High levels of extracellular K+ ensure proper development and prolong survival of cerebellar granule neurons in culture. We find that when switched from a culture medium containing high K+ (25 mM) to one containing a low but more physiological K+ concentration (5 mM), differentiated granule neurons degenerate and die. Death induced by low K+ is due to apoptosis (programmed cell death), a form of cell death observed extensively in the developing nervous system and believed to be necessary for proper neurogenesis. The death process is accompanied by cleavage of genomic DNA into internucleosome-sized fragments, a hallmark of apoptosis. Inhibitors of transcription and translation suppress apoptosis induced by low K+, suggesting the necessity for newly synthesized gene products for activation of the process. Death can be prevented by insulin-like growth factor I but not by several other growth/neurotrophic factors. cAMP but not the protein kinase C activator phorbol 12-myristate 13-acetate can also support survival in low K+. In view of the large numbers of granule neurons that can be homogeneously cultured, our results offer the prospect of an excellent model system to study the mechanisms underlying apoptosis in the central nervous system and the suppression of this process by survival factors such as insulin-like growth factor I.

Cerebellar granule cells deprived of depolarizing concentration of extracellular potassium, [K+]o, undergo apoptosis. We here report that this apoptotic process is associated with an immediate and permanent decrease in the levels of free intracellular calcium, [Ca2+]i. Although forskolin and IGF-1 are both able to prevent apoptosis, only forskolin is able to counteract the instantaneous decrease of [Ca2+]i. However, the early effect of forskolin on [Ca2+]i is lost after longer incubation in low [K+]o. The calcium antagonist nifedipine is able to inhibit the survival effect of high [K+]o, while not affecting forskolin and IGF-1 promoted survival, as assessed by viability and genomic DNA analysis. Accordingly, the L-type calcium channels agonist Bay K8644 significantly enhanced the survival of low KCl treated neurons. To temporally characterize the signal transduction events and the essential transcriptional step in cerebellar granule cells apoptosis, we determined the time course of the rescue capacity of high [K+]o, forskolin, IGF-1, and actinomycin D. Addition of high KCl, forskolin, or IGF-1 6 hr after the initial KCl deprivation saves 50% of cells. Remarkably, 50% of neurons loss the potential to be rescued by actinomycin D after only 1 hr in low [K+]o. Finally, we show that the survival promoting activities of high [K+]o, forskolin, and IGF-1 do not require RNA synthesis. We conclude that [Ca2+]i is involved in the survival promoting activity exerted by high [K+]o but not in those of forskolin and IGF-1, and that all three agents, although rescuing neurons from apoptosis through distinct mechanisms of action, do not necessitate RNA transcription.

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as`glutamate neurotoxicity' (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome c release is described in detail, with the released cytochrome c playing a role in the cell defense mechanism against neurotoxicity. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early factors in the failure of the microtubule network that occurs during neuronal apoptosis. Furthermore, this study introduces new insights into the mechanism(s) that generate the truncated forms of tau present in Alzheimer's disease.