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 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. 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 studied 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. Evidence indicated that mitochondrial overactivation is a determinant of early retinal pathology. The rd/rd murine model lines have mutations that lead to photoreceptor degeneration and are widely used to study IRDs. To investigate the mechanisms of ciliopathy in rod photoreceptor degeneration, we utilized the rd16 murine line with mutations in the cilia-related gene, Cep290 (Cep290rd16/rd16). Transcriptomes of rod photoreceptors showed significant abnormal trends with peak changes in oxidative stress response and mitochondrial metabolism occurring by postnatal day (P) 6. Seahorse metabolic assays revealed increases in mitochondrial ATP production in retinal tissues between P2 and P6, coinciding with elevated protein expression of oxidative phosphorylation complexes and mitophagy factors. These findings are indicative of convergent mechanisms in IRD. Mechanisms of diet and nutritional influence on retinal health and diseases We compared retinal and liver transcriptomes from WT mice fed isocaloric high- and low-glycemic diet over short- and long-terms. Both the retina and liver showed alterations in housekeeping processes with overexpression of inflammation markers a well-known phenotype in aging-related diseases. We observed upregulated fatty acid biogenesis with long-term high-glycemic diet, indicative of hepatic steatosis. Our results show significant impact of dietary glycemic exposure on highly metabolic organs like the retina and liver. In collaboration with NHLBI, we investigated the effect of dietary supplementation of very long chain poly unsaturated fatty acid (VLC-PUFA). The retina, among a few other organs, are uniquely enriched for >24C n:3-n:6 VLC-PUFA, that are generally absent from our diet. In vivo production is carried out by the enzyme Elovl4, a target of inherited retinal and macular degenerations. Our RNA-seqs study revealed activation of cytochrome P450 family 4A enzymes and PPAR delta network in mice liver, in response to VLC-PUFA supplementation. Altered pathways are reminiscent of efficient fatty acid oxidation and peroxisomal processes. Whole exome and whole genome sequencing of AMD families We have obtained whole exome and whole genome sequencing data for over 107 large multigenerational AMD families. Segregation of variants and linkage analysis has been completed. In a large familial AMD cohort, we have identified ultra-rare variants in complement factors 8A and 8B and 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 To further identify putative functional genes and pathways for AMD, we have profiled genome-wide DNA methylation from 152 postmortem retinas and identified 37,453 variants associated in cis (<1 Mb) with CpG sites known as methylation quantitative trait loci (meQTLs). Gene ontology analysis of mGenes identified genes involved in regulation of GTPase activity, actin filament and synaptic signaling. We used these genetic associations to infer potential causal relationships between gene expression and DNA methylation. Integrating AMD GWAS results with eQTL or mQTL using multiple colocalization methods revealed previously reported 9 GWAS loci including CFH, ARMS2/HTRA1, or LINC01004. Furthermore, integration of expression with methylation identified 13,747 expression quantitative trait methylation (eQTMs) and target genes of eQTM are enriched in oxidative phosphorylation, electron transport chain and translational initiation. The high correlation of genetic effects between methylation and gene expression highlights the potential to identify target genes in AMD for functional studies. Epitranscriptome landscapes of human macula To determine the epitranscriptome landscape in human retina and compare the difference in the process of AMD, we employ highly sensitive and accurate ultra-high performance liquid chromatographytandem mass spectrometry (UHPLC-MS) to detect RNA modifications in human retinae. We detected 31 nucleotide modifications and in which the m3C, m7G and mnm5U levels are significantly increased in advanced AMD group compared with normal control. Disease modeling and gene therapy of CRX-LCA and NPHP5-LCA We have 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. Fibroblasts from 3 families affected by NPHP5-LCA were examined for cilia defects. Pronounced elongation of primary cilia was observed in two siblings carrying biallelic truncation mutations in NPHP5. Skin biopsies from these two patients and a familial control were used to derive iPSCs, which were differentiated into RPE cells and retinal organoids. Consistently, cilia extension phenotype and diminished CEP290 were also observed in patient RPE. Retinal organoids showed impaired outer segment development with mislocalization of opsins and other phototransduction proteins and loss of CEP290 protein. This phenotype was rescued by administration of an AAV vector encoding correct copy of NPHP5 gene, suggesting a potential future therapeutic avenue. Drug candidates for photoreceptor survival in retinal degenerative diseases We previously identified 5 drug candidates that can sustain photoreceptors in mouse and human retinal organoids derived from iPSCs carrying mutations in the CEP290-LCA. 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.