This proposal represents a direct continuation of our ongoing investigation of the therapeutic role of the neurotrophic agent, testosterone propionate (TP), in enhancing nerve regeneration. The model system we have been utilizing is the hamster facial motoneuron (HFMN), a population of peripheral neurons which contain androgen receptors. Accomplishment of the aims of the previous grant has established that TP significantly accelerates the rate of facial nerve regeneration without shortening the delay before sprouting occurs, TP triggers the nerve cell body response to injury by accelerating the onset and increasing the magnitude of the nucleolar reaction, intrinsic sex differences exist in facial nerve regeneration, and the effects of androgens on facial nerve regeneration are sex-specific. How this neurotrophic agent differentially regulates the molecular program of regenerating motoneurons will be the focus of this continuation application.
The specific aims are to test the following 5 hypotheses. First, TP accelerates the rate of facial nerve regeneration through differential regulation of cytoskeletal gene expression following axotomy. The effects of TP on the levels of cytoskeletal mRNAs encoding proteins essential for axonal regrowth will be determined in axotomized HFMN using in situ hybridization and northern blot analyses with the appropriate cytoskeletal cDNA probes. Second, TP accelerates the rate of facial nerve regeneration through an acceleration of the rate of slow component b of axonal transport. Stereotaxic injections of labeled amino acids into the facial nuclear groups will be done and the effects of TP on the transport and quantity of labeled cytoskeletal proteins within segments of regenerating axons assessed at defined timepoints after facial nerve injury. Third, TP-induced acceleration of the onset of the facial nerve cell body response to injury occurs through the rapid transcriptional activation of the ribosomal RNA gene. Tandem in situ hybridization with 2 ribosomal DNA probes specific to the initial transcript (precursor) or stable (product rRNA will be done at selected timepoints after injury to determine if rRNA gene expression in axotomized HFMN is transcriptionally regulated by TP. Fourth, the gender- specific effects of TP on hamster facial nerve regeneration involve sex differences in androgen receptor mRNA levels and/or regulation in HFMN. In situ hybridization with androgen receptor mRNA probes will be done to determine if intrinsic differences in androgen receptor mRNA levels between male and female HFMN exist, as well as to establish whether sex- specific regulation of receptor mRNA levels occurs following axotomy, with/without TP. Fifth, TP can alter, in a positive manner, the regenerative response pattern in androgen receptor-containing central motoneurons. In situ hybridization with rDNA and betaII-tubulin cDNA probes will be used to determine if testosterone can trigger a regenerative response pattern in injured hamster rubrospinal neurons similar to that observed in HFMN. These studies should provide information relevant to the mechanism of steroid action in neuronal injury, and new information concerning differences in the intrinsic molecular programs of injured PNS vs. CNS neurons.
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