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Although pathogenesis of neuronal ischemia is incompletely understood, evidence indicates apoptotic neuronal death after ischemia. Bcl-2, an anti-apoptotic and neuroprotective protein, interacts with calcineurin in non-neuronal tissues. Activation of calcineurin, which is abundant in the brain, may play a role in apoptosis. Using co-immunoprecipitation experiments in biopsy-derived, fresh human cortical and hippocampal slices, we examined possible interactions between calcineurin and Bcl-2. Calcineuin-Bcl-2 interactions increased after exposure in vitro to excitotoxic agents and conditions of hypoxia/aglycia. This interaction may shuttle calcineurin to substrates such as the inositol-1,4,5-tris-phosphate receptor because under these experimental conditions interactions between calcineurin and inositol-1,4,5-tris-phosphate receptor also increased. A specific calcineurin inhibitor, FK-520, attenuated insult-induced increases in calcineurin-Bcl-2 interactions and augmented caspase-3 like activity. These data suggest that Bcl-2 modulates neuroprotective effects of calcineurin and that calcineurin inhibitors increase ischemic neuronal damage., (Copyright 2003 IBRO)

Calcineurin, a calmodulin-dependent protein phosphatase, regulates transcription and possibly apoptosis. Previous studies demonstrated that in baby hamster kidney-21 cells after co-transfection calcineurin interacts with Bcl-2, thereby altering transcription and apoptosis. Using co-immunoprecipitation and subcellular fractionation techniques, we observed that calcineurin occurred as a complex with Bcl-2 in various regions of rat and mouse brain. The calcineurin-Bcl-2 complex was identified in mitochondrial, nuclear, microsomal and cytosol fractions. In vitro induction of hypoxia and aglycia or N-methyl-D-aspartate treatment markedly altered both extent of complex formation and its subcellular localization. These observations suggest that Bcl-2 either sequesters calcineurin, that calcineurin dephosphorylates Bcl-2, or that Bcl-2 shuttles calcineurin to specific substrates. Calcineurin also co-immunoprecipitated with the inositol-tris-phosphate receptor. This interaction increased after in vitro hypoxia/aglycia. In Bcl-2 (-/-) mice, interactions between calcineurin- and inositol-tris-phosphate receptor occurred less frequently than in wild-type mice under both control and hypoxic conditions. Experiments involving cell-free systems, as well as brain slices treated with thapsigargin or with N-methyl-D-aspartate suggested that calcium and calmodulin activation of calcineurin leads to interactions between calcineurin and Bcl-2. These data indicate that during times of cellular stress and damage, Bcl-2 targets activated calcineurin to specific compartments and substrates., (Copyright 2003 IBRO)

The distribution of the enzyme protein-O-carboxylmethyltransferase (EC 2.1.1.24) has been investigated in the rat brain using both immunohistochemical and biochemical techniques. The enzyme, which carboxylmethylates free aspartic and glutamic acid residues of protein substrates, was localized in neurons, but not other cell types throughout the brain. The highest immunoreactivity was detected throughout the cortex, followed by the hippocampus, the corpus striatum, the thalamus and the amygdala. Immunoreactive cells were detected in other brain regions but were not as prominent as those regions listed above. The distribution of immunoreactivity in the hippocampus was most striking, with considerable labelling of the pyramidal and granule cells in all regions. Numerous pyramidal cells were labelled in the cerebral cortex, with some ascending processes exhibiting immunoreactivity. The corpus striatum was uniformly labelled, suggesting that the enzyme was not localized to any specific neurotransmitter system. The antisera employed in this study was generated against purified bovine brain protein-O-carboxylmethyltransferase and Western immunoblot analysis showed cross reactivity against both rat brain and human erythrocyte forms of the enzyme. Enzyme activity and methyl acceptor protein capacity were examined in 1.5 mm coronal sections of rat brain. The regions with highest enzyme activities were found in cross-sections containing cortex and corpus striatum or cortex and hippocampus. The lowest enzyme activities were noted in slices of brainstem and cerebellum, areas exhibiting low amounts of immunoreactive protein-O-carboxylmethyltransferase. Methyl acceptor protein capacity was highest in slices of cortex and corpus striatum, cortex and hippocampus and was lowest in slices of brainstem and cerebellum. These results demonstrate that protein-O-carboxylmethyltransferase has an unique neuronal pattern of distribution in the rodent central nervous system, and suggest that the carboxylmethylation of proteins may be of functional significance in these neurons.

Trimethyltin is a neurotoxicant which produces a distinct pattern of neuronal cell death following peripheral administration of a single dose (8 mg/kg, i.p.) in rats. The cupric-silver degeneration stain was used to produce an atlas documenting the distribution and time course of trimethyltin-induced neuronal damage in adult, male Long-Evans rats. Animals were examined at survival times of 1, 2, 3, 4, 5, 7, 10 and 18 days after intoxication. The earliest degeneration was observed at day 1 in the intermediate and ventral divisions of the lateral septal nucleus, followed by development of degeneration on days 2-4 in neuron populations including the septohippocampal nucleus, septohypothalamic nucleus, anterior olfactory nucleus, bed nucleus of the stria terminalis, endopiriform nucleus, parafascicular nucleus, superior colliculus, interstitial nucleus of the posterior commissure, inferior colliculus, pontine nuclei, raphe nuclei, pars caudalis of the spinal trigeminal nucleus, the caudal aspect of nucleus tractus solitarius, dorsal vagal motor nucleus, granule cells in the dentate gyrus, pyramidal cells in CA fields of the hippocampus, and of neurons in the subiculum, pyriform cortex, entorhinal cortex and neocortex (mainly layer Vb and VI). This was followed by degenerative changes on days 5-7 in other structures, including the amygdaloid nuclei, the ventral posterolateral and ventral posteromedial thalamic nuclei and the periaqueductal gray. The distribution of terminal degeneration from these neurons indicate that specific pools of cells are affected in each structure, and the time course suggests somatofugal degeneration. The trimethyltin damage was also assessed with immunocytochemical visualization of a neuronotypic protein, protein-O-carboxyl methyltransferase and a radioimmunoassay for glial fibrillary acidic protein. Protein-O-carboxyl methyltransferase immunoreactivity was altered in neuronal populations damaged by trimethyltin, but did not appear to be either as sensitive or selective an assay of neuronal damage as the silver stain, especially at short survival times. Glial fibrillary acidic proteins were dramatically elevated 21 days after trimethyltin intoxication, particularly in areas of extensive damage. These studies revealed advantages and problems encountered in the use of each technique in assessing neurotoxic effects, forming a basis for discussion of the relative merits of using a battery of specific molecular probes for neurotoxicity evaluations.