Metformin lowers Ser-129 phosphorylated α-synuclein levels via mTOR-dependent protein phosphatase 2A activation.

Phospho-Ser129 α-synuclein is the modified form of α-synuclein that occurs most frequently within Parkinson's disease pathological inclusions. Here we demonstrate that the antidiabetic drug metformin significantly reduces levels of phospho-Ser129 α-synuclein and the ratio of phospho-Ser129 α-synuclein to total α-synuclein. This effect was documented in vitro in SH-SY5Y and HeLa cells as well as in primary cultures of hippocampal neurons. In vitro work also elucidated the mechanisms underlying metformin's action. Following metformin exposure, decreased phospho-Ser129 α-synuclein was not strictly dependent on induction of AMP-activated protein kinase, a primary target of the drug. On the other hand, metformin-induced phospho-Ser129 α-synuclein reduction was consistently associated with inhibition of mammalian target of rapamycin (mTOR) and activation of protein phosphatase 2A (PP2A). Evidence supporting a key role of mTOR/PP2A signaling included the finding that, similar to metformin, the canonical mTOR inhibitor rapamycin was capable of lowering the ratio of phospho-Ser129 α-synuclein to total α-synuclein. Furthermore, no decrease in phosphorylated α-synuclein occurred with either metformin or rapamycin when phosphatase activity was inhibited, supporting a direct relationship between mTOR inhibition, PP2A activation and protein dephosphorylation. A final set of experiments confirmed the effectiveness of metformin in vivo in wild-type C57BL/6 mice. Addition of the drug to food or drinking water lowered levels of phospho-Ser129 α-synuclein in the brain of treated animals. These data reveal a new mechanism leading to α-synuclein dephosphorylation that could be targeted for therapeutic intervention by drugs like metformin and rapamycin.

Increased α-synuclein phosphorylation and nitration in the aging primate substantia nigra.

Post-translational modifications of α-synuclein occur in the brain of patients affected by Parkinson's disease and other α-synucleinopathies, as indicated by the accumulation of Lewy inclusions containing phosphorylated (at serine 129) and nitrated α-synuclein. Here we found that phospho-Ser 129 and nitrated α-synuclein are also formed within dopaminergic neurons of the monkey substantia nigra as a result of normal aging. Dopaminergic cell bodies immunoreactive for phospho-Ser 129 and nitrated α-synuclein were rarely seen in adult mature animals but became significantly more frequent in the substantia nigra of old primates. Dual labeling with antibodies against phospho-Ser 129 and nitrated α-synuclein revealed only limited colocalization and mostly stained distinct sub-populations of dopaminergic neurons. Age-related elevations of modified protein paralleled an increase in the number of neurons immunoreactive for unmodified α-synuclein, supporting a relationship between higher levels of normal protein and enhanced phosphorylation/nitration. Other mechanisms were also identified that likely contribute to α-synuclein modifications. In particular, increased expression of Polo-like kinase 2 within neurons of older animals could contribute to phospho-Ser 129 α-synuclein production. Data also indicate that a pro-oxidant environment characterizes older neurons and favors α-synuclein nitration. Aging is an unequivocal risk factor for human α-synucleinopathies. These findings are consistent with a mechanistic link between aging, α-synuclein abnormalities and enhanced vulnerability to neurodegenerative processes.

Decreased susceptibility to oxidative stress underlies the resistance of specific dopaminergic cell populations to paraquat-induced degeneration.

The vulnerability of different dopaminergic cell populations to damage caused by the herbicide paraquat was assessed by stereological counts of tyrosine hydroxylase-positive and calbindin-D28k-immunoreactive neurons in A9 (substantia nigra pars compacta) and A10 (ventral tegmental area and other cell groups). In saline-treated control mice, tyrosine hydroxylase-immunoreactive neurons represented 80% and 45% of the total neuronal population in A9 and A10, respectively, and the number of calbindin-D28k-positive neurons was five times greater in A10 than A9. Sequential injections with paraquat resulted in a significant loss of dopaminergic neurons in A9. In contrast, tyrosine hydroxylase-positive cells in A10 were spared from paraquat-induced degeneration. Furthermore, expression of calbindin-D28k was consistently associated with neuronal resistance to the herbicide in both A9 and A10. Paraquat exposure also induced oxidative stress as indicated by an increase in the number of midbrain cells positive for 4-hydroxy-2-nonenal, a marker of lipid peroxidation. Co-localization studies revealed that calbindin-D28k immunoreactivity overlapped with tyrosine hydroxylase labeling and that, after paraquat administration, (i) the vast majority of midbrain 4-hydroxy-2-nonenal-immunoreactive cells were dopaminergic (tyrosine hydroxylase-immunoreactive), (ii) tyrosine hydroxylase/4-hydroxy-2-nonenal-positive neurons were much more prevalent in A9 than A10, and (iii) all calbindin-D28k-containing neurons were characterized by lack of lipid peroxidation (4-hydroxy-2-nonenal immunoreactivity). Results in this paraquat model emphasize that, despite sharing a similar dopaminergic phenotype, different groups of midbrain neurons vary dramatically in their vulnerability to injury. Data also indicate that these differences are attributable, at least in part, to a varying susceptibility of dopaminergic cell populations to oxidative stress.

Enhanced striatal opioid receptor-mediated G-protein activation in L-DOPA-treated dyskinetic monkeys.

Long-term l-3,4-dihydroxyphenylalanine (L-DOPA) treatment in Parkinson's disease leads to dyskinesias in the majority of patients. The underlying molecular mechanisms for L-DOPA-induced dyskinesias (LIDs) are currently unclear. However, the findings that there are alterations in opioid peptide mRNA and protein expression and that opioid ligands modulate dyskinesias suggest that the opioid system may be involved. To further understand its role in dyskinesias, we mapped opioid receptor-stimulated G-protein activation using [35S]guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) autoradiography in the basal ganglia of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned squirrel monkeys administered water or L-DOPA. Subtype-selective opioid receptor G-protein coupling was investigated using the mu-opioid agonist [D-Ala, N-Me-Phe, Gly-ol]-enkephalin, delta-agonist SNC80 and kappa-agonist U50488H. Our data show that mu-opioid receptor-mediated G-protein activation is significantly enhanced in the basal ganglia and cortex of L-DOPA-treated dyskinetic monkeys, whereas delta- and kappa-receptor-induced increases were limited to only a few regions. A similar pattern of enhancement was observed in both MPTP-lesioned and unlesioned animals with LIDs suggesting the effect was not simply due to a compromised nigrostriatal system. Opioid receptor G-protein coupling was not enhanced in non-dyskinetic L-DOPA-treated animals, or lesioned monkeys not given L-DOPA. The increases in opioid-stimulated [35S]GTPgammaS binding are directly correlated with dyskinesias. The present data demonstrate an enhanced subtype-selective opioid-receptor G-protein coupling in the basal ganglia of monkeys with LIDs. The positive correlation with LIDs suggests this may represent an intracellular signaling mechanism underlying these movement abnormalities.

Increases in striatal preproenkephalin gene expression are associated with nigrostriatal damage but not L-DOPA-induced dyskinesias in the squirrel monkey.

Changes in preproenkephalin expression in the caudate and putamen have been linked to the development of L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias in primate models of Parkinson's disease, although not all investigators have been able to confirm this association. Because nigrostriatal damage per se is associated with increases in striatal preproenkephalin mRNA levels, it is difficult to know if changes in transcript levels are a result of lesioning or concurrent L-DOPA treatment and resulting dyskinesias. To circumvent these difficulties, we measured striatal preproenkephalin mRNA levels in monkeys with L-DOPA-induced dyskinesias both with and without lesions of the nigrostriatal system. The latter model is not confounded by morphological and biochemical changes resulting from nigrostriatal damage. Monkeys were gavaged with L-DOPA (15 mg/kg) twice daily for a 2-week period and killed 3 days after treatment. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment alone resulted in an increase in preproenkephalin mRNA levels as previously shown. However, striatal transcript levels were similarly elevated in dyskinetic MPTP-lesioned animals treated with L-DOPA. In unlesioned animals, preproenkephalin mRNA levels were also similar in control and L-DOPA-treated dyskinetic monkeys. Because drug-induced changes in mRNA may not be sustained for a prolonged period after treatment, a second series of experiments were done in which animals were killed 3-4 h after the last dose of L-DOPA, but the results were similar to those obtained after 3 days. These data show that, while elevations in striatal preproenkephalin mRNA levels are associated with nigrostriatal damage, they are not linked to the development of L-DOPA-induced dyskinesias. These results thus question the importance of preproenkephalin mRNA in the pathogenesis of this disabling complication of L-DOPA therapy in Parkinson's disease.

Nicotine administration reduces striatal MPP+ levels in mice.

Nicotine administration has previously been shown to attenuate nigrostriatal damage in animal models of Parkinson's disease, including the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse. The present experiments were done to determine whether nicotine may be exerting its effects by altering striatal levels of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of MPTP. Mice were injected with nicotine (0.33-1 mg/kg i.p.) 10 min prior to MPTP (30 mg/kg s.c.) followed by three subsequent doses of nicotine at 15-min intervals according to a dose schedule previously shown to be neuroprotective. The mice were sacrificed 1.5, 4 and 8 h after MPTP administration and striatal MPP+ levels measured. Nicotine administration (0.33-1.0 mg/kg) resulted in a time-dependent decline in striatal MPP+ levels that was significantly enhanced over that in saline injected animals. Experiments done to examine the effect of age showed that the decrease was observed in older (8-10 months) but not young (6-8 weeks) mice, a finding which may explain some of the variability in the effect of nicotine in the MPTP-induced model of nigrostriatal degeneration. In summary, these results suggest that nicotine may exert its neuroprotective action against nigrostriatal degeneration, at least in part, by decreasing striatal MPP+ levels.

Levodopa induces dyskinesias in normal squirrel monkeys.

This study assessed whether or not levodopa induces dyskinesias in normal (ie, unlesioned) squirrel monkeys. All six animals treated twice daily with levodopa (15 mg/kg with carbidopa by oral gavage) for two weeks developed choreoathetoid dyskinesias, whereas none of the vehicle-treated animals displayed any abnormal movements. These dyskinesias did not merely reflect a generalized motor activation as locomotion was actually suppressed. The present data demonstrate that preexisting nigrostriatal damage is not necessary for the development of levodopa-induced dyskinesias.

Lack of nigral pathology in transgenic mice expressing human alpha-synuclein driven by the tyrosine hydroxylase promoter.

alpha-Synuclein has been identified as a major component of Lewy body inclusions, which are one of the pathologic hallmarks of idiopathic Parkinson's disease. Mutations in alpha-synuclein have been found to be responsible for rare familial cases of Parkinsonism. To test whether overexpression of human alpha-synuclein leads to inclusion formation and neuronal loss of dopaminergic cells in the substantia nigra, we made transgenic mice in which the expression of wild-type or mutant (A30P and A53T) human alpha-synuclein protein was driven by the promoter from the tyrosine hydroxylase gene. Even though high levels of human alpha-synuclein accumulated in dopaminergic cell bodies, Lewy-type-positive inclusions did not develop in the nigrostriatal system. In addition, the number of nigral neurons and the levels of striatal dopamine were unchanged relative to non-transgenic littermates, in mice up to one year of age. These findings suggest that overexpression of alpha-synuclein within nigrostriatal dopaminergic neurons is not in itself sufficient to cause aggregation into Lewy body-like inclusions, nor does it trigger overt neurodegenerative changes.

Acute exposure to organochlorine pesticides does not affect striatal dopamine in mice.

The purpose of this study was to evaluate the possible association between the risk of developing Parkinson's disease (PD) and exposure to organochlorine pesticides in the mouse model. Animals were treated with a single subcutaneous injection of either dieldrin (40 and 80 mg/kg) or 2,4-dichlorophenoxyacetic acid (100 and 200 mg/kg, 2,4-D) and levels of dopamine (DA) and DA metabolites were measured in the striatum at the 7-day time point. Dieldrin exposure did not affect the striatal concentrations of DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Administration of 2,4-D did not produce any changes with the exception of a slight (15%), but statistically significant decrease in DOPAC using the higher dose of the pesticide. No neurochemical signs of dopaminergic injury were found following the combined treatment with either dieldrin or 2,4-D plus diethyldithiocarbamate (DDC), a compound known to potentiate the effects of the dopaminergic toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, neither dieldrin nor 2,4-D caused additional damage in animals previously lesioned with MPTP. Data failed to support the hypothesis that acute exposure to organochlorine compounds or synergistic interactions involving these pesticides may cause significant damage to dopaminergic terminals and therefore contribute to nigrostriatal degeneration in PD.

Increased striatal dopamine turnover following acute administration of rotenone to mice.

Because of the potential role of mitochondrial dysfunction in nigrostriatal degeneration in Parkinson's disease, the effects of rotenone (an inhibitor of mitochondrial NADH dehydrogenase and a naturally occurring toxicant) on the levels of striatal dopamine (DA) and DA metabolites were evaluated after acute and subchronic administration to mice. Systemic acute treatment with relatively high doses of rotenone did not affect DA concentration, but caused a significant increase in both DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). DOPAC and HVA changes were measured at 1 day and were reversed within 1 week, paralleling the time course of rotenone-induced increase in striatal lactate levels. Subchronic administration with a relatively mild dose of rotenone did not significantly alter the striatal levels of DA and DOPAC, while it slightly reduced HVA concentration. No neurochemical signs of dopaminergic damage were seen when mice were co-exposed to rotenone and diethyldithiocarbamate, a compound known to enhance nigrostriatal injury caused by the neurotoxicant 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP). Also, rotenone did not cause additional injury to animals previously lesioned by MPTP. Taken together, data indicate that rotenone is not capable of causing overt dopaminergic toxicity under the testing paradigms used in this study. Rather, an increase in DA turnover, as indicated by a higher (DOPAC+HVA)/DA ratio, seems to be associated to rotenone-induced striatal energy impairment.

Autoradiographic analysis of dopamine receptor-stimulated [(35)S]GTPgammaS binding in rat striatum.

Autoradiographic analysis of [(35)S]GTPgammaS binding was used to investigate functional activation of dopamine receptors in rat striatum. Dopamine-stimulated [(35)S]GTPgammaS binding was observed with a maximal increase of 38% over basal activity. A similar stimulatory response was obtained with the D(2) agonist quinpirole, but not SKF-238393, a D(1) agonist. The effect of dopamine was blocked by the D(2) antagonist raclopride, but was unaffected by SCH-23990, a D(1) antagonist. There appeared to be a differential distribution of dopamine-stimulated [(35)S]GTPgammaS binding, with the lowest activity obtained in the medial portion of the caudal striatum. These results demonstrate, using an autoradiographic approach, (i) that dopamine stimulated [(35)S]GTPgammaS binding in the rat striatum occurs through activation of D(2) receptors, and (ii) that the effects of dopamine activation vary in different areas of the rat striatum.

Autoradiographic analysis of N-methyl-D-aspartate receptor binding in monkey brain: effects of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine and levodopa treatment.

The anatomic distribution of N-methyl-D-aspartate receptors was investigated in the squirrel monkey brain using quantitative autoradiography with [125I]MK-801 as the radioligand. A heterogeneous distribution of [125I]MK-801 binding sites was observed, with the most intense expression in the outer cortex, hippocampus, olfactory tubercle, caudate and putamen. High levels were also observed in the thalamus, nucleus accumbens and inner cortex, with moderate levels in the claustrum. Relatively low expression levels were detected in the subthalamic nucleus with no apparent binding in the globus pallidus and the substantia nigra. Characterization of striatal [125I]MK-801 binding yielded a B(max) of 63.5 fmol/mg tissue and K(d) of 0.53 nM in the caudate, with similar values for the putamen. Experiments were subsequently performed to compare striatal [125I]MK-801 binding in the following four experimental groups: (i) control animals injected with saline; (ii) monkeys treated with levodopa; (iii) animals rendered parkinsonian after exposure to the neurotoxicant 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine; and (iv) dyskinetic monkeys treated with both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and levodopa. No changes were observed in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-lesioned animals compared with the saline control group. However, administration of levodopa to either unlesioned or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys resulted in a significant decrease in [125I]MK-801 binding in both the caudate and putamen. The data indicate that levodopa exerts a modulatory effect on the striatal glutamatergic system and suggest that a down-regulation of N-methyl-D-aspartate receptors by levodopa, combined with a deficiency in nigrostriatal dopamine function, may play a role in the development of levodopa induced dyskinesias.

Relationship among nigrostriatal denervation, parkinsonism, and dyskinesias in the MPTP primate model.

Presynaptic denervation is likely to play an important role in the pathophysiology of dyskinesias that develop after levodopa administration to patients with Parkinson's disease. In this study, the thresholds of nigrostriatal damage necessary for the occurrence of parkinsonism and levodopa-induced involuntary movements were compared in squirrel monkeys lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Animals treated with a regimen of MPTP that caused parkinsonism displayed > or =95% striatal dopamine depletion, 90% reduction of striatal dopamine uptake sites, and 70% nigral neuronal loss. Levodopa administration ameliorated the parkinsonian signs of these monkeys but also induced dyskinesias. A separate group of animals was treated with a milder MPTP regimen that caused 60%-70% striatal dopamine depletion, a 50% decrease in dopamine transporter, and 40% loss of dopaminergic nigral neurons. While these monkeys displayed no behavioral signs of parkinsonism, they all became dyskinetic after levodopa administration. The priming effect of levodopa, that is, the recurrence of dyskinesias in animals previously exposed to the drug, was compared in severely versus mildly lesioned monkeys. When severely injured parkinsonian animals underwent a second cycle of levodopa treatment, they immediately and consistently developed involuntary movements. In contrast, the recurrence of dyskinesias in primed monkeys with a partial nigrostriatal lesion required several levodopa administrations and remained relatively sporadic. The data indicate that moderate nigrostriatal damage which does not induce clinical parkinsonism predisposes to levodopa-induced dyskinesias. Once dyskinesias have been induced, the severity of denervation may enhance the sensitivity to subsequent levodopa exposures.

Expression of D(3) receptor messenger RNA and binding sites in monkey striatum and substantia nigra after nigrostriatal degeneration: effect of levodopa treatment.

D(3) receptors are prominently localized in the primate caudate-putamen, and D(3) receptor agonist properties may offer an advantage in Parkinson's disease therapy. In the present experiments, we investigated the relationship between D(3) receptor mRNA, D(3) receptor sites and the dopamine transporter in monkey basal ganglia by comparing their distribution in the brain of control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys (Samirai sciureus). In control monkeys, D(3) receptor mRNA appears to be widely expressed throughout the brain, with a distribution similar to that observed in both man and rodent. D(3) receptors are present in areas which express mRNA but also in some which do not, an observation which suggests they may be both pre- and postsynaptic in the monkey brain. Chronic MPTP administration, which selectively destroys the nigrostriatal system, resulted in a 70 to 99% depletion of the dopamine transporter in the basal ganglia. Autoradiographic analysis showed that after MPTP treatment there was a significant decline in D(3) receptors in the caudate, but not putamen, globus pallidus, substantia nigra or other dopaminergic regions. D(3) receptor mRNA expression was not changed in any region after nigrostriatal lesioning. Two weeks of L-3,4-dihydroxyphenylalanine (levodopa, L-DOPA) treatment, which alleviated Parkinsonism but also induced dyskinesias, reversed the MPTP-induced decline in caudate D(3) receptors. These results show that there is a selective decline in D(3) receptors in the caudate after nigrostriatal degeneration, which is reversed by L-DOPA treatment. Since the majority of dopaminergic nerve terminals were destroyed after MPTP lesioning, the reversal in D(3) receptors after L-DOPA treatment may represent an increase in caudate postsynaptic receptors, which could conceivably contribute to an imbalance in striatal circuitry and the development of dyskinesias.

Investigating levodopa-induced dyskinesias in the parkinsonian primate.

Research into the cause of dyskinesias arising from levodopa treatment has been vexingly limited, partly due to the lack of an inexpensive and widely available animal model. Rodents do not develop levodopa-induced dyskinesias in a clinically recognizable form. However, nonhuman primates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism readily develop levodopa-induced dyskinesias that are virtually indistinguishable from those seen in patients with Parkinson's disease. We have developed and validated a five-point Global Primate Dyskinesia Rating Scale to accurately measure these dyskinesias. Monkeys with MPTP-induced parkinsonism were then investigated to evaluate the relationship between dyskinesias, parkinsonism and severity of the nigrostriatal lesion. All parkinsonian animals were responsive to levodopa, and developed dyskinesias within 2-3 days of levodopa administration. Monkeys treated with only a single injection of MPTP also developed dyskinesias, even though they were not parkinsonian. It would appear that there is a different threshold of striatal dopamine depletion for parkinsonism and dyskinesias in the monkey. Finally, three hypotheses, put forward to explain the genesis of dyskinesias, are reviewed, and various experimental approaches suggested for each.

Combined assessment of tau and neuronal thread protein in Alzheimer's disease CSF.

OBJECTIVE: Comparative study of CSF levels of tau and AD7C-neuronal thread protein (NTP) in patients with AD and control subjects. BACKGROUND: AD is characterized by neurofibrillary tangles composed of the abnormally hyperphosphorylated microtubule-associated protein tau. AD7C-NTP is a proposed AD marker expressed at early stages of neurofibrillary degeneration. METHODS: Enzyme-linked immunosorbent assays specific for tau and AD7C-NTP. CSF samples were obtained from 35 demented patients (25 with antemortem clinical diagnosis of probable AD, 5 with neuropathologic diagnosis of definite AD, 5 with Lewy body pathology), 29 nondemented patients with PD, and 16 elderly healthy control subjects. Receiver operating characteristics (ROC) and multivariate discriminant analysis for AD versus controls. Correlational analysis of CSF tau and AD7C-NTP and of each marker with Mini-Mental State Examination (MMSE) scores was performed. RESULTS: Levels of both tau and AD7C-NTP were significantly elevated in the AD patients compared with control subjects. ROC analysis showed that CSF tau distinguished between patients with AD and nondemented control subjects with 63% sensitivity and 89% specificity, AD7C-NTP with 70% sensitivity and 87% specificity. Combined evaluation of both markers with discriminant analysis raised the specificity to 93% at a 63% sensitivity level. Both markers positively correlated with each other within the AD group, but not among control subjects. CSF levels of AD7C-NTP, but not of tau, showed a small but significant inverse correlation (r = -0.43) with MMSE scores of AD patients. CONCLUSIONS: CSF levels of tau and AD7C-NTP may be useful biomarkers for AD.

7-Nitroindazole prevents 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-induced ATP loss in the mouse striatum.

The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is dependent upon the MAO-B (monoamine oxidase type B)-catalyzed production of 1-methyl-4-phenylpyridinium ion (MPP(+)) and is likely to involve a perturbation of energy metabolism. Protection against MPTP neurotoxicity has been shown by treating mice with 7-nitroindazole (7-NI), a reversible inhibitor of both MAO-B and neuronal nitric oxide synthase (nNOS) activity. The objective of the present study was to evaluate (i) the relationship between the neuroprotective effect of 7-NI and MPTP-induced energy deficiency, and (ii) the role of nitric oxide production as a potential mechanism for energy perturbation after MPTP exposure. Maximum protection against striatal dopamine depletion and nigral neuronal loss was achieved when 7-NI (50 mg/kg, i.p.) was administered to C57BL/6 mice immediately before and after MPTP (50 mg/kg, s.c.). This short-term regimen of 7-NI administration parallels the time when MPTP exposure causes energy failure. 7-NI also completely prevented the loss of striatal ATP that occurs in mice during the initial hours after MPTP administration. In contrast, N(G)-nitro-L-arginine (two injections of 50 mg/kg each, given i.p. 20 and 4 h prior to MPTP), another NOS inhibitor, failed to affect MPTP-induced ATP depletion. Taken together, data indicate that (i) a temporal and causal relationship exists between the neuroprotective effect of 7-NI and its ability to counteract ATP reduction, and (ii) MAO-B rather than NOS inhibition is the mechanism by which 7-NI counteracts MPTP-induced ATP depletion.

Impaired glutamate clearance as a consequence of energy failure caused by MPP(+) in astrocytic cultures.

Astrocytes are the site of bioactivation of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) into its toxic 1-methyl-4-phenylpyridinium (MPP(+)) metabolite. The mechanism by which MPP(+) is capable of decreasing astrocytic glutamate uptake was evaluated in this study using primary cultures of astrocytes. Addition of glutamate to these cultures was followed by its efficient clearance from the extracellular space. However, when astrocytes were preincubated with MPP(+), glutamate clearance was significantly impaired. This effect was concentration-dependent, became more pronounced by prolonging the incubation in the presence of MPP(+) and occurred at a time when cell membrane integrity was still preserved. No evidence was found that reactive oxygen species contributed to MPP(+)-induced decrease in glutamate clearance. Indeed, neither the spin trapping agent alpha-phenyl-tert-butyl nitrone, the lazaroid antioxidant U-74389G, nor the disulfide-reducing agent dithiothreitol was capable of restoring glutamate net uptake. The effect of MPP(+) on glutamate clearance: (i) was accompanied by a decrease in cellular ATP; (ii) could be enhanced by withdrawing glucose from the incubation medium or by inhibiting glycolysis with 2-deoxyglucose, and (iii) could be reproduced using the mitochondrial complex I inhibitor rotenone. Taken together, these results indicate that, by acting as a mitochondrial poison, MPP(+) impairs energy metabolism of astrocytes and significantly reduces their ability to maintain low levels of extracellular glutamate.

Absence of mutations in the coding region of the alpha-synuclein gene in pathologically proven Parkinson's disease.

A missense mutation of the alpha-synuclein gene has been associated with parkinsonism in a large Italian kindred. Recently, alpha-synuclein was also identified in Lewy bodies. Using reverse transcribed-polymerase chain reaction (RT-PCR) technique, we sequenced the entire coding region of the alpha-synuclein gene using brain tissue from 24 pathologically proven Parkinson's disease cases. No mutations were found in any of the patients, suggesting that a mutation at the coding region of the alpha-synuclein gene is unlikely to be responsible for nigrostriatal degeneration in typical sporadic Parkinson's disease.