Sonoda, K.-H. | Search bioRxiv

bioRxiv2023

Characterization and contribution of RPE senescence to Age-related macular degeneration in Tnfrsf10 knock out mice

BackgroundRetinal pigment epithelial cells (RPE) play vital role in the pathogenesis of age-related macular degeneration (AMD). Our laboratory has shown that RPE cellular senescence contributed to the pathophysiology of experimental AMD, and SASP members are involved in this process. Recently, we presented confirmatory evidence to earlier GWAS studies that dysregulation of tumor necrosis factor receptor superfamily 10A (TNFRSF10A) dysregulation leads to AMD development and is linked to RPE dysfunction. This study aims to investigate the contribution of RPE senescence to AMD pathophysiology using TNFRSF10A silenced human RPE (hRPE) cells and Tnfrsf10 KO mice. MethodsSub-confluent primary hRPE cells and TNFRSF10A silenced hRPE were exposed to stress-induced premature senescence with H2O2 (500 M, 48h), and senescence-associated markers ({beta}gal, p16, and p21) were analyzed by RT-PCR and WB analysis. The effect of H2O2-induced senescence in non-silenced and silenced hRPE on OXPHOS and glycolysis was determined using Seahorse XF96 analyzer. Male C57BL/6J Tnfrsf10 KO (Tnfrsf10-/-) mice were used to study the regulation of senescence by TNFRSF10A in vivo. Expression of p16 and p21 in control and KO mice of varying ages were determined by RT-PCR, WB, and immunostaining analysis. ResultsThe senescence-associated p16 and p21 showed a significant (p < 0.01) upregulation with H2O2 induction at the gene (1.8- and 3-fold) and protein (3.2- and 4-fold) levels in hRPE cells. The protein expression of p16 and p21 was further significantly increased by co-treatment with siRNA (p < 0.05 vs. H2O2). Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (pmol/min/total DNA) increased with senescence induction by H2O2 for 48h in control RPE, and knockdown of TNFRSF10A caused a further increase in OCR and ECAR. In addition, co-treatment with PKC activator significantly improved all parameters. Similarly, in vivo studies showed upregulation of p16 and p21 by RT-PCR, WB, and immunostaining analysis in RPE/choroid of Tnfrsf10 KO mice. When subjected to examination across distinct age groups, namely young (1-3 months), middle (6-9 months), and old (12-15 months) mice, a discernible age-related elevation in the expression of p16 and p21 was observed. ConclusionsOur findings suggest that TNRSF10A is a regulator of regulates in RPE senescence. Further work on elucidating pathways of senescence will facilitate the development of new therapeutic targets for AMD.

pathology↗

bioRxiv2019

Systematic detection of Mendelian and non-Mendelian variants associated with retinitis pigmentosa by genome-wide association study

To uncover genetic basis of autosomal recessive retinitis pigmentosa (ARRP), we applied 2-step genome-wide association study (GWAS) in 640 Japanese patients prescreened with targeted re-sequencing. Meta-GWAS identified three independent peaks at P < 5.0x10-8, all within the major ARRP gene EYS. Two were each tagged by a low frequency variant (allele frequency < 0.05); a known founder Mendelian mutation (c.4957dupA, p.S1653Kfs*2) and a presumably hypomorphic non-synonymous variant (c.2528G>A, p.G843E). c.2528G>A newly solved 7.0% of Japanese ARRP cases, improving genetic diagnosis by 26.8% and simultaneously serving as a new attractive target for genome editing gene therapy. The third peak was tagged by an intronic common variant, representing a novel disease-susceptibility signal. GWAS successfully unraveled genetic causes of a rare "monogenic" disorder for the first time, which provided unexpected insights into significant contribution of non-Mendelian genetic factors and identified a novel high frequency variant directly linked to development of local genome therapeutics.

genetics↗

bioRxiv2018

Frequent variants in the Japanese population determine quasi-Mendelian inheritance of rare retinal ciliopathy

Hereditary retinal degenerations (HRDs) are Mendelian diseases characterized by progressive blindness and caused by ultra-rare mutations. In a genomic screen of 331 unrelated Japanese patients, we identify a disruptive Alu insertion and a nonsense variant (p.Arg1933*) in the ciliary gene RP1, neither of which are rare alleles in Japan. p.Arg1933* is almost polymorphic (frequency = 0.6%, amongst 12,000 individuals), does not cause disease in homozygosis or heterozygosis, and yet is significantly enriched in HRD patients (frequency = 2.1%, i.e. a 3.5-fold enrichment; p-value = 9.2x10-5). Familial co-segregation and association analyses show that p.Arg1933* can act as a Mendelian mutation, in trans with the Alu insertion, but might also cause disease in association with two alleles in the EYS gene in a non-Mendelian pattern of heredity. Our results suggest that rare conditions such as HRDs can be paradoxically determined by relatively common variants, following a quasi-Mendelian model linking monogenic and complex inheritance.

genomics↗