title: Retinal ganglion cell death and neuroprotection: Involvement of the CaMKIIalpha gene.
abstract: The purpose of this study is to determine if calcium/calmodulin-dependent protein kinase-II (CaMKII) plays a role in neuronal cell death and if inhibition of this kinase affords some neuroprotection in the RGC-5 retinal ganglion cell line. The RGC-5 cells were treated with glutamate at various concentrations for increasing increments of time. Cytotoxicity was assayed by measuring the lactate dehydrogenase (LDH) leakage from non-viable cells and TUNEL assays. The involvement of caspase-3, Bcl-2 and caspase-8 in glutamate-induced cytotoxicity was determined by immunoblots and/or real time RT-PCR. In addition, the autocamtide-2-related inhibitory peptide (AIP), a specific inhibitor of CaMKII, was used to determine the involvement of CaMKII in glutamate-induced RGC-5 cell death. Application of increasing concentrations of glutamate to RGC-5 cells caused a dose-dependent increase in the level of cell death after 24 h. There was a glutamate-stimulated increase in the expression of caspase-8 and caspase-3 and a corresponding decrease in Bcl-2. The active fragment of caspase-3 increased in glutamate-treated cells. An early transient increase in the expression of CaMKIIalpha(B) gene and a corresponding CaMKIIalpha nuclear translocation was found in glutamate-treated cells. Treatment with AIP blocked the activation of caspase-3 and protected RGC from glutamate-mediated cell death but did not alter the glutamate-enhanced expression levels of caspase-8 or caspase-3. This report shows the likely involvement of a transcript of the CaMKIIalpha gene in the cytotoxicity response of RGC-5 cells similar to previous reports in the neural retina. AIP is shown to be a neuroprotectant for RGC-5 cells as was reported for the neural retina.
authors: Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, 500 S. Preston Street, Louisville, KY 40202, USA.
authors: Wei
authors: W
authors: Fan
authors: Neeraj
authors: N
authors: Agarwal
authors: Maneesh D
authors: MD
authors: Kumar
authors: Nigel G F
authors: NG
authors: Cooper
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D002352:Carrier Proteins / Q000494:pharmacology; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D002452:Cell Count / Q000379:methods; D002460:Cell Line; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005455:Fluorescent Antibody Technique / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D018698:Glutamic Acid / Q000633:toxicity*; D020287:In Situ Nick-End Labeling / Q000379:methods; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D012165:Retinal Ganglion Cells / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D012333:RNA, Messenger; D019938:Cyclin D1; D018698:Glutamic Acid; D007770:L-Lactate Dehydrogenase; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases