The primary goal of this project is to study the gene expression, metabolism, and functions of neuronal intermediate filament proteins (e.g., neurofilament (NF) proteins) in the developing and adult nervous system. For this purpose, we have characterized sensory neuron tissue cultures derived from fetal (E15~E20) and postnatal (>PN2) dorsal root (DRG) and trigeminal ganglia (TGG). The neurons in these ganglia were analyzed for their expression of seven target genes in vivo and in vitro. These included neurofilament~L, ~M, ~H, peripherin, alpha~tubulin, calcitonin gene~related peptide (CGRP) and substance P. Analysis for mRNA was done by Northern blot and in situ hybridization histochemistry, and of proteins and peptides by Western blot and immunocytochemistry. The results show robust expression of all seven target genes in vivo and in culture. We found that the TGG, like the DRG, contains two major cell types, distinguishable by their size and intermediate filament subtype expression: a population of relatively large cells that express NF~L (61%) and a population of relatively smaller cells that expresses peripherin (35%) with approximately 5% of cells coexpressing both proteins. In situ hybridization studies of embryonic DRG show that during development, NF~L mRNA and protein are up~regulated while peripherin protein is down~regulated in the NF~L~ immunoreactive (IR) population. Peripherin mRNA remains at high levels in this population throughout development into the adult, despite the lack of peripherin immunoreactivity. We have also developed and characterized an in vitro system of DRG neurons to study the regulation of the genes of interest in response to growth factors and target tissues. DRG neurons were cultured from E15, 17, 20 and PN2 rats in the presence of NGF to examine the expression of the above genes by in situ hybridization. NF~L and peripherin mRNAs are expressed in every neuron of the E15 and E17 cultures. Between E20 and PN2, peripherin mRNA expression persists, while the number of neurons expressing NF~L mRNA is drastically reduced. E15 cultures treated with skeletal muscle extract show increased levels of NF~L mRNA. These results suggest that factors in addition to NGF are present in skeletal muscle extract and may allow the NF~L phenotype class to be expressed in vitro. In a second project we initiated studies to test the hypothesis that axonal protein synthesis occurs in the squid giant axon. Protein biosynthesis~immunoprecipitation experiments confirmed robust biosynthesis of NF proteins in squid stellate ganglia. Although, we found squid NF protein mRNA in axoplasm, we failed to detect any NF biosynthesis in the giant axon.
