Neuronal connectivity and function undergo activity-dependent changes that are central to learning, memory and development. The long-term goal of the proposed research is to understand how activity changes synapses. Recent work suggests gene transcription may make activity-dependent synaptic changes permanent. At many synapses, glutamate receptors initiate plasticity through ion fluxes, second messengers and gene transcription. The proposed research tests the hypothesis that glutamate induces gene expression through a unique pathway important for synaptic plasticity. We will use the immediate early gene, c-fos, to probe pathways of glutamate- induced gene expression. Others found that glutamate induces c-fos partly through the N-methyl-D-aspartate (NMDA)-receptor subtype, however, the role of other glutamate receptor subtypes is unclear. It is also unclear whether Ca2+ influx through the NMDA-receptor is sufficient to induce c- fos or whether the channel itself serves some signaling function. Certain NMDA subunits undergo tyrosine phosphorylation and contain peptide domains that may transmit signals to other cytoplasmic proteins.
Specific aims and methods are: 1) We propose to test the role of glutamate receptor subtypes in endogenous hippocampal c-fos expression with agonists and antagonists of specific glutamate receptors, second messengers, protein kinases and phosphatases as measured by Northern blotting. Parallel correlative experiments will test the role of cytoplasmic Ca2+ rises for c-fos expression using the Ca2+ -sensitive dye fura-2 and video microscopy. 2) We will test the sufficiency of NMDA receptors for c-fos induction by expressing functional NMDA receptors in PC 12 cells; demonstrating the channels using patch clam,p and Ca2+ imaging methods; and testing for glutamate-induced c-fos expression with Northern immunoblotting. We will deduce gene elements that mediate NMDA-induced c- fos by cotransfecting vectors with intact or mutated c-fos promoters. We will immunocytochemically test for active upstream signaling factors using antiphosphoprotein antibodies. 3) We will test the idea that the NMDA channel itself transmits signals by measuring glutamate-induced NMDA receptor phosphorylation in transfected PC12 cells; identifying specific tyrosine phosphorylation sites by tryptic peptide and phosphoamino analysis and by testing their importance using site-directed mutagenesis and transfections. Other candidate cytoplasmic domains of the NMDA receptor will be tested similarly. The proposed research should improve understanding of glutamate receptor signaling, NMDA receptor structural biology, NMDA gene expression and mechanisms of glutamate-mediated plasticity. Clinically, glutamate kills neurons at high concentrations and may play a role in the pathogenesis of neurodegenerative diseases, epilepsy and stroke. Elucidating mechanisms of glutamate induced gene transcription and plasticity may lead to new treatments for diseases associated with cellular hyperexcitability and glutamate toxicity.
| Curtis, J; Finkbeiner, S (1999) Sending signals from the synapse to the nucleus: possible roles for CaMK, Ras/ERK, and SAPK pathways in the regulation of synaptic plasticity and neuronal growth. J Neurosci Res 58:88-95 |
| Tao, X; Finkbeiner, S; Arnold, D B et al. (1998) Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron 20:709-26 |
| Finkbeiner, S; Greenberg, M E (1998) Ca2+ channel-regulated neuronal gene expression. J Neurobiol 37:171-89 |
| Finkbeiner, S; Tavazoie, S F; Maloratsky, A et al. (1997) CREB: a major mediator of neuronal neurotrophin responses. Neuron 19:1031-47 |
| Finkbeiner, S; Greenberg, M E (1997) Spatial features of calcium-regulated gene expression. Bioessays 19:657-60 |
