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Cultured cerebellar granule neurons die by apoptosis when switched from medium containing elevated potassium (K+) and serum to serum-free medium containing low K+. Although cell death begins at about 16 hr, commitment to death occurs within 6 hr after the lowering of K+. We have used this paradigm to examine the role of metabotropic glutamate receptor (mGluR) genes in the regulation of neuronal survival. We find that the expression of one of the mGluR genes, the type-4 gene, is associated with increased neuronal survival. Lowering of K+ leads to an 80% decrease in mGluR-4 mRNA expression within 6 hr. Downregulation of mGluR-4 messenger RNA (mRNA) does not occur if low K+-induced death is prevented by treatment with insulin-like growth factor I or adenosine 3',5'-cyclic monophosphate. If transcription is inhibited by actinomycin D, the difference in mGluR4 mRNA expression between cells switched to high-K+ medium and those switched to low-K+ medium is dramatically reduced, suggesting that decreased mGluR-4 gene transcription rather than increased mRNA breakdown is mainly responsible for the apoptosis-associated decrease in mGluR4 levels. Blockade of transcription also reduces mGluR4 mRNA expression in healthy neurons by more than 50% within 4 hr, suggesting that the mGluR4 mRNA has a relatively short half-life. In pharmacological experiments, we observe that the specific group III mGluR agonists such as L-amino-4-phosphobutyric acid and O-phospho-L-serine inhibit low K+-induced apoptosis. On the other hand, a selective mGluR4 antagonist, (RS)-alpha-cyclopropyl-4-phosphono-phenylglycine, induces apoptosis even in the presence of elevated K+. These results indicate that elevated mGluR4 expression or the activation of this receptor promotes survival and that an inhibition of such survival mechanisms contributes to apoptosis.

Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+ (25 mM KCl) to medium containing lower levels of K+ (5 mM KCl). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp x (O-methyl) x fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp x fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10-25 microM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp x chloromethyl ketone (YVAD x cmk), an interleukin-1beta converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp x fluoromethyl ketone (DEVD x fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD x cmk provided only modest (<20%) protection and only at the highest concentration (100 microM) tested, suggesting that interleukin-1beta converting enzyme and/or closely related caspases were not involved. In comparison, DEVD x fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD x fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+-deprived granule neurons.

Cultured cerebellar granule neurons die by apoptosis when switched from a medium containing an elevated level of potassium (K+) to one with lower K+ (5 mM). Death resulting from the lowering of K+ can be prevented by insulin-like growth factor (IGF-1). To understand how IGF-1 inhibits apoptosis and maintains neuronal survival, we examined the role of phosphoinositide 3-kinase (PI 3-kinase). Activation of PI 3-kinase has been shown previously to be required for NGF-mediated survival in the PC12 pheochromocytoma cell line. We find that in primary neurons, IGF-1 treatment leads to a robust activation of PI 3-kinase, as judged by lipid kinase assays and Western blot analysis. Activation of PI 3-kinase is likely to occur via tyrosine phosphorylation of the insulin receptor substrate protein. Treatment with two chemically distinct inhibitors of PI 3-kinase, wortmannin and LY294002, reduces PI 3-kinase activation by IGF-1 and inhibits its survival-promoting activity, suggesting that PI 3-kinase is necessary for IGF-1-mediated survival. Death resulting from PI 3-kinase blockade is accompanied by DNA fragmentation, a hallmark of apoptosis. Furthermore, neurons subjected to PI 3-kinase blockade can be rescued by transcriptional and translation inhibitors, suggesting that IGF-1-mediated activation of PI 3-kinase leads to a suppression of "killer gene" expression. In sharp contrast to IGF-1, elevated K+ does not activate PI 3-kinase and can maintain neuronal survival in the presence of PI 3-kinase inhibitors. Therefore, survival of granule neurons can be maintained by PI 3-kinase dependent (IGF-1-activated) and independent (elevated K+-activated) pathways.