neuroanatomy of the primate netic resonance imaging spectroscopy showed that the le­ basal ganglia much more closely resembles that of man, and sions were accompanied by focal accumulations of lactate it is possible to produce chorea, the cardinal clinical feature in the basal ganglia which preceded lesions detectable with of HD. Chronic administration of 3-NP to primates re­

T 2 weighted imaging, indicating that the energy defect pre­ sulted in an apomorphine-inducible movement disorder cedes the neuronal degeneration. The lesions were accom­ which closely resembles that seen in HD (Brouillet et al., panied by a depletion of markers for intrinsic striatal neu­ I 993a). The animals showed orifacial dyskinesia, dystonia, rons; however, concentrations of the striatal afferent mark­ dyskinesia of the extremities, and choreiform movements. ers dopamine and 3.4-dihydroxphenylacetic acid were Both a clinical rating scale and a quantitative analysis of 8 DAWSON ET AL. tangential velocity of individual movement velocities con­ positron em1ss10n tomography with 6-fluoro-L-dopa re­ firmed that the 3-NP-treated animals had a significant in­ vealed marked dysfunction of dopaminergic transmission crease in choreiform and dystonic movements. in the posterior regions of the basal ganglia. similar to that Histologic evaluation showed that the lesions were strik­ observed in Parkinsonism (Rosenberg et al., 1989). Also, ingly reminiscent of those which occur in HD. There was a chronic cyanide exposure has been associated with motor depletion of both Nissl-stained and calbindin-stained neu­ neuron disease, hereditary optic atrophy, and tobacco am­ rons. In contrast medium-sized aspiny neurons stained with blyopia. Kato ct al. ( 1985) proposed that a disorder in cya­ NADPH-diaphorase and large striatal neurons were nide metabolism may predispose to motor neuron disease. spared, similar to findings which occur in HD. In addition Cyanide interacts with several central transmitter sys­ Golgi studies showed proliferative changes in the dendrites tems and altered neurotransmitter function may play a role of spiny neurons similar to changes in HD. The patch-ma­ in cyanide toxicity. Cyanide depletes )'-aminobutyric acid trix compartmentalization of the striatum, which is pre­ and elevates glutamate in rat brain (Persson et al., 1985). served in HD, was also spared by the lesions. Last, there was Dopaminergic systems appear to be highly susceptible to sparing of the nucleus accumbens. which is spared in HD. cyanide. A lethal dose of cyanide depletes striatal dopamine These findings therefore show that chronic 3-NP lesions in (DA) in rats (Cassel and Persson. 1992) and local perfusion primates can produce both an apomorphine-inducible of cyanide in the striatum stimulates release of DA as mea­ movement disorder and histologic findings which replicate sured by in vivo microdialysis (Matsumoto ct al.. 1993). In those of HD. the PC 12 cell model, cyanide produces a calcium-depen­ Beal and co-workers have recently carried out studies us­ dent release and depletion of DA and altered DA synthesis ing magnetic resonance imaging of HD patients in vivo. and metabolism have been described (Kanthasamy el al.. These studies show that there are marked increases in lac­ 1991 ). In rats, lethal doses of cyanide partially inhibit brain tate concentrations both in the basal ganglia and in the ce­ DOPA decarboxylase as measured by increased levels of rebral cortex (Jenkins el al., 1993). These findings provide DOPA (Persson et al.. 1985). Cyanide also reacts with the direct evidence for impairment of energy metabolism in DA metabolite 3,4-dihydroxyphenyl acetaldehyde to gen­ HD and therefore suggest that 3-NP neurotoxicity which erate a neurotoxic cyanohydrin (Kanthasamy et al., I 994b). acts through secondary excitotoxic mechanisms may be a The role of this compound in the cyanide-induced neuro­ relevant model for the disease process. toxic syndrome remains to be determined. A recently characterized mouse model of cyanide-in­ CYANIDE-INDUCED EXCITOTOXICITY AND duced dopaminergic toxicity may prove useful in studying NEURODEGENERA TION the delayed neurodegeneration produced by cyanide (Kan­ thasamy et al.. l 994a). Following treatment with cyanide Cyanide is well known for its rapid, lethal action in which over a 7-day period, mice exhibited marked decreases in its primary target organ is the central nervous system (Way, striatal and hippocampal DA levels which were accompa­ 1984). Cyanide rapidly inhibits cytochrome oxidase. which nied by significant peroxidation of lipids in these brain ar­ lowers the cell's energy charge and disrupts homeostasis. eas. Tyrosine hydroxylase (TH) immunohistochemical ex­ During cyanide intoxication the appearance of neurological amination showed a reduced number of TH-positive cells dysfunction occurs within seconds. It has been assumed in the substantia nigra. indicating a loss of dopaminergic that the extreme sensitivity to cyanide results from the neurons. Over one-third of the mice exhibited decreased lo­ brain's low anaerobic capacity and limited energy reserve. comotor activity and akinesia which were suppressed by L­ However. it is becoming incre. singly apparent that in addi­ dopa. These behavioral deficits are likely associated with tion to disruption of oxidative phosphorylation. cyanide striatal DA deficiency since they are reversed by L-dopa produces other actions in the CNS which can manifest as treatment. This animal model is presently being used to degeneration of select brain areas. study the mechanisms underlying the selective dopaminer­ Several clinical reports have shown that the development gic toxicity of cyanide. of a delayed, progressive Parkinsonism and dystonia can Spencer et al. ( 1992) proposed that cyanide is involved in follow acute cyanide intoxication (Uitti et al .. 1985: Carella basal ganglia disease through either an excitotoxic effect or el al.. 1988: Grandas el al., 1989; Rosenberg et al., 1989; disruption of energy metabolism. Defects in mitochondrial Valenzuela et al.. 1992). In patients surviving a severe, oxidative phosphorylation have been associated with Par­ acute intoxication, several days to weeks after the intoxica­ kinson's disease (Shoffner et al.. 1991 ). Toxicants that in­ tion, dystonia developed which in some cases progressed hibit oxidative phosphorylation, including MPTP, carbon to a severe Parkinson-like state. Levodopa therapy pro­ monoxide, and manganese, can produce basal ganglia dys­ duced variable improvement. Magnetic resonance imaging function associated with locomotor impairment. Cyanide showed extensive bilateral lesions of the basal ganglia and inhibits cytochrome oxidase, the terminal enzyme in mito- EXCITOTOXINS. AGING. AND ENVIRONMENTAL NEUROTOXINS 9 chondrial oxidative metabolism, producing a decrease in intracellular Ca2 + is increased to excessive levels by cyanide cellular ATP levels (Lee et al., 1988). A rapid drop in neu­ and homeostatic processes are unable to buffer the increase, ronal ATP would result in altered ionic handling and sub­ the ion may activate several critical membranous and cyto­ sequent breakdown in transmembrane ionic homeostasis. plasmic events in concert (Maduh et al., 1990). Unregu­ It has been proposed that mitochondria of sensitive brain lated activation of these Ca2+-sensitive reactions can lead to areas may be highly sensitive to cyanide, resulting in altered cell dysfunction and eventually, if not controlled, threaten bioenergetics and predisposing to neuronal injury. survival of the cell. In a variety of cell types, cyanide-in­ The role of cytochrome oxidase inhibition as the primary duced activation of endonucleases. phospholipases, and 2 biochemical lesion in cyanide toxicity remains controver­ proteases is associated with elevated cytosolic Ca +. Prote­ sial. It is well established that at physiologically active con­ ase inhibitors attenuate formation of cyanide-induced cell centrations cyanide inhibits a number of enzymes which surface blebs, suggesting that cytoskeletal proteins may be are more sensitive to cyanide than cytochrome oxidase (Ar­ substrates for these enzymes. In different cell models, cya­ delt et al., 1989). Petterson and Cohen (1985) reported a nide activates phospholipase A2 (PLA 2) to produce plasma disparity between the dose-response relationship of cya­ membrane phospholipid breakdown and mitochondrial nide lethality and brain and heart cytochrome oxidase inhi­ dysfunction. In PC 12 cells PLA 2 activation by cyanide re­ bition. Yamamoto ( 1989) demonstrated in mice rendered sults from both influx ofCa2 + and mobilization of intracel­ 2 unconscious by cyanide that no significant decrease in brain lular Ca + stores (Yang et al., 1994). Cyanide also stimu­ ATP levels occurred. In transverse slices of guinea pig hip­ lates generation of inositol triphosphate through an interac­ pocampus, cyanide rapidly depressed synaptic transmission tion with the glutamate/metabotropic receptor (Yang and between the Schaffer collateral commissural fiber and CA I Isom, 1994). pyramidal cells (Aitken and Braitmen, 1989). This effect of Cyanide-induced oxidative stress and subsequent peroxi­ cyanide reversed within seconds of washout, suggesting that dation of membrane lipids may be important in cellular cyanide has a direct action on neurons not mediated by damage. Exposure of cells to cyanide produces a Ca2+ -de­ metabolic inhibition. pendent generation of intracellular hydroperoxides, result­ Morphological and biochemical responses to cyanide are ing in lipid peroxidation. Accompanying the generation of related temporally to a rise in cytosolic free Ca2 + and in neu­ hydroperoxides is an inhibition of the brain's antioxidant rons the elevated Ca2+ initiates a series of intracellular cas­ defense mechanisms (Ardelt et al., 1989). Cyanide inhibits cades which can lead to cell dysfunction and death. In elec­ brain catalase, superoxide dismutase, and glutathione per­ trically excitable cells, destablization of Ca2 + homeostasis oxidase. In animals intoxicated with cyanide, brain lipid by cyanide results from several actions (Johnson et al., peroxidation is increased and significant lipid peroxidation 1986 ). ATP depletion is linked to increased Ca conduc­ occurs in the striatum (Kanthasamy et al., I 994a). Oxida­ tance through activation of voltage-sensitive Ca channels tive damage to select brain areas appears to play a role in (VSCC). In brain the Land N type of voltage-sensitive Ca2+ the neurotoxicity of cyanide. channels may be involved in Ca2+-mediated neural injury. Activation of Ca2 +-dependent endonucleases occurs in VSCC blockers can attenuate cyanide-induced influx of cyanide-mediated neuronal death. In terminally differen­ Ca2+ through these channels. Receptor-operated Ca chan­ tiated PC 12 cells, cyanide induces an apoptotic cell death nels play a role in the response since cyanide activates the characterized by internucleosomal fragmentation of DNA glutamate/NMDA receptor in primary hippocampal neu­ and chromatin margination which was prevented by an en-. rons, producing an immediate influx of Ca2+ (Patel et al., donuclease inhibitor (Mills and Isom, 1994 ). It remains to 1992). This may result from a direct interaction of cyanide be determined if this form of neuronal death plays a role in with the receptor and/or may be indirectly related to cya­ the cyanide-induced brain lesions. nide-stimulated release of glutamate which then activates Considering the multiple and complex activity of cya­ the receptor. The influx ofCa2+ through the NMDA recep­ nide, it is proposed that cyanide induces a rise in neuronal tor-gated channel contributes to the cytotoxic response to cytosolic Ca2+ which then activates a cascade of biochemi­ cyanide since death of primary hippocampal neurons is cal events culminating in the signs, symptoms, and lesions blocked with NMDA receptor blockers (Patel et al., 1993). characteristic of cyanide toxicity. Cyanide rapidly depletes It is apparent that cyanide can initiate excitotoxicity; its role energy reserves due to cytochrome oxidase inhibition re­ in cyanide-mediated neuronal degeneration remains to be sulting in an ionic disequilibrium across plasma mem­ determined. branes. Activation of VSCC and NMDA receptors pro­ The relationship of cyanide-induced cell injury and ele­ duces a rapid influx of Ca2+ which is accompanied by mo­ vation of cytosolic free Ca2+ has been studied in detail. Ele­ bilization of intracellular Ca2+ stores. Elevated cytosolic vation of neuronal Ca2+ triggers intracellular Ca2+-depen­ free Ca2+ initiates a series of intracellular reactions and the dent processes and signaling pathways. It appears that when neuron eventually loses its homeostatic ability. These Ca2+- 10 DAWSON ET AL. dependent cascades trigger release of neurotransmitters, dizing species and thus disrupting the redox equilibrium lipid peroxidation, and activation of destructive enzymes within dopaminergic neurons. Indeed, dopamine turnover (lipases, proteases, and endonucleases); activation of involves its oxidation via monoamine oxidase (MAO), multiple processes then leads to neuronal injury. In sum­ which generates hydrogen peroxide (H202). Dopamine mary, cyanide produces multiple biochemical and func­ may also undergo autoxidation with the formation of toxic tional insults which account for the sensitivity of the CNS quinoncs as well as H 20 2 (Graham, 1978). The ability of to this agent. dopamine to induce oxidative stress has been supported ex­ perimentally by the work of Cohen and colleagues showing PARKINSON'S DISEASE AND EXCITOTOXICITY increased levels of oxidized glutathione following pharma­ cologically induced increased dopamine turnover both in Parkinson's disease is one of the most common ncurode­ vivo and in 1•itro (Spina and Cohen, l 989). generative disorders, affecting approximately I% of the MPTP poisoning mimics most features of Parkinson's population over age 50. Clinical features of the disease in­ disease as closely as anyone could have anticipated at the clude the classic triad of tremor, rigidity, and bradykinesia. time of its discovery in 1983 (Langston cl al., 1983; Burns From the neuropathologic and neurochemical point of ct al., 1983). It has therefore become a widely used model view, Parkinson's disease is characterized by the degenera­ for research concerning the nigrostriatal system, providing tion of pigmented neurons in the substantia nigra and the the initial hint that an impairment of mitochondrial respi­ depletion of dopamine in the striatum, the area of the brain ratory chain activity could play a role in the pathogenesis of to which nigral neurons project their axons. The cause of cell loss in Parkinson's disease. MPTP is converted by death of dopaminergic neurons in the brain of patients with MAO B to its 1-methyl-4-phenylpyridinium (MPP+) me­ Parkinson's disease remains unknown, although both ge­ tabolite (Chiba cl al.. 1984), which represents the ultimate netic and environmental factors have been implicated mediator of its neurotoxicity (Markey cl al., 1984; Langston (Tanner, 1994) and different mechanisms of cytotoxicity ct al.. 1984). MPP' is actively taken up by dopaminergic have been hypothesized. Among these mechanisms, the one neurons (Javitch ct al.. 1985) and it is also accumulated into which has attracted the attention of scientists for the longest mitochondria where it acts as an inhibitor of complex I period of time is oxidative stress (Fahn and Cohen, 1992). (Ramsay and Singer, 1986; Nicklas el al.. 1985). MPTP/ More recently, nigrostriatal degeneration has been related MPP+ toxicity has been directly linked to a failure of energy to an impairment of mitochondrial respiratory activity supplies in a number of in l'itro model systems (Di Monte (Beal et al.. 1993), and pharmacologic and toxicologic evi­ et al., 1986; Denton and Howard. 1987), and, more re­ dence has also suggested an involvement of excitotoxicity cently, the depletion of dopamine caused by MPTP in the (Albin and Greenamyre, 1992; Carlsson and Carlsson, mouse striatum has been found to be preceded by a selective 1990). Thus, it appears that no single mechanism may be decrease in ATP levels (Chan ct al .. I 993b). regarded as the sole cause of neuronal death in Parkinson's The third prototype model for striatal damage is that of disease, whereas, more likely, nigral cell loss may arise from methamphetamine toxicity. When administered in re­ a concerted sequence of toxic events. peated doses or in one relatively large dose, methamphet­ In light of these considerations, the following discussion amine causes dramatic damage to nigrostriatal neurons concerning the role of excitotoxicity in Parkinson's disease both in rodents and nonhuman primates, as evidenced by will emphasize the relationships between EAA-induced long-lasting depiction of striatal dopamine, decreases in ty­ damage and other biochemical and pharmacologic alter­ rosine hydroxylase activity, the number of high-affinity do­ ations. In particular, the mechanisms of action of different pamine uptake sites, and histochemical observation of neurotoxic agents will be reviewed as models of how excito­ nerve terminal degeneration in the striatum (Ellison et al., toxicity may contribute to nigrostriatal degeneration. 1978; Seiden ct al.. 1975; Wagner cl al., 1980). In 1989. Sonsalla and colleagues reported that the depletion of do­ Neurotoxic Afodels ofParkinson '.1· Disease pamine and tyrosine hydroxylase activity caused by meth­ As already mentioned, at least three different processes amphetamine in the mouse striatum could be prevented by have been suggested to cause neurodegeneration in Parkin­ (+ )MK-80 I. an antagonist of the NMDA receptor. This son's disease, and, interestingly, the actions of three toxic finding, which has since been confirmed and extended by agents have become prototype models for such neurotoxic further studies (Muraki ct al., 1992; Fuller cl al., 1992). not mechanisms: namely, dopamine for oxidative stress, MPTP only indicates that EAAs are involved in the mechanism of for abnormal mitochondrial activity, and methamphet­ methamphetamine neurotoxicity, but also supports a role amine for excitotoxicity. Dopamine not only is the neuro­ of excitotoxicity in nigrostriatal degenerative processes. transmitter utilized by neurons in the nigrostriatal pathway, Toxicity models should help us to unravel the precise na­ but may also act as an endogenous toxin by generating oxi- ture of events underlying a pathologic condition. However, EXCITOTOXINS, AGING, AND ENVIRONMENTAL NEUROTOXINS 11

OXIDATIVE et al., 1994). A significant and rather selective decrease in ATP was found in the stria tum of mice injected with meth­ amphetamine. This decrease seemed to be correlated with / .'::~::_ ~ NEURONAL dopamine depletion, since pretreatment of mice with 2-de­ METH oxyglucose, an inhibitor of glucose uptake and utilization, "'- .. TOXICITY / DAMAGE potentiated both ATP and dopamine loss caused by meth­ amphetamine. Taken together, these findings suggest that ~ENERGY/ different toxic events, including excitotoxicity, oxygen rad­ LOSS ical production, and energy failure, may all play a role in FIG. 2. Schematic representation of the possible mechanism of action nigrostriatal damage. The theory schematized in Fig. 2 ap­ of methamphetamine (METH), primarily involving excitotoxicity, with pears therefore to be supported by experimental evidence, the additional contributions of oxidative stress and energy loss leading to making the methamphetamine model particularly valuable damage of the dopaminergic neurons of the nigrostriatal pathway. not only for studies on the role of excitotoxicity in nigrostri­ atal degeneration, but also for evaluating the interactions between excitotoxicity and other toxic mechanisms. if the mechanisms of toxicity of dopamine, MPTP, and If this hypothesis of "interactive" mechanisms of nigro­ methamphetamine are each viewed as separate models for striatal damage explains methamphetamine neurotoxicity, the pathogenesis of cell loss in Parkinson's disease, more could it also be applied to the dopamine and/or MPTP questions are raised than addressed. First, we may question models? In particular, is there evidence for a role of excito­ the validity of the models themselves, and then we may still toxicity in these models? The interaction between dopa­ argue about the respective roles that oxidative stress, im­ mine and EAAs has become a much debated topic in pairment of mitochondrial function, and excitotoxicity neuropharmacology. It has recently been suggested that do­ play in Parkinson's disease. Information gathered from the paminergic terminals in the striatum are part of a cortico­ dopamine, MPTP, and methamphetamine models could striato-thalamo-cortical negative feedback loop, with dopa­ also lead to a different interpretation, however, namely that mine release causing the release of EAAs (Carlsson and an initial insult may then trigger a series of toxic events Carlsson, 1990). It is possible therefore that EAAs may con­ which ultimately cause nigrostriatal damage. For example, tribute to cell damage in conditions of increased dopamine as schematized in Fig. 2 and discussed in greater detail be­ turnover and, together with oxidative stress, may play a low, methamphetamine neurotoxicity may depend upon toxic role in the dopamine model of nigrostriatal degenera­ EAA-induced alterations, but may also involve other toxic tion. mechanisms. The involvement ofEAAs in the MPTP model is contro­ versial due to conflicting results of studies which have used Excitotoxicity and Nigrostriata/ Damage NMDA antagonists as protective agents against the toxic In addition to excitotoxicity, oxidative stress is likely to effects of MPTP (Sonsalla et al.. 1989, 1992; Storey et al., be a consequence ofmethamphetamine exposure. Using in­ 1992; Turski et al., 1991; Zuddas et al., 1992). The reasons tracerebral microdialysis, Nash and Yamamoto (1992) for these conflicting data remain to be elucidated, but are have found an increase in striatal glutamate release after likely to include differences in the role of EAAs in various repeated administration ofmethamphetamine to rats. Glu­ animal species (i.e., rodents vs primates), in various brain tamate release and, more in general, excitation of the regions (i.e., the striatum vs the substantia nigra), and at NMDA receptor may result in the production of oxygen different time points after exposure to MPTP and/or radicals (Lafon-Caza! et al., 1993), suggesting one possible NMDA antagonists. For example, ( + )MK-80 I has been link between excitotoxicity and oxidative stress. This link is found to prevent completely striatal dopamine depletion at further supported by the fact that methamphetamine neu­ early time points (i.e., 1.5, 4, and 8 hr) after administration rotoxicity appears to be strictly dependent upon the pres­ ofMPTP to mice, whereas no protective effect was observed ence and release of striatal dopamine which, as previously at later times (Chan et al., l 993a). Another factor that may discussed, may itself lead to oxidative stress. Indeed, meth­ contribute to the action ofNMDA antagonists as well as to amphetamine causes an increase in extracellular dopamine the conflicting data on their effects on MPTP neurotoxicity levels, as assessed by in vivo microdialysis (O'Dell et al., is the fact that ( + )MK-80 I has been reported to modify the 1991 ), and its toxic effects can be prevented by blocking rate of elimination of MPP+ from the mouse striatum dopamine synthesis with a-methyl-tyrosine (Gibb and Ko­ (Chan et al., I 993a). gan, 1979). Although the interactions of NMDA antagonists with Recent work has also revealed that a failure of energy sup­ MPTP in different experimental conditions are not com­ plies may occur after exposure to methamphetamine (Chan pletely clear, the overall evidence presented to date makes 12 DAWSON ET AL. it conceivable that excitotoxicity participates in the cascade sonian brain is relatively less documented, although initial of MPTP-induced toxic events. In particular, it is possible studies using animal models of Parkinson ism have already that increased extracellular levels of EAAs may result from led to pilot clinical trials of NMDA antagonists as antiPar­ the failure of energy supplies caused by MPP' accumula­ kinsonian drugs (Montastruc et al., 1992). It has been tion into mitochondria and the consequent inhibition of shown, for example, that (I) NMDA blockers potentiate the oxidative phosphorylation. Indeed, energy impairment antiParkinsonian action oflevodopa ( Klockgether and Tur­ may lead to decreased reuptake and/or increased release of ski, 1990), (2) that ( + )MK80 I stimulates locomotor activ­ EAAs (Nicholls and Atwell, 1990). The hypothesis of a re­ ity in mice previously depleted of monoaminergic stores lationship between excitotoxicity and mitochondrial ab­ (Carlsson and Carlsson, 1990), and (3) that microinjections normalities, supported by the MPTP model. has gained ofEAAs into different areas of the basal ganglia of rats cause wide interest among neuroscientists not only as a mecha­ electromyographic activity reflecting Parkinsonian rigidity nism for nigrostriatal damage, but also as a more general (Klockgether and Turski. 1993). It is also noteworthy that feature of the pathogenesis of neurodegenerative disease the role of EAA receptors may vary depending upon recep­ (Albin and Greenamyre. 1992; Beal, I 992). tor subtypes and sites within the basal ganglia (Klockgether and Turski. 1993), suggesting that nigrostriatal damage Implication/in· Parkinson '.1· Disease may be the result of molecular changes more complex and selective than previously thought and emphasizing the need Two main conclusions can be drawn from the dopamine, for more detailed pharmacologic and toxicologic studies. MPTP, and methamphetamine models of neurotoxicity, The high selectivity ofEAA-mediated neurotransmission namely that ( 1) a series of insults rather than a particular may contribute to an essential feature ofneurodegenerative toxic mechanism is likely to underlie nigrostriatal degener­ disorders such as Parkinson's disease, namely their discrete ation and that (2) these insults are likely to involve excito­ and characteristic patterns of neuronal death. Such selectiv­ toxicity, which could either represent an initial primary ity may be further enhanced ifEAA-induced cell damage is event or the consequence of other neurotoxic changes. associated with other neurotoxic mechanisms which may These conclusions are compatible with findings of studies themselves target specific populations of neurons. Other on Parkinson's disease, providing evidence in favor of the critical features of neurodegenerative disorders are their de­ occurrence of oxidative stress, mitochondrial abnormali­ layed onset and gradually progressive evolution. Both of ties, and excitotoxicity. these characteristics may result from the development of Oxidative stress has been implicated in Parkinson's dis­ metabolic abnormalities which may render neurons pro­ ease because of findings in the substantia nigra of patients gressively more susceptible to excitotoxicity. For example, showing, for example, ( 1) increased levels of nonheme iron a failure of energy supplies may become increasingly evi­ (Dexter et al .. l 989b) as well as products of lipid peroxida­ dent as a result of the aging process (Di Monte et al .. 1993) tion (Dexter et al., l 989a) and (2) impaired antioxidant de­ and may "predispose" to the toxic effects of EAAs (Henne­ fense mechanisms in the forms of decreased levels of re­ berry et al.. 1989). Thus, excitotoxicity could explain a duced glutathione (Perry et al.. 1982) and decreased activi­ number of features of neurodegenerative disorders, leading ties of glutathione peroxidase (Kish et al.. 1985 ). Defects in to the hypothesis that it may represent the final common oxidative phosphorylation have also been found in different pathway of neuronal death (Albin and Greenamyre, 1992). tissues of patients, leading to the hypothesis that Parkin­ This hypothesis also points to alterations in energy metab­ son's disease may be a mitochondrial disorder (Di Monte, olism as the most likely mechanism which would enhance 1991 ). While data concerning a possible failure in mito­ the susceptibility to EAA toxicity as well as the selectivity chondrial respiratory chain activity in the skeletal muscle of neuronal damage (Beal, 1992). A similar scenario seems remain controversial (Di Mauro, 1993), more consistent re­ particularly plausible for Parkinson's disease, consistent sults have been obtained by comparing specimens from the with findings from the Parkinsonian brain as well as from substantia nigra of patients vs control subjects (Schapira et the toxic models of nigrostriatal damage reviewed in this al .. 1990; Mizuno et al .. 1994 ). Indeed, abnormalities in the paper. nigral tissue appear to be rather selective for mitochondrial complex I. Evidence supporting a role of both oxidative stress and mitochondrial impairment in Parkinson's disease SUMMARY should not be surprising not only in view of our previous discussion, but also because mitochondria represent a pri­ A central theme that emerges from this review is that ex­ mary target for oxidative damage and, vice versa, because citotoxicity is inextricably intertwined with neuronal bioen­ impairment of mitochondrial activity may itself generate ergetics and mitochondrial function. Neurotoxicants that oxidizing species (Di Monte et al .. 1992). disrupt cellular energy metabolism may interact synergisti­ The occurrence of EAA-induced damage in the Parkin- cally with age-associated decrements in glucose utilization EXCITOTOXl'.\JS. AGING. AND ENVIRONMENTAL NEUROTOXl'.\JS 13 and mitochondrial function. Oxidative stress and reduced tochondrial energy metabolism underlie the pathology of neurodegener­ intracellular energy levels can interact to increase both ex­ ative diseases'' Trends J\'cumsci. 16, 125-131. tracellular GLU concentrations and GLU receptor-medi­ Benvenistc. H .. Jorgensen. M. B.. Sanberg. M .. Christensen. T.. Hagherg. H .. and Diemer, N. H. ( 1989). lschemic damage in hippocampal CA I is ated excitotoxicity (Coyle and Puttfarcken, 1993; Henne­ dependent on glutamate release and intact innervation from CA3 . .I berry et al.. 1989). Age-related or genetic alterations in an­ Cerch. IJ/ood Flo\\' :\IC'/i/h. 9, 629-639. tioxidant defense mechanisms and/or mitochondrial Bernath. S. ( 1991 ). Calcium-independent release of amino acid neuro­ function may render individuals susceptible to a variety of transmitters: Fact or artifact. /'mg. Nc11rohiol. 38, 57-91. environmental neurotoxins. Bondy. S. C. ( 1995). The relation of oxidative stress and hyperexcitation Three major points clearly emerge from the integration for neurological disease. /'me Soc. Ew Biol ..\led 208, 337-345. of the various perspectives presented here: first, the value of Bondy, S. C.. and LeBel. C. P. ( 1993). The relationship hetween excitotm.­ using neurotoxicants as models for elucidating the patho­ icity and oxidative stress in the central nerrnus system. Free Rad Biol. Med 14, 633-642. genic mechanisms underlying both rare and common neu­ Bose. R., Schnell, C. L.. Pinsky, C.. and Zitko. V. ( 1992). Effects of excito­ rological diseases: second, the high degree of overlap that toxins on free radical indices in mouse brain. 1iJ.ricol. l.C'll. 60, 211- exists between prooxidant and excitatory events: and last, 219. the fact that, while many diverse agents and diseases can Bossi. S. R .. Simpson, J. R .. and Isacson. 0. ( 1993). Age dependence of cause selective damage to the nervous system, these specific striatal neuronal death caused hy mitochondrial dysfunction. NcuroRc­ pathologies may be underlaid by a few ubiquitous vulnera­ JIOrt 4, 73- 76. ble features found in all neural tissues. Excitatory amino Brouillet. E .. Hantraye. P .. Dolan. R., Leroy-Willig, A .. Bottlaender, M .. acids acting as final common mediators of neuronal death Isacson, 0 .. Maziere, M .. Ferrante, R. J., and Beal. M. F. (I 993a). Chronic administration of 3-nitropropionic acid induced selective stria­ appear to be one of those vulnerable features that interact tal degeneration and abnormal choreiform movements in monkeys. Soc. with hoth pre- and postsynaptic mechanisms that are al­ 1Vc11ro.1ci .. Jhstr. 19, 409. tered hy the aging process. Brouillet. E .. Jenkins. B. G .. Hyman. B. T .. Ferrante. R. J.. Kowall. N. W .. Srivastava. R., Samanta Roy, D .. Rosen. B. R .. and Beal. M. F. ( l 993b). Age-dependent vulnerability of the striatum to the mitochondrial toxin ACKNOWLEDGMENTS 3-nitropropionic acid . ./. Nrnmchem. 60, 356-359. Brown. A. W., Aldrigde. W. N .. Street, B. W .. and Verchoyle, R. D. ( 1979). The authors acknowledge the efforts of Linda Panchou and Ana Morgan The behavioral and neuropathologic sequelac of intoxication by tri­ in the technical preparation of this manuscript. The authors also thank methyltin compounds in the rat. Am . ./. Pat/10/. 97, 59-82. Chantell Green for her assistance in coordinating this symposium. The Burke. S. P .. and Nadler, J. V. ( 1989). Effects of glucose deficiency on glu­ authors also thank Dr. Michael J. Meldrum for his critical reading of the tamate/aspartate release and excitatory synaptic responses in the hippo­ manuscript. This work was supported hy grants from the NIH (ES04 I 40) campal CA I area in l'itm. Brain Res. 500, 333-342. and the University of Florida (Research Development Award). Burns, R. S .. Chiueh, C. C.. 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