ACTIVE P21RAS IS SUFFICIENT FOR RESCUE OF NGF-DEPENDENT RAT SYMPATHETIC NEURONS

We have examined whether p21Ras proteins can rescue nerve growth facto r-deprived rat sympathetic neurons from death, to test further our hyp othesis that p21Ras is a central mediator in the nerve growth factor-t o-survival signalling pathway. After crosslinking [I-125]nerve growth factor to live neurons, two forms of Trk (molecular weight similar to 140,000 and 115,000) were immunoprecipitated with anti-Trk antibodies. Nerve growth factor induced tyrosine phosphorylation of both Trk form s and at least two additional proteins. When these phosphorylations we re prevented by staurosporine (in a protein kinase C-independent manne r) the neurons died. However, neurons were rescued from death due to s taurosporine treatment by intracellular loading of oncogenic Ha Ras(va l12) protein. Both Ha-Ras(val12) and cellular Ha-Pas proteins maintain ed survival for several days in the absence of nerve growth factor and mimicked other actions of nerve growth factor, inducing rapid c-Fos p rotein expression and robust neurite outgrowth. Conversely, Fab fragme nts of neutralizing antibodies to p21Ras which blocked the capacity of nerve growth factor to promote neuron survival were also found to inh ibit the early expression of c-Fos protein in these neurons. The close correspondence observed between the timing of onset of c-Fos responsi veness and acquisition of nerve growth factor-dependence in embryonic day 17 sympathetic neurons, and the coordinate increase found in both parameters until embryonic day 19 indicates that c-Fos protein express ion is a good biochemical indicator of the presence of a functional ne rve growth factor-to-survival signal transduction pathway. Nevertheles s, expression of c-Fos is not sufficient for survival since phorbol es ters induce c-Fos with no effect on survival. These data strengthen ou r proposal that p21Ras proteins are crucial anti-apoptotic mediators o f survival in rat sympathetic neurons by demonstrating that p21Ras is both necessary and sufficient to rescue neurons which are disabled fro m signalling through Trk receptors.