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Susan Bolch, Frank M Dyka, Manoj Kulkarni, Rachel Stupay, Chiab Simpson, W Clay Smith, William W Hauswirth, Astra Dinculescu; The expression pattern and pathological effects of CLRN1 mutant proteins in the mouse retina. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3053.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in clarin-1 (CLRN1) gene are the cause of Usher syndrome type III (USH3A), an autosomal recessive disorder manifested as combined loss of vision and hearing. Our goal here was to examine in-vivo the localization of several AAV vector-delivered HA-tagged CLRN1 mutants following subretinal injections in adult C57BL/6 mice, and to assess their effects on retinal function and morphology.
We generated AAV2 vectors expressing the following HA-tagged CLRN1 mutant proteins under the control of the ubiquitous smCBA promoter: A123D, C40G, L154W, N48K, and R207X. The vectors (108 vector genomes) were delivered subretinally into one eye of C57BL/6 mice. Retinal function was assessed by electroretinography (ERG) under scotopic and photopic conditions. Transgene expression was detected by immunohistochemistry using an anti-HA antibody and by Western blotting. The cellular localization of each CLRN1-HA mutant was examined by confocal microscopy. To examine CLRN1 mutant glycosylation, we treated retinal extracts overexpressing each mutant with PNGase F, an enzyme that removes the N-linked oligosaccharide side chains from glycoproteins.
The L154W CLRN1 mutation, which causes nonsyndromic recessive RP with no hearing impairment, was detected at the plasma membrane, in contrast to all other mutants examined. This mutant was glycosylated in-vivo, as determined by the shift in mobility upon PNGase treatment. Expression of A123D, C40G, and N48K CLRN1 mutant proteins led to their mislocalization and accumulation in the ER, and a dramatic loss in both retinal function and photoreceptors. At 1 month post-injection, C40G CLRN1 expression led to the most severe effects on both retinal function and morphology. Thus, for the mixed rod-cone ERGs, AAV-smCBA-C40G CLRN1 injected eyes had abnormally reduced amplitudes with mean values being reduced to 25% of mean untreated eyes (103.8±15.43 μV versus 418.8 ±27.85 μV, respectively, p=0.0002, n=6). Histological analysis revealed that there was severe thinning of both the inner and outer retina in this case.
This work demonstrates that although USH3 is a recessive disorder caused by lack of functional CLRN1 at the plasma membrane, there are differences in the behavior of different CLRN1 mutants, potentially impacting protein binding properties and mechanisms of disease.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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