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Astra Dinculescu, Lei Xu, Susan N Bolch, Frank M Dyka, Ping Zhu, Hugh McDowell; Functional Consequences and Cellular Distribution of C1QTNF5 Mutants in Murine Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4899.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the globular domain of C1QTNF5 protein cause an autosomal dominant disorder known as late-onset retinal degeneration (L-ORD), leading to widespread chorioretinal atrophy, central and peripheral vision loss. Here, we examine the expression pattern and functional consequences of pathogenic C1QTNF5 mutants following their specific expression in murine RPE.
We generated AAV2 vectors expressing three C1QTNF5 mutants associated with L-ORD: S163R, G216C and P188T, all HA tagged at the C-terminal end. We have also generated a construct expressing a C1QTNF5-S163R-GFP fusion protein for direct visualization of fluorescent transgene expression. AAV vectors were subretinally delivered to adult C57BL/6 wild-type mice. Full field ERG analysis was performed under scotopic and photopic conditions. Mutant protein expression was detected by immunohistochemistry and Western Blotting using an anti-HA antibody. Histological examination of AAV-injected eyes was performed by haematoxylin and eosin staining of paraffin sections.
Previous studies showed that L-ORD disease associated with S163R mutant leads to characteristic thick basal RPE deposits in patients. We detect similar widespread RPE basal deposits in all AAV-S163R injected mouse eyes, consistent with our published data (PMID:26513502). These deposits consist of mislocalized mutant S163R protein, as determined by immunostaining, and can reach up to 40μm in thickness. The S163R-GFP fusion protein is also misrouted towards basal RPE membrane. In contrast, the AAV-expressed P188T mutant accumulates at the apical RPE membrane, where it forms aggregates at the RPE/photoreceptor interface, while the G216C mutant accumulates in the RPE cytoplasm. Both P188T and G216C mutants lead to severely diminished scotopic ERG a-wave amplitudes (2- and 3-fold decrease in AAV-P188T and AAV-G216C injected versus untreated eyes, respectively, p<0.001) and loss of RPE microvilli even early in the disease process, in contrast to S163R, which causes a slow, progressive photoreceptor loss.
Each L-ORD mutant displays a unique, abnormal expression pattern in mouse RPE, leading to RPE and photoreceptor dysfunction. In contrast to S163R C1QTNF5 protein, none of the C1QTNF5 mutants examined lead to basal deposits. These distinct behaviors suggest that each mutant triggers retinal degeneration by different mechanisms.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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