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Kevin Breen, Alejandra Bosco, David Calkins, Monica Vetter; CX3CR1 signaling modulates retinal ganglion cell degeneration in glaucoma. Invest. Ophthalmol. Vis. Sci. 2013;54(15):776.
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© ARVO (1962-2015); The Authors (2016-present)
Microglia become chronically activated in multiple neurodegenerative diseases including glaucoma, but how activation is regulated and impacts disease is unclear. Healthy neurons express signals to limit microglia activation, which are lost in degenerating neurons. Neurons constitutively express fractalkine (CX3CL1, FKN), a chemokine that binds to the CX3CR1 receptor (FKNR) on microglia to balance activation. Because loss of FKNR results in overactivation, here we explore the role of fractalkine signaling in glaucoma. We hypothesize that retinal ganglion cells (RGCs) express FKN, and that loss of FKNR signaling increases microglia activation and neurodegeneration in the DBA/2J (D2) mouse model of glaucoma.
We determined whether RGCs express FKN by in-situ hybridization on cryosections from 1 month-old (mo) Thy1+/cfp D2 mice, followed by CFP immunostaining to detect RGCs. We asked if FKNR loss affects RGC degeneration, comparing 10mo D2 (n=9) versus FKNR null retinas (CX3CR1gfp/gfp D2 n=20). We collected confocal images of entire wholemount inner retinas, immunostained for the RGC marker Brn3 or pNF (phospho-neurofilament). We tracked somal pNF accumulation in RGCs as a readout for axonal transport decline, and counted Brn3+ cells in 8 radial sectors at 3 eccentricities, classifying them as “no/mild”, “moderate”, or “severe damage” if average density was >400, 200-400 and <200 RGCs/mm2, respectively.
FKN mRNA expression was highest in the RGC layer and in CFP+ RGCs, and lower in other retinal layers. We observed enhanced RGC pathology in null FKNR D2 retinas. 58% of retinal sectors in FKNR null D2 (n=153 sectors) were severely depleted of Brn3+ RGCs vs. 27% (n=59 sectors) in D2. Thus sectors with moderate and mild RGC loss were reduced (43% in FKNR null D2 vs. 73% in D2). The entire retina in 9/20 FKNR null D2 eyes showed severe Brn3+ depletion versus only 1/9 in D2 retinas. FKNR null D2 retinas also had ~4x more RGCs showing abnormal somal pNF build up (average of 1.7 ± 0.5 SEM/mm2) relative to D2 (0.4 ± 0.1 SEM/mm2).
RGCs express FKN, and loss of FKN signaling leads to increased retinal disease severity in RGCs of 10mo D2 mice. Ongoing experiments will assess the effect of FKNR loss and FKN overexpression on microglial activation and severity of optic nerve pathology. Overall, our findings support a contribution of microglia activation to RGC degeneration in glaucoma.
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