Phagosomal and mitochondrial alterations in RPE may contribute to KCNJ13 retinopathy

Abstract

Mutations in KCNJ13 are associated with two retinal disorders; Leber congenital amaurosis (LCA) and snowflake vitreoretinal degeneration (SVD). We examined the retina of kcnj13 mutant zebrafish ( obelix td15 , c.502T > C p.[Phe168Leu]) to provide new insights into the pathophysiology underlying these conditions. Detailed phenotyping of obelix td15 fish revealed a late onset retinal degeneration at 12 months. Electron microscopy of the obelix td15 retinal pigment epithelium (RPE) uncovered reduced phagosome clearance and increased mitochondrial number and size prior any signs of retinal degeneration. Melanosome distribution was also affected in dark-adapted 12-month obelix td15 fish. At 6 and 12 months, ATP levels were found to be reduced along with increased expression of glial fibrillary acidic protein and heat shock protein 60. Quantitative RT-PCR of polg2 , fis1 , opa1 , sod1/2 and bcl2a from isolated retina showed expression changes consistent with altered mitochondrial activity and retinal stress. We propose that the retinal disease in this model is primarily a failure of phagosome physiology with a secondary mitochondrial dysfunction. Our findings suggest that alterations in the RPE and photoreceptor cellular organelles may contribute to KCNJ13 -related retinal degeneration and provide a therapeutic target.

Clinical and preclinical therapeutic outcome metrics for USH2A-related disease

“…The samples were embedded in Tissue-Tek O.C.T embedding medium (VWR, Radnor, PA, USA) using dry ice and 12 μm sections were collected onto Superfrost ® PLUS slides (VWR) using a Leica CM1850 cryostat. The RNAscope assay was performed using the RNAscope Fluorescent Multiplex Detection v2 kit (ACD) as previously described ( 52 ). The ush2a target probe and odc1 and dapB control probes were designed and provided by ACD.…”

“…Cryosections were prepared from adult enucleated eyes or whole larvae and immunostained as previously described ( 10 , 52 ). Primary antibodies used were 4D2 (1:200; Abcam, Cambridge, UK; ab98887), anti-UV opsin, anti-blue opsin, anti-red opsin (1:200; gifted by Professor David Hyde, University of Notre Dame), anti-usherin-C (1:500; Novus Biological, Littleton, CO, USA; 27 640 002), anti-usherin-N (1:200; gifted by Dr Jennifer Phillips, University of Oregon), anti-LC3 (1:200; Thermo Fisher Scientific, Waltham, MA, USA; PA1-16930), anti-centrin (1:500; Merck-Millipore; 04-1624) or anti-acetylated tubulin (1:200; Sigma-Aldrich, St Louis, MO, USA; T7451).…”

Retinal pigment epithelium and age‐related macular degeneration: A review of major disease mechanisms

“…61 Since the energy demands of the cell dictates the mitochondrion count, RPE cells have an enriched mitochondrial population to meet the energy demands of the outer retina. 62 Early studies treating RPE cells with H 2 O 2 to induce oxidative stress showed preferential damage of mitochondrial DNA and subsequently provided a rationale for a model of AMD that is mitochondrion-based. 63 Recently, there is increasing evidence of an age-related decline in mitochondrial function in people with AMD.…”

“…RPE also metabolizes fatty acids to produce b‐hydroxybutarate, which can be used as an alternate energy source 61 . Since the energy demands of the cell dictates the mitochondrion count, RPE cells have an enriched mitochondrial population to meet the energy demands of the outer retina 62 …”

Data-Independent Acquisition-Based Quantitative Proteomic Analysis of m.3243A>G MELAS Reveals Novel Potential Pathogenesis and Therapeutic Targets

“…So it was controversial whether autophagy played protective or harmful effects. Defects in phagosome was related to a variety of cellular metabolic process, including abnormal lipid metabolism, oxidative stress injure and mitochondrial dysfunction [31][32][33] but autophagosome accumulation could impair cell bioenergetics and also induce cell death 34 . We proposed that the response to autophagosome varied widely, depending on the severity of mitochondrial de ciency.…”