Retinal localization of the glutamate receptor GluR2 and GluR2-regulating proteins in diabetic rats

Impaired glutamatergic activity and synaptic dysfunction contributing to excitotoxicity and neuronal degeneration has been observed in the diabetic retina. Here we analyzed the expression changes and trafficking abnormalities of the AMPA glutamate receptor 2 subunit (GluR2) and its regulators protein kinase Cα (PKCα) and PKC-interacting protein 1 (PICK1) in the rat retina during the early phases of streptozotocin-(STZ-) induced diabetes. Diabetes was induced in Long Evans rats by injection of STZ. Two and six weeks after induction of diabetes, immunohistochemistry and in situ hybridization were performed on retinal paraffin sections to investigate the expression and localization of GluR2 and its regulators PKCα and PICK1. The cellular distribution and trafficking of these proteins in retinae were also investigated by subcellular fractionation and western blotting. While no significant changes were observed for GluR2 transcripts, we observed a strong increase in GluR2 immunoreactivity, predominantly in the ganglion cell layer (GCL) and the inner plexiform layer (IPL), as early as two weeks of diabetes. GluR2/3 immunoreactivity was further increased from the GCL to OPL after 6 weeks of diabetes. Increased expression of a phosphorylated non-synaptic population of GluR2 was detected in the GCL, the IPL and in distinct photoreceptor cells within the outer nuclear layer (ONL) of diabetic animals. Further, the PICK1 retinal distribution was unchanged two and six weeks after onset of diabetes and in both control and diabetic rat retinae the PKCα immunoreactivity remained the same. However, phosphorylated PKCα immunoreactivity was increased in diabetic retina as compared to control and peaked after 6 weeks of diabetes. Activated PKCα was almost completely lost in all membrane fractions and primarily recovered in the cytosolic fraction. These results are consistent with PKCα being re-localized in the diabetic retina. The observations indicate a diabetes-dependent increase in the activation of PKCα and a disturbed GluR2 regulation by altered internalization and recycling.