The key target of this study was the tau protein kinase II system (TPK II) involving the catalytic subunit cdk5 and the regulatory component p35. TPK II is one of the tau phosphorylating systems in neuronal cells, thus regulating its functions in the cytoskeletal dynamics and the extension of neuronal processes. This research led to demonstration that the treatment of rat hippocampal cells in culture with fibrillary L L-amyloid (AL L) results in a significant increase of the cdk5 enzymatic activity. Interestingly, the data also showed that the neurotoxic effect of 1^20 W WM AL L on primary cultures markedly diminished with co-incubation of hippocampal cells with the amyloid fibers plus the cdk5 inhibitor butyrolactone I. This inhibitor protected brain cells against AL L-induced cell death in a concentration dependent fashion. Moreover, death was also prevented by a cdk5 antisense probe, but not by an oligonucleotide with a random sequence. The cdk5 antisense also reduced neuronal expression of cdk5 compared with the random oligonucleotide. The studies indicate that cdk5 plays a major role in the molecular path leading to the neurodegenerative process triggered by the amyloid fibers in primary cultures of rat hippocampal neurons. These findings are of interest in the context of the pathogenesis of Alzheimer's disease.z 1999 Federation of European Biochemical Societies.

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We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca 2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.

ABBREVIATIONS: Aβ, β-amyloid peptide; AD, Alzheimer's disease; CNh, neuronal cell line derived from the cerebral cortex of a normal mouse; CTb, neuronal cell line derived from the cerebral cortex of a trisomy 16 mouse; FBS, fetal bovine serum; PBS, phosphate buffered saline; 2,4-DNP, 2,4-dinitrophenol; 3-NP, 3nitrophenol; 4-AA, 4-anisidine; NGF, nerve growth factor; [Ca 2+ ] i , intracellular Ca 2+ levels; PMSF, phenylmethylsulfonylfluoride.