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Brittany N. Weibley, Travis S. D'Cruz, Alistair J. Barber, Penn State Hershey Eye Center; Synaptophysin Mono-ubiquitination In Explant Retinas. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5939.
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Synaptophysin (Syn) is an abundant presynaptic neurotransmitter vesicle-associated transmembrane protein, previously shown to be significantly depleted in rat retinas after 1-2 months of STZ-diabetes. Syn plays an important role in neurotransmitter vesicle docking and recycling at the presynaptic terminal. Cell culture studies suggest the Syn is primarily degraded by the proteasome after poly-ubiquitination, but this has not been replicated in whole retina tissue. The aim of this study was to determine the mechanism of degradation of Syn in the rat retina.
Diabetes was induced in male Sprague-Dawley rats by injection of streptozotocin (STZ, 65 mg/ml, i.p.) and rats were housed for 1-2 months. Whole retinas dissected from rats were incubated in DMEM culture media and treated with cycloheximide to stop protein synthesis. Relative synaptophysin content was measured after various time periods to determine its rate of degradation. Synaptophysin was also immunoprecipitated and samples blotted for mono- and poly-ubiquitin. Samples were also treated with proteasome inhibitors (MG132 and NEM) and lysosome inhibitor (E64) to determine the primary route of degradation.
There was significantly less synaptophysin in retinas from STZ-diabetic rats compared to controls after 1 and 2 months of hyperglycemia (p<0.05). Immunoprecipitation of Syn and Blotting Syn immunoprecipitates for ubiquitin revealed only a single ubiquitin band at a molecular weight consistent with mono-ubiquitin. Incubation with E64 increased the intensity of the mono-ubiquitin band, while incubation with proteasome inhibitors did not.
Previous cell culture studies suggest that Syn is degraded primarily through the proteasome, but it is more likely that Syn is mono-ubiquitinated and degraded through a lysosomal pathway in the intact retina. The difference may be due to the relative lack of differentiation of Syn-expressing cells in culture compared to neurons in adult retinas.
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