Long-term exposure to high glucose increases the content of several exocytotic proteins and of vesicular GABA transporter in cultured retinal neural cells.

Diabetic retinopathy is a leading cause of vision loss and blindness. Increasing evidence has shown that the neuronal components of the retina are affected even before the detection of vascular lesions. Hyperglycemia is considered the main pathogenic factor for the development of diabetic complications. Nevertheless, other factors like neuroinflammation, might also contribute for neural changes. To clarify whether hyperglycemia can be the main trigger of synaptic changes, we evaluated whether prolonged elevated glucose per se, mimicking chronic hyperglycemia, is able to change the content and distribution of several exocytotic proteins and vesicular glutamate and GABA transporters in retinal neurons. Moreover, we also tested the hypothesis that an inflammatory stimulus (interleukin-1ß) could exacerbate the effects induced by exposure to elevated glucose, contributing for changes in synaptic proteins in retinal neurons. Rat retinal neural cells were cultured for 9 days. Cells were exposed to elevated D-glucose (30 mM) or D-mannitol (osmotic control), for 7 days, or were exposed to interleukin-1ß (10 ng/ml) or LPS (1 µg/ml) for 24 h. The protein content and distribution of SNARE proteins (SNAP-25, syntaxin-1, VAMP-2), synapsin-1, synaptotagmin-1, rabphilin 3a, VGluT-1 and VGAT, were evaluated by western blotting and immunocytochemistry. The protein content and immunoreactivity of syntaxin-1, synapsin-1, rabphilin 3a and VGAT increased in retinal neural cells exposed to high glucose. No changes were detected when cells were exposed to interleukin-1ß, LPS or mannitol per se. Particularly, exposure to interleukin-1ß for 24 h did not exacerbate the effect of high glucose on the content and immunoreactivity of exocytotic proteins, suggesting the primordial role of hyperglycemia for neuronal changes. In summary, prolonged exposure to elevated glucose alters the total content of several proteins involved in exocytosis, suggesting that hyperglycemia per se is a fundamental factor for neuronal changes caused by diabetes.

Diabetes causes transient changes in the composition and phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and interaction with auxiliary proteins in the rat retina.

PURPOSE: The impairment of glutamatergic neurotransmission has been associated with diabetic complications in the central nervous system, such as diabetic retinopathy. Here, we investigated the effect of elevated glucose exposure and diabetes on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor composition, subunit phosphorylation, and the association of the GluA2 subunit with accessory proteins in the retina. METHODS: The subunit composition of AMPA receptors and the association of the GluA2 subunit with modulatory proteins were evaluated with coimmunoprecipitation in retinal neural cell cultures and in the retina of experimentally induced-diabetic rats. The phosphorylation status of AMPA receptor subunits was evaluated with western blotting. RESULTS: In retinal neural cell cultures, elevated glucose did not significantly alter the composition of AMPA receptors, namely, the interactions between the GluA1, GluA2, and GluA4 subunits, but reduced GluA2 association with GRIP1. Moreover, elevated glucose did not cause changes on the level of GluA1 phosphorylated at serine residues 831 and 845. Diabetes induced early transitory changes in the interaction between AMPA receptor subunits GluA1, GluA2, and GluA4. At 8 weeks of diabetes, the content of GluA1 phosphorylated at serine 831 or serine 845 in the retina increased, compared to age-matched controls. CONCLUSIONS: Taken together, these results suggest that diabetes induces dynamic changes in AMPA receptor subunit composition, which could affect glutamatergic transmission in the rat retina.