Modification of synaptic neurotransmission at glutamatergic synapses and activation of Ca+2- dependent second messenger systems contribute to the processes of learning and memory, and the neuronal dysfunction observed following stroke and ischemia, focal epilepsies, and Alzheimer's disease. In addition, these systems play an important, yet poorly understood, role in neuronal development, regeneration, and mitosis. A multifunctional type II Ca+2 and calmodulin-dependent protein kinase (CaM KII) and a substrate for this kinase, the microtubule-associated protein MAP-2 have been implicated in these processes. In the present proposal, Primary neuronal cultures and transformed cells will be used to examine potentially important physiological factors that regulate expression and targeting of CaM KII isoforms and MAP-2 variants during neuronal development. Cerebellar granule cells expressing primarily NMDA or non-NMDA receptor- channels will be used to test the hypothesis that chronic activation of NMDA receptors induces expression of a """"""""forebrain-like"""""""", isoform of CaM KII. Preliminary data indicate that MAP-2 protein expression appears normal and does not differ in these two phenotypes, whereas CaM KII subunit mRNA expression is altered. During neuronal maturation, the phosphorylation state and expressed variant of MAP-2 are developmentally regulated similar to intact brain. A second series of experiments will examine factors regulating expression of MAP-2 and CaM KII in transformed neuronal cultures where a novel mode of MAP-2 mRNA regulation has been observed. These experiments will ultimately provide insights into the complex relationship between phosphorylation of MAP-2 by Ca+2-dependent protein kinases and synaptic function. A combination of molecular biological, immunochemical and biochemical approaches will be used to answer the following specific questions: Do cerebellar granule cells expressing primarily the NMDA glutamate receptor subtype express a """"""""forebrain-like"""""""" isoform of CaM KII? Do cerebellar granule cells expressing primarily the non-NMDA glutamate receptor subtype express a """"""""cerebellar-like"""""""" isoform of CaM KII? Is there a difference in the subcellular distribution of CaM KII in cerebellar granule cells expressing NMDA versus non-NMDA receptors? How is MAP-2 mRNA regulated in actively dividing NG108 cells?
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