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HUI XU, Nduka Enemchukwu, Yingbin Fu; Genetic deletion of M-opsin prevents “M-cone” degeneration in a mouse model of Leber congenital amaurosis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4516.
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Mutations in either RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal recycling and cause Leber congenital amaurosis (LCA), the most severe retinal dystrophy in early childhood. In two compatible murine LCA models (Lrat–/– and Rpe65–/–), common pathologic features include: 1) mislocalization of both M- and S-opsins; 2) M-opsin was degraded whereas S-opsin accumulated; 3) S-opsin enriched ventral/central cones degenerate more rapidly than M-opsin enriched dorsal cones. We have shown previously in Lrat–/– model that S-opsin accumulates and aggregates in cones leading to rapid degeneration of ventral/central cones. The objective here is to investigate the mechanism of dorsal cone (“M-cone”) degeneration in a LCA mouse model. Our hypothesis is that M-opsin degradation associated proteasome stress is involved in “M-cone” degeneration.
We first generated M-opsin (Opn1mw) knockout mice by CRISPR/Cas9. Since cones degenerate rapidly in Lrat–/– mice in the presence of S-opsin (Opn1sw), we bred Opn1mw–/– mice into the Lrat–/–Opn1sw–/– (a LCA model to study “M cones”) background to analyze the preventive effect by M-opsin deletion. The expression and localization of cone opsins (M- and S-opsin) were detected by western blot and immunohistochemistry. Cones were labeled with mouse cone arrestin antibody and counted at 1-, 6-, and 12-month. Proteasome stress was assessed by the accumulation ubiquitinated proteins in cones using an anti-ubiquitin antibody.
M-opsin (Opn1mw) was disrupted by 1-bp insertion at second exon. M-opsin was not detectable in Opn1mw–/– retina by either western blot or Immunohistochemistry. The number of dorsal cones was decreased to 73.87% (N=4, P< 0.01) at 6-month and 54.73% (N=4, P<0.001) at 12-month in Lrat–/–Opn1sw–/– mice compared with Wild-Type(WT), indicating a slow degeneration of dorsal cones. In contrast, the number of dorsal cones of Opn1mw–/–Lrat–/– Opn1sw–/– is similar to that in control Opn1mw–/–Opn1sw–/– and WT mice (i.e. no degeneration). Ubiquitin labeling shows strong ubiquitin signal in dorsal cones of Lrat–/– Opn1sw–/– mice, but it was markedly decreased in dorsal cones of Opn1mw–/–Lrat–/–Opn1sw–/–mice.
Deletion of M-opsin alleviates proteasome stress in Lrat–/–Opn1sw–/– mice, suggesting that M-opsin degradation associated proteasome stress plays a key role in “M-cone” degeneration in Lrat–/–mice.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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