Mechanisms, modeling and therapies of retinal and macular neurodegeneration

National Eye Institute

Investigators

Linked publications, trials & patents

Abstract

Mitochondria and metabolic defects leading to photoreceptor cell death in retinal degenerative diseases Extensive genetic and phenotypic heterogeneity associated with retinal and macular diseases warrants the design of gene-agnostic therpaies. A better understanding of pre-degeneration molecular cues in photoreceptor cells would uncover novel targets that can be used to treat pathologies caused by multiple disease genes. Multi-omics investigation of retinas from rd1 mice, a widely used model of inherited retinal degeneration (IRD) for mutant Pde6brd1/rd1, uncovered progressive metabolic abnormalities and mitochondrial defects leading to onset of degeneration in rods. Parallel examination using oxygen consumption rate assays and ultrastructure analysis confirmed the omics findings. Interestingly, significant dysregulation of mitochondrial genes was also noted in the pre-degeneration stages of rd16 mice (Cep290rd16/rd16), where the disease gene is unrelated to that of rd1. However, rd16 rods also presented significant dysregulation of autophagy and mitophagy factors. Overlapping molecular trends are indicative of convergent etiological mechanisms among genetically distinct diverse IRDs. We optimized mitochondrial stress assays and the glycolytic rate assay to study mitochondrial respiration and glycolysis in fresh dissected ex vivo retinal tissues using the new generation of Agilent Seahorse XFe24 analyzer. We are also studying the effect of IRD mutations on mitochondrial dynamics and found that early retinas show increased mitochondrial fusion, ETC complexes, and altered levels of mitophagy marker parkin. We will further compare mitochondrial signaling in mouse models to identify novel therapeutic targets. We tested the hypothesis that augmented HSP70 expression in degenerating retina may have a protective effect. We over-expressed the Crx-driving HSP70 protein in photoreceptors of three mouse models of inherited retinal degeneration and observed a divergent effect in different models. Thus, the effect of HSP70 overexpression is determined by the nature of the mutation. Whole exome sequencing (WES) of patients with IRDs Elucidating the genetic variations leading to disease development and progression is the first step to be able to provide patients with both accurate genetic counseling and development of therapeutic strategies. WES analyses of IRD patients have identified known and novel potentially disease-causing mutations in about 50% of our IRD cohort. Whole exome and whole genome sequencing of AMD families We have obtained whole exome and whole genome sequencing data for over 125 large multigenerational AMD families. Segregation of variants and linkage analysis has been completed. In a large familial AMD cohort we were able to validate several of the previously reported genes (e.g., TIMP3, C9 and CFH) and identify novel gene candidates contributing to the development and pathology of AMD. Methylation quantitative trait loci (mQTLs) associated with age-related macular degeneration Previously, we have profiled the transcriptome of the human peripheral retina and identified expression quantitative trait loci (eQTLs) with respect to AMD. To further identify putative functional genes and pathways for AMD, we have profiled genome-wide DNA methylation quantitative trait loci (mQTLs). We used these genetic associations to infer potential causal relationships between gene expression and DNA methylation. Integrating AMD GWAS results with eQTL or mQTL revealed previously reported 17 GWAS loci including ARMS2/HTRA1, PILRB/PILRA, C3 and NPLOC4/TSPAN10. The high correlation of genetic effects between methylation and gene expression highlights the potential to identify target genes in AMD for functional studies. Transcriptome landscape of human macula We generated transcriptomic profiles of 185 AMD macula samples and with only control samples for reference transcriptome we identified 12,975 protein coding and 955 noncoding genes. Cis-eQTL analysis identified 5,959 eGenes and enrichment of best eQTL showed enrichment of secretory and mitochondrial genes. Integration of AMD GWAS with eQTL proposed potential target genes and regulatory mechanisms at 6 AMD loci including ACAD10, RDH5/CD63 and PILRB/PILRA. We will further perform colocalization analysis using various tools to identify novel genes and pathways associated with AMD. Disease modeling and gene therapy of CRX-LCA and NPHP5-LCA We established retinal organoid disease models to study the molecular mechanisms underlying severe early onset vision loss in LCA caused by mutations in CRX and NPHP5 genes with the objective of developing novel treatment approaches. Retinal organoids were differentiated from induced pluripotent stem cells (iPSCs) derived from two patients carrying heterozygous autosomal dominant mutations in CRX. Treatment of patient retinal organoids by delivering an additional correct copy of CRX resulted in partial restoration of Rhodopsin and L/M cone opsin expression as assessed by histology and single cell RNA sequencing. RPE and retinal organoids derived from iPSCs of two siblings carrying biallelic truncation mutations in NPHP5 showed a striking cilia elongation phenotype. Furthermore, inner/outer segment-like region was less developed in the patient organoids compared to healthy control. This phenotype was rescued by administration of an AAV vector encoding correct copy of NPHP5 gene, suggesting a potential future therapeutic avenue. Modulation of epigenome and cellular pathways in the aging retina by diet and micronutrient supplementation Nutrients are proposed to regulate age-related epigenetic processes to impact retinal health. To elucidate the role of dietary vitamins, micronutrients, and glycemic index, we are generating transcriptome and epigenome profiles of aging mouse retina. With a grant from the NIH Office of Dietary Supplements, one-year old mice are currently being adapted on experimental diets for a period of nine months. After completion of treatment, a battery of high throughput experiments including whole genome DNA methylation, ATAC sequencing, and RNA sequencing would be performed on retinas from treated animals. Drug candidates for photoreceptor survival in retinal degenerative diseases In collaboration with NCATS, we identified 5 drug candidates that can sustain photoreceptors in mouse and human retinal organoids derived from iPSCs carrying mutations in the CEP290-LCA. This year, we focused on underlying mechanisms of drug candidates in maintaining photoreceptor survival. Time series RNA seq of patient derived retinal organoids demonstrated CEP290-LCA associated pathway changes to occur after Day 90 of differentiation. A combination of histological assessments and transcriptomic analyses directed the selection and dosage strategy of one of the drugs that showed efficacy in rescuing retinal degeneration in patient organoids. Bioinformatic analyses suggested autophagy remodulation a key pathways of drug action. Further investigations revealed misregulation of autophagic flux in patient stem cell-derived organoids and in the rd16 retina, resulting in downregulation of autophagy adaptor p62. Restoration of p62 level by specific autophagy inhibitors reduced the level of HDAC6 and improved cellular microtubule networks and biogenesis of the outer segment. Our work indicates a critical role of autophagy misregulation in photoreceptor dysfunction and provides new insights into druggable targets to maintain photoreceptor survival.