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Travis S. D'Cruz, Brittany N. Weibley, Scot R. Kimball, Alistair J. Barber, Penn State Hershey Eye Center; Diabetes Increases 35S-methionine Incorporation Into Synaptophysin In Explant Retinas. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5936.
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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. The aim of this study was to test the hypothesis that diabetes alters the rate of synthesis and degradation of Syn in the retina.
Diabetes was induced in male Sprague Dawley rats by injection of streptozotocin (STZ, 65mg/ml, i.p.) and rats were housed for 1-2 months. 35S-methionine (35S-met) incorporation was measured in whole explant retinas from STZ-diabetic and aged-matched control rats. Retinas were incubated with 35S-met in 37°C DMEM for 30 minutes. For pulse-chase experiments, 35S-met was washed out and retinas were incubated in regular DMEM with cycloheximide for 0, 30, and 60 minutes. Incorporated 35S-met in Syn and total protein was measured by immunoprecipitation and autoradiography.
Total 35S-met incorporation in the whole retina was significantly increased after 1 month of STZ diabetes (p<0.005) but was not altered after 2 months, compared with age-matched controls. 35S-met incorporation into Syn was significantly increased in retinal explants after 1 (p<0.0005) and 2 months (p<0.01) of STZ diabetes compared to age-matched controls. When normalized to total incorporation, 35S-met Syn was increased only after 2 months of STZ diabetes. In the pulse-chase study, 35S-met Syn was reduced faster in 2 month STZ retinas than in age-matched controls (p<0.01).
The data suggest that diabetes increases the rate of synthesis of Syn in the rat retina. Diabetes also increases the rate of Syn degradation, which begins within minutes after synthesis. The previously reported reduction of total Syn content likely results from highly accelerated protein degradation.
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