The decrement in dopamine levels exceeds the loss of dopaminergic neurons in Parkinson's disease (PD) patients and experimental models of PD. This discrepancy is poorly understood and may represent an important event in the pathogenesis of PD. Herein, we report that the ratelimiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is a selective target for nitration following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (MPP ؉ ). Nitration of TH also occurs in mouse striatum after MPTP administration. Nitration of tyrosine residues in TH results in loss of enzymatic activity. In the mouse striatum, tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 hr post MPTP injection. Striatal TH was not nitrated in mice overexpressing copper͞zinc superoxide dismutase after MPTP administration, supporting a critical role for superoxide in TH tyrosine nitration. These results indicate that tyrosine nitration-induced TH inactivation and consequently dopamine synthesis failure, represents an early and thus far unidentified biochemical event in MPTP neurotoxic process. The resemblance of the MPTP model with PD suggests that a similar phenomenon may occur in PD, inf luencing the severity of parkisonian symptoms.Parkinson's disease (PD) is a common neurodegenerative disorder characterized by disabling motor abnormalities attributed to a profound deficit in dopamine (1). The decline in dopamine level has been thought to arise solely from the severe loss of dopaminergic neurons in the nigrostriatal pathway. However, the dopamine deficit in the affected regions of the brain significantly exceed the loss of dopaminergic neurons (2, 3), suggesting that dopamine synthesis is impaired before cellular demise. Support for this hypothesis comes from studies of experimental models of PD demonstrating that the reduction in dopamine metabolism-related markers such as tyrosine hydroxylase (TH) and dopamine transporter is far greater than the loss of neuronal cell bodies (4-6). Because the severity of PD symptoms correlates with the magnitude of dopamine deficit, elucidating mechanisms that impair dopamine synthesis and metabolism in neurons that undergo selective degeneration in PD may have important therapeutic implications.There is experimental evidence from studies of humans and animals in support of the hypothesis that oxidative stress contributes to the pathogenesis of PD (7). Studies performed in the MPTP model of PD suggest that peroxynitrite, a reactive species formed by the nearly diffusion-limited reaction of nitric oxide with superoxide, may be a mediator of nigrostriatal damage in PD (8-10). The potential role of peroxynitrite in the pathogenesis of PD is further supported by demonstrating that exposure of the monoamine-producing PC12 cells to peroxynitrite induced a dose-dependent alteration in dopamine synthesis that was not due to cell death or the oxidation of dopamine (11)....

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Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. Tyrosine hydroxylase (TH) 1 (EC 1.14.16.2) is a non-heme iron, tetrahydrobiopterin-dependent protein that catalyzes the conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA) and represents the rate-limiting step in the biosynthesis of catecholamines (1). Loss of ability to synthesize catecholamines is an important step in the development of Parkinson's disease (PD) and other neurodegenerative diseases (2-6). Early loss of TH activity followed by a decline in TH protein is thought to contribute to the dopamine deficiency and phenotypic expression in PD and the MPTP mouse model (4). Tyrosine hydroxylase is a selective target for nitration following administration of the parkinsonian toxin MPTP to mice and following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (7). Nitration of one or more tyrosine residues of TH was temporally associated with loss of enzymatic activity. The magnitude of inactivation was proportional to the number of TH molecules that were nitrated in PC12 cells. In the mouse striatum, the tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 h post-MPTP injection (7).However, a recent report indicated that exposure of recombinant purified TH to peroxynitrite in vitro results not only in nitration of tyrosine residues but also in the formation of covalently linked dimers and oxidation of cysteine residues (8). The same report also indicated that cysteine oxidation rather than tyrosine nitration is responsible for the loss of TH enzymatic activity (8). Cysteine, methionine, tryptophan, and tyrosine appear to be the principal amino acids in proteins modified by peroxynitrite in vitro (9 -14). To resolve the apparent differences, the reaction of peroxynitrite with recombinant purified rat TH in vitro was re-examined, and no evidence of cysteine oxidation was found. Oxidation of one cysteine residue per molecule of TH was observed only at high peroxynitrite concentrations, and three cysteine residues were oxidized in partially unfolded protein. Amino acid analysis failed to show any * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Neuronal injury resulting from mechanical deformation is poorly characterized at the cellular level. The immediate structural consequences of the mechanical loading lead to a variety of inter-and intra-cellular signaling events that interact on multiple time and length scales. Thus, it is often difficult to establish cause-and-effect relationships such that appropriate treatment strategies can be devised. In this report, we showed that treating mechanically injured neuronal cells with an agent that promotes the resealing of disrupted plasma membranes rescues them from death at 24 h post-injury. A new in vitro model was developed to allow uniform mechanical loading conditions with precisely controlled magnitude and onset rate of loading. Injury severity increased monotonically with increasing peak shear stress and was strongly dependent on the rate of loading as assessed with the MTT cell viability assay, 24 h post-injury. Mechanical injury produced an immediate disruption of membrane integrity as indicated by a rapid and transient release of LDH. For the most severe injury, cell viability decreased approximately 40% with mechanical trauma compared to sham controls. Treatment of cells with Poloxamer 188 at 15 min post-injury restored long-term viability to control values. These data establish membrane integrity as a novel therapeutic target in the treatment of neuronal injury.

The mechanisms of cell death and the progressive degeneration of neural tissue following traumatic brain injury (TBI) have come under intense investigation. However, the complex interactions among the evolving pathologies in multiple cell types obscure the causal relationships between the initial effects of the mechanical trauma at the cellular level and the long-term dysfunction and neuronal death. We used an in vitro model of neuronal injury to study the mechanisms of cell death in response to a well-defined mechanical insult and found that the majority of dead cells were apoptotic. We have previously reported that promotion of membrane repair acutely with the non-ionic surfactant poloxamer 188 (P188) restored cell viability to control values at 24 h postinjury. Here, we showed that P188 significantly inhibits apoptosis and prevents necrosis. We also examined the role of mitogen-activated protein kinases (MAPKs) in cell death. There was a rapid, transient activation of extracellular signal-regulated kinases, c-Jun N-terminal kinase, and p38s after mechanical insult. Of these, activation of the proapoptotic p38 was the greatest. Treatment with P188 inhibited p38 activation; however, direct inhibition of p38 by SB203580, which selectively inhibits the activity of the p38 MAPK, provided only partial inhibition of apoptosis and had no effect on necrosis. These data suggest that multiple signaling pathways may be involved in the long-term response of neurons to mechanical injury. Furthermore, that the membrane resealing action of P188 provides such significant protection from both necrosis and apoptosis suggests that acute membrane damage due to trauma is a critical precipitating event that is upstream of the many signaling cascades contributing to the subsequent pathology.

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Experimental evidence has implicated oxidative stress in the development of Parkinson's disease, amyotrophic lateral sclerosis, and other degenerative neuronal disorders. Recently, peroxynitrite, which is formed by the nearly diffusion‐limited reaction of nitric oxide with superoxide, has been suggested to be a mediator of oxidant‐induced cellular injury. The potential role of peroxynitrite in the pathology associated with Parkinson's disease was evaluated by examining its effect on DOPA synthesis in PC12 pheochromocytoma cells. Peroxynitrite was generated from the compound 3‐morpholinosydnonimine (SIN‐1), which releases superoxide and nitric oxide simultaneously. Exposure of PC12 cells to peroxynitrite for 60 min greatly diminished their ability to synthesize DOPA without apparent cell death. The inhibition was due neither to the formation of free nitrotyrosine nor the oxidation of DOPA by peroxynitrite. The inhibition in DOPA synthesis by SIN‐1 was abolished when superoxide was scavenged by the addition of superoxide dismutase. These data indicated that neither nitric oxide nor hydrogen peroxide generated by the dismutation of superoxide is responsible for the SIN‐1‐mediated inhibition of DOPA production. The inhibition of DOPA synthesis at high concentration of SIN‐1 persisted even after removal of SIN‐1. The inactivation of the tyrosine hydroxylase may be responsible for the significant decline in DOPA formation by peroxynitrite. Inactivation of tyrosine hydroxylase may be part of the initial insult in oxidative damage that eventually leads to cell death.

Age-related cataract is a condition characterized by multiple mechanisms and multiple risk factors. The mechanisms that bring about a loss in transparency include oxidation, osmotic stress, and chemical adduct formation. Risk factors for cataract include diabetes, radiation (ultraviolet B, x-ray), certain pharmaceutical substances, certain nutritional states, and possibly acute episodes of dehydration. Interaction occurs between and among mechanistic factors and risk factors. Thus nutrition must be considered as one part of a tapestry of intertwined events and responses. Certain experimental models for nutritional cataract have been useful for study of the cataractogenic process but are probably not important factors in the human disease. Little current evidence supports significant roles in human senile cataract for imbalances of tryptophan or other amino acids, deficiencies of calcium or selenium, or excessive intake of selenium. Overconsumption of galactose is likely to be hazardous only in subjects with genetic inability to metabolize this sugar. Vitamins with antioxidant potential (riboflavin, vitamin E, vitamin C, carotenoids) deserve further research scrutiny to ascertain their significance in cataract etiology. Excessive caloric intake needs to receive added emphasis as a factor contributing to cataract. Diabetes increases the likelihood of cataract three- to four-fold. Obesity, defined as more than 20% overweight, is considered a major risk factor for non-insulin-dependent, or type II, diabetes (69, 73). Weight control can be recommended as a prudent, safe, economic, and effective means of lowering risk probability for diabetes and the associated complication of cataract.