The goal of this proposal is to identify molecular mechanisms by which neurotrophic factors regulate ion channel expression during neuronal differentiation. Neurotrophic factors are crucial to nervous system development as evidenced by the severe defects that arise when genes encoding neurotrophins or their receptors are deleted. These factors have also been implicated in a spectrum of neurological disorders, including familial dysautonomia, diabetic neuropathy, amyotrophic lateral sclerosis, epilepsy, Parkinson's disease, Huntington's disease, and Alzheimer's disease, and are potential candidates for their treatment. Thus, there is tremendous interest in defining the actions of these factors and the mechanisms underlying their actions, both as a means of revealing fundamental aspects of nervous system development and function, and in determining their therapeutic potential. Previously, we have found that among the important actions of neurotrophic factors is their regulation of neuronal sodium (Na) channel expression. The tightly controlled expression of Na channels is critical to the ability of neurons to generate and propagate action potentials, and as such is central to information transfer in the nervous system. We have shown that, unlike many of the responses to neurotrophic factors, Na channel induction is ras- independent, and as such provides an avenue for identifying the molecular components of an ill-defined, yet obviously important, mechanism of growth factor action. To further determine how nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) shape the electrophysiological characteristics of neurons, Na channel regulation will be analyzed in cells with selective alterations in signalling molecules implicated in previous studies. Specifically, pharmacological approaches will be used to inhibit ras activation, and then patch clamp recording, Northern blot analysis, and RNAse protection assays used to determine the influence of this manipulation on the induction of Na channel expression in embryonic striatal neurons by BDNF. The effect of NGF on rat pheochromocytoma (PC12) cells transfected or microinjected with genes encoding constitutively active or interfering forms of c-src, c-fyn, and c-yes, and the effect of BDNF on embryonic striatal neurons of """"""""knockout"""""""" mice with deletions in the genes encoding either c-src, c-yes, or c-fyn, will be determined using patch clamp recording, RNAse protection, and Northern blot analysis. RNAse protection and patch clamp recording will also be used to analyze PC12 cells transfected or microinjected with genes encoding constitutively active or interfering forms of the B-Raf, Raf-l, MEKK1, and MAP kinases. Finally, some of the first insight into regulation of Na channel beta1 subunit expression will be obtained through use of Northern blot and RNAse protection analysis, patch clamp recording, epitope-tagged beta1 subunits, and antibodies specific for alpha subunits. Experiments will determine if neurotrophins regulate beta1 and alpha subunit expression through similar mechanisms, if altered beta1 expression influences neurotrophin-mediated Na channel induction, and if manipulation of beta1 expression has selective effects on the neurotrophin induction of type I or type II alpha subunits that are expressed in the same neuronal cell. The results of these studies will be important both for identifying molecular pathways by which neurotrophic factors induce differentiation, and for revealing fundamental mechanisms governing neuronal ion channel expression and electrical excitability.
