Mitochondrial functional impairment in skin fibroblasts of patients with ARL3-related retinitis pigmentosa

Abstract

Purpose : To explore the influence of ARL3 mutations (G353T and A91G) on mitochondrial function.

Methods : Sanger sequencing was used to find mutation sites. Mitochondrial aerobic respiration level was detected by mitochondrial stress test using Seahorse X96. ROS and mitochondrial membrane potential (MMP) levels reflect mitochondrial damage. Flow cytometry was performed to evaluate apoptosis rate. The protein level of ARL3 and NOX5 was measured using western blot.

Results : Sanger sequencing found two mutations (G353T and A91G) of ARL3 in two RP probands. Western blot showed that ARL3 protein stability was decreased significantly with the two mutations. Mitochondrial stress tests demonstrated that mitochondrial aerobic respiration level of fibroblasts derived from patients bearing ARL3^A91G or ARL3^A91G+G353T was downregulated. Interestingly, compared with ARL3^WT, ARL3^G353T has no effect on respiratory capacity in 293T cells, although ARL3^A91G+G353T inhibit mitochondrial aerobic respiration level. ROS and MMP detection also indicated that ARL3^A91G and ARL3^A91G+G353T were associated with mitochondrial damage, but G353T mutation have no influence. Finally, flow cytometry results showed higher apoptosis rates in fibroblasts with ARL3^A91G or ARL3^A91G+G353T.

Conclusions : This study demonstrated that ARL3 variants with single mutation of A91G or compound mutation of A91G+G353T were associated with mitochondrial damage and apoptosis, which may result in death of photoreceptor. Results proposed a possible mechanism of retinitis pigmentosa caused by ARL3 mutations and provided theoretical basis for finding new therapeutic methods.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.