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G. C. Munguba, M. Tapia, A. S. Camp, M. Risco, S. K. Bhattacharya, R. K. Lee; Differential VGLUT3 Isoform Expression in Glaucomatous DBA/2J Mice. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6095.
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Glutamate mediated excitotoxicity has been implicated in glaucomatous retinal ganglion cell (RGC) death. Glutamate, the major neurotransmitter in the retina, is loaded into synaptic vesicles by a family of proteins known as Vesicular Glutamate Transporters (VGLUTs). DBA/2J mice develop glaucoma in association with elevated IOP. We identified differential expression patterns of VGLUT3 isoforms in DBA/2J retinas compared to non-glaucomatous mouse strains, suggesting a possible role for VGLUT3 in glaucoma pathogenesis.
Pooled whole retinas from DBA/2J or C57BL/6J mice were homogenized and prepared for Western blot (WB) analysis. Globes were fixed in formaldehyde and sectioned for immunofluorescense (IF). Sectioned retinas were subjected to Trilogy (Cell Marque, CA) antigen retrieval and double-labeled with VGLUT(1,2,3) and EAAT1 antibodies.
VGLUT3 IF expression was undetectable in DBA/2J retinas, yet robust expression was observed in C57BL/6J, BALB/cJ, and C3H/HeJ retinas. WB analyses of C57BL/6J retinas show high expression of an expected ~65kDa VGLUT3 isoform in heart, moderate expression in kidney, liver, and weak expression in retina and brain. Although a 65kDa VGLUT3 signal was detected for many tissues, strong signal was present in a ~140kDa band restricted to the CNS (brain and retina). DBA/2J animals express very similar levels of the 65kDa band in all tissue samples, while the 140kDa band was present only in the brain.
IF of DBA/2J retinas demonstrate a lack of VGLUT3 immunoreactivity in the inner plexiform layer. VGLUT3 is expressed in presumptive "dual function" neurons containing more than one neurotransmitter, and its expression in tissues such as liver has been speculated to serve a protective buffering function. WB analyses of DBA/2J tissues show similar levels of the 65kDa VGLUT isoform compared to C57BL/6J samples, while the 140kDa isoform is only present in brain. These results provide a mechanism for improper signaling and/or a loss of retinal excitotoxicity control in the DBA/2J model and implicate VGLUT3 in the pathogenesis of RGC death in glaucoma.
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