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Active p21Ras is sufficient for rescue of NGF-dependent rat sympathetic neurons
C D Nobes1, J B Reppas, A Markus
1Department of Human Anatomy, University of Oxford, U.K.
Abstract:
We have examined whether p21Ras proteins can rescue nerve growth factor-deprived rat sympathetic neurons from death, to test further our hypothesis that p21Ras is a central mediator in the nerve growth factor-to-survival signalling pathway. After crosslinking [125I]nerve growth factor to live neurons, two forms of Trk (molecular weight approximately 140,000 and 115,000) were immunoprecipitated with anti-Trk antibodies. Nerve growth factor induced tyrosine phosphorylation of both Trk forms and at least two additional proteins. When these phosphorylations were prevented by staurosporine (in a protein kinase C-independent manner) the neurons died. However, neurons were rescued from death due to staurosporine treatment by intracellular loading of oncogenic Ha-Ras(val12) protein. Both Ha-Ras(val12) and cellular Ha-Ras proteins maintained survival for several days in the absence of nerve growth factor and mimicked other actions of nerve growth factor, inducing rapid c-Fos protein expression and robust neurite outgrowth. Conversely, Fab fragments of neutralizing antibodies to p21Ras which blocked the capacity of nerve growth factor to promote neuron survival were also found to inhibit the early expression of c-Fos protein in these neurons. The close correspondence observed between the timing of onset of c-Fos responsiveness 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 expression is a good biochemical indicator of the presence of a functional nerve growth factor-to-survival signal transduction pathway. Nevertheless, expression of c-Fos is not sufficient for survival since phorbol esters induce c-Fos with no effect on survival. These data strengthen our proposal that p21Ras proteins are crucial anti-apoptotic mediators of survival in rat sympathetic neurons by demonstrating that p21Ras is both necessary and sufficient to rescue neurons which are disabled from signalling through Trk receptors.
Insights
p21Ras proteins are crucial for rat sympathetic neuron survival, acting as central mediators in nerve growth factor signaling. They are both necessary and sufficient to rescue neurons from death, mimicking nerve growth factor
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Nerve growth factor (NGF) is essential for sympathetic neuron survival.
- The signaling pathway mediating NGF's survival effects is not fully understood.
- p21Ras proteins are hypothesized to be central mediators in this pathway.
Purpose of the Study:
- To investigate the role of p21Ras proteins in mediating NGF-induced survival of rat sympathetic neurons.
- To determine if p21Ras can rescue neurons from death when NGF signaling is disrupted.
- To explore the relationship between p21Ras, c-Fos expression, and neuron survival.
Main Methods:
- Immunoprecipitation of Trk receptors and associated proteins after NGF stimulation.
- Assessment of neuronal survival following staurosporine treatment (inhibitor of phosphorylation).
- Intracellular loading of oncogenic Ha-Ras(val12) protein to rescue neurons.
- Analysis of c-Fos protein expression and neurite outgrowth.
- Inhibition of p21Ras activity using neutralizing antibodies.
Main Results:
- NGF induced tyrosine phosphorylation of Trk and other proteins, essential for neuron survival.
- Staurosporine-induced neuronal death was rescued by intracellular Ha-Ras(val12) protein.
- Both Ha-Ras(val12) and cellular Ha-Ras proteins promoted survival and mimicked NGF actions (c-Fos expression, neurite outgrowth).
- Neutralizing antibodies to p21Ras blocked NGF-induced survival and c-Fos expression.
- c-Fos expression correlated with NGF-dependence but was not sufficient for survival.
Conclusions:
- p21Ras proteins are crucial anti-apoptotic mediators in rat sympathetic neuron survival.
- p21Ras is both necessary and sufficient to rescue neurons when Trk receptor signaling is impaired.
- p21Ras functions as a central mediator in the NGF-to-survival signaling pathway.