The regulatory mechanisms which control the process of myelination can be characterized by at least two phases. The first phase includes the regulatory mechanisms by which Schwann cells control the biosynthesis of myelin specific components in the absence of myelin assembly, and the second phase involves the regulatory mechanisms by which Schwann cells control the assembly of the myelin membrane. Both phases will be evaluated in this grant proposal by examining the crush injured adult rat sciatic nerve where there is myelin assembly and comparing it to the permanently transected adult rat sciatic nerve where there is no myelin assembly. The experimental approach involves the following specific aims. 1) To determine the temporal sequence of events associated with the biosynthesis of the major myelin glycoprotein (Po) from peripheral nerve in the crush injured nerve and the permanently transected nerve to assess whether the molecular weight shift and the progressive increase in mannose incorporation into Po are involved in the down regulation mechanism in the biosynthesis of this glycoprotein and whether these events also play a role in the subsequent regulation of myelin assembly. 2) To determine if there's an association between completion of the post-translational processing events for Po glycoprotein and the subsequent initiation of myelin assembly by evaluating the permanently transected nerve after treating the adult Schwann cells at 35 days after nerve transection in culture with thyroid hormone, axolemma membrane preparations, or corticosteroids. 3) To evaluate other post-translational processing events besides glycosylation in the biosynthesis and assembly of Po glycoprotein in both the crush injured nerve and the permanently transected nerve. Specifically, acylation, sulfation, and phosphorylation will be evaluated to determine the sequence of post-translational modifications relative to the glycosylation events. 4) To evaluate to discrepancies that exist in the literature regarding a lack of Schwann cell expression of myelin specific components when embryonic or neonatal Schwann cell expression of myelin specific components when embryonic or neonatal Schwann cells are grown in culture with our findings concerning the in vivo expression of basal levels of Po glycoprotein in the adult permanently transected nerve. The expression of Po glycoprotein in culture will be assessed through an evaluation of the following techniques: a) Radioiodinated lectin binding after SDS-pore gradient electrophoresis, b) radioactive precursor incorporation into this glycoprotein, c) endoglycosidase-H digestion and d) serial lectin affinity chromatography of pronase digested radioactive labeled glycopeptides obtained after electrophoretic separation.
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