Localization and characterization of stannin: relationship to cellular sensitivity to organotin compounds.

The cDNA encoding the protein stannin was isolated previously via subtractive hybridization, using differential expression after trimethyltin (TMT) intoxication, as a basis for isolating mRNA which may be expressed in TMT-sensitive cells. Initial characterization revealed a novel gene product which was differentially expressed in several tissues sensitive to TMT. In the current study, biochemical and molecular techniques were used to quantitate stannin expression at the cellular and subcellular levels. Northern blot analysis showed that the stannin 3.0 kb mRNA transcript was present, in decreasing amounts, in: spleen, hippocampus, neocortex, cerebellum, striatum, midbrain, kidney and lung. Liver, heart, skeletal muscle and testis showed no detectable expression of stannin mRNA. Immunoblot analysis using antipeptide antisera raised against stannin indicated a high level of expression in spleen, followed by brain and kidney. Stannin mRNA was present during early brain development and consolidated by post-natal day (PND) 20 to patterns and levels seen in adults. In situ hybridization studies showed widespread neuronal expression of stannin mRNA at PND 1, which shifted to a restricted pattern of expression in specific regions by PND 20. Stannin was partially purified from rodent brain and spleen using cation exchange, sizing and hydrophobic interaction chromatography. It behaved as a monomer throughout purification. Stannin was also expressed in a baculovirus system, using a series of constructs containing the entire cDNA, 1.0 kb of DNA flanking the open reading frame, and a 400 bp open reading frame minimal construct. While all constructs expressed stannin, the best expression was seen with the entire cDNA. Based on current findings, we suggest that stannin expression is necessary but not sufficient for TMT toxicity.

Alterations in hippocampal expression of SNAP-25, GAP-43, stannin and glial fibrillary acidic protein following mechanical and trimethyltin-induced injury in the rat.

A set of well-defined antisera against neuronal and glial proteins were used to characterize patterns of protein expression in rat hippocampus following transection of the fimbira-fornix and perforant pathways or after administration of the selective neurotoxicant trimethyltin (8 mg/kg, i.p.). SNAP-25 (synaptosomal protein, mol. wt 25,000) is a neuron-specific, developmentally regulated presynaptic protein, stannin is a protein enriched in cells sensitive to trimethyltin, and GAP-43 (growth-associated protein, mol. wt 43,000) is associated with axonal growth and regeneration. Glial fibrillary acidic protein is an astrocyte-specific intermediate filament protein and a marker for reactive gliosis. SNAP-25 immunoreactivity was altered following both neurotoxicant and mechanical injury. Three days after fimbria-fornix/perforant path lesions, there was a loss of SNAP-25 immunoreactivity in hippocampal efferent pathways and in the lesioned entorhinal cortex. By day 12, there was evidence of reinnervation of hippocampal subfields by SNAP-25-immunopositive commissural afferent fibers. On day 3, immunoblots showed the appearance of SNAP-25a, a developmental isoform produced by alternative splicing of nine amino acids in exon 5, in lesioned tissues. This isoform declined by day 12 and was not found in contralateral control hippocampus or non-lesioned brain regions. Stannin immunoreactivity was unchanged, while GAP-43 was prominent on day 12 post-lesion. Glial fibrillary acidic protein immunoreactivity indicated gliosis near the site of pathway transection. In contrast, trimethyltin induced a marked loss of stannin immunoreactivity in hippocampal neurons seven days after injection. Trimethyltin increased glial fibrillary acidic protein staining in the hippocampus and other damaged regions. SNAP-25 immunoreactivity was markedly increased in mossy fibers and other hippocampal fields seven days following trimethyltin. Immunoblot analysis showed that only the adult SNAP-25b isoform was expressed after trimethyltin intoxication. These data suggest that SNAP-25 is a useful marker for presynaptic damage. Furthermore, reexpression of developmental isoforms of SNAP-25a may precede functional reinnervation when the postsynaptic target remains intact.

Developmental expression of neuronal calmodulin-binding proteins in rat brain.

The developmental patterns of calmodulin-binding proteins (CaM-BPS) in rat brain were examined using biotinylated calmodulin overlays of one- and two-dimensional gels. Hippocampus showed the earliest onset of CaM-BP expression (postnatal day 5; PND5), followed by cerebral cortex and striatum, both of which had detectable levels of CaM-BPs by PND7. Cerebellum had the latest onset of CaM-BP expression; CaM-BPs were not detectable until PND9. Very few CaM-BPs were present in brain before PND5 and all regions reached near adult levels by PND20. However, several unique CaM-BPs were seen in embryonic brain and these proteins may have an important role in developing neurons. These data suggest an orderly, complex expression of CaM-BPs which increases during times of synaptogenesis and synaptic maturation.