We are studying the kinase-mediated signaling pathways that promote neuronal survival and prevent apoptosis in primary cultures of granule neurons. Relevant neurotrophins are insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF). Glutamate is the predominant neurotransmitter that regulates neuronal activity through its interaction with different glutamate receptor-channels, referred to herein as N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Binding of these agents to their respective receptors in the neuronal plasma membrane initiates activation of intracellular signaling cascades that regulate the transcription of pro-apoptotic or anti-apoptotic genes. The balance between these gene products ultimately determines cell fate. Early in granule cell development, before neurons have formed synaptic connections, they rely exclusively on neurotrophic factors, present in the serum that bathes them, for survival. We have novel evidence that particular isoforms of the family of phospholipid-sensitive protein kinases (PKCs), designated as atypical PKCs (aPKCs), are critical components of the neurotrophin-dependent survival pathway. We are quite enthusiastic about these data since almost nothing is currently known about the neuronal functions of aPKCs. Moreover, preliminary data indicate that inhibition of aPKCs reduces phosphorylation on serine-133 of the nuclear transcription factor CREB. One goal of this application is to extend our studies examining the linkage between neurotrophin-dependent activation of aPKCs. phosphorvlation of CREB. and survival. In many neuronal populations, inadequate electrical stimulation by excitatory neurotransmitters or inadequate trophic factor make cell death more likely. A """"""""Ca2+ set-point hypothesis"""""""" postulates that the bulk cytosolic Ca2+ activity determines the degree to which immature neurons require trophic factor to suppress the mechanism responsible for apoptosis (Koike et al., 1989). Accordingly, many types of neurons grown in dissociated culture can be rescued from death, induced by neurotrophin deprivation, by inclusion of agents in the media that produce a sustained elevation in Ca2+. These agents include elevated KCI acting through voltage-sensitive Ca2+ channels (VSCCs) and NMDA acting directly upon NMDA receptor channels (NRs). We have novel evidence that a Ca2+ and calmodulin-dependent kinase, designated as CaM KIV, and its substrate CREB are critical components of this Ca2+-dependent signaling pathway. Immunocytochemical evidence also indicated that these treatments induce the redistribution of CaM KIV from the nucleus to the cytosol. A second goal of this application is to extend our studies examining the linkage between calcium, CaM KIV phosphorvlation of CREB, and survival. Phosphorylation and activation of CREB may mediate induction of anti-apoptotic genes, such as bc1-2, or repression of pro-apoptotic genes, such as bax. A third goal in this application is to compare the expression levels of bc1-2 and bax rnRNAs under culture conditions promoting survival or apoptosis.
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