Abstract
Purpose:
Previous research has shown that the neurotransmitter vesicle protein, synaptophysin, is depleted in the retinas of diabetic rats. We recently demonstrated that diabetes alters the post-translational processing of synaptophysin by increasing mannose glycosylation leading to an accumulation of mannose-rich synaptophysin. In this study we hypothesized that accumulation of mannose-rich synaptophysin is due to reduced activity of α-mannosidase, the enzyme responsible for de-mannosylation of synaptophysin, resulting in reduced expression of the mature form of the protein. We tested this hypothesis by determining the effect of diabetes on α-mannosidase activity, and investigating whether inhibition of this enzyme in control retinas affects synaptophysin expression and turnover similar to diabetes.
Methods:
Diabetes was induced in male Sprague-Dawley rats by streptozotocin (STZ, 65 mg/kg, i.p.). After four weeks of hyperglycemia the retinas were explanted for in vitro study. The drug swainsonine was used to inhibit α-mannosidase activity in explanted control retinas and the relative amount of synaptophysin was measured by western blot analysis. The relative amount of mannose-rich synaptophysin was also determined by western blotting after incubating lysates with endoglycosidase H (endo-H). Relative degradation of synaptophysin was measured by western blotting retinas treated with cycloheximide.
Results:
Retinas from STZ-diabetic rats had significantly more mannose-rich (endo-H sensitive) synaptophysin compared to controls (p<0.05), while there was significantly less α-mannosidase activity (p<0.05). Control retinas treated with swainsonine contained significantly less synaptophysin compared to untreated (p<0.001) while there was significantly more endo-H sensitive synaptophysin (p<0.05). Swainsonine also significantly increased the degradation of endo-H sensitive synaptophysin (p<0.05).
Conclusions:
Inhibition of α-mannosidase in control retinas had the same affect on synaptophysin mannosylation, expression and degradation as diabetes. These results suggest that the amount of mature synaptophysin expressed in the retina is determined by the activity of α-mannosidase during the late stages of post-translational processing. Reduced activity of α-mannosidase offers a mechanism for the diminished expression of retinal synaptophysin in diabetes.
Keywords: 499 diabetic retinopathy •
616 neurotransmitters/neurotransmitter systems •
514 enzymes/enzyme inhibitors