title: Retinal localization of the glutamate receptor GluR2 and GluR2-regulating proteins in diabetic rats.
abstract: 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 Calpha (PKCalpha) 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 PKCalpha 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 PKCalpha immunoreactivity remained the same. However, phosphorylated PKCalpha immunoreactivity was increased in diabetic retina as compared to control and peaked after 6 weeks of diabetes. Activated PKCalpha was almost completely lost in all membrane fractions and primarily recovered in the cytosolic fraction. These results are consistent with PKCalpha being re-localized in the diabetic retina. The observations indicate a diabetes-dependent increase in the activation of PKCalpha and a disturbed GluR2 regulation by altered internalization and recycling.
authors.affiliation: Department of Ophthalmology, University of Düsseldorf, Moorenstr. 5, D-40225 Düsseldorf, Germany. Irina.Semkova@med.uni-duesseldorf.de
authors.forename: Irina
authors.initials: I
authors.lastname: Semkova
authors.forename: Markus
authors.initials: M
authors.lastname: Huemmeke
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Ho
authors.forename: Barbara
authors.initials: B
authors.lastname: Merkl
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Abari
authors.forename: Mats
authors.initials: M
authors.lastname: Paulsson
authors.forename: Antonia M
authors.initials: AM
authors.lastname: Joussen
authors.forename: Markus
authors.initials: M
authors.lastname: Plomann
mesh_terms: D000818:Animals; D001786:Blood Glucose / Q000032:analysis; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D003930:Diabetic Retinopathy / Q000378:metabolism*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D018091:Receptors, AMPA / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000378:metabolism; D055211:Retinal Photoreceptor Cell Inner Segment / Q000378:metabolism; D013347:Subcellular Fractions
chemical_list: D001786:Blood Glucose; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D009687:Nuclear Proteins; C498298:PICK1 protein, rat; D018091:Receptors, AMPA; D051571:Protein Kinase C-alpha; C104722:glutamate receptor ionotropic, AMPA 2