Mechanisms, modeling and therapies of retinal and macular neurodegeneration

National Eye Institute

Investigators

Linked publications, trials & patents

Abstract

Aging of human retina: Transcriptome, eQTLs and mQTLs While age is a major factor contributing to the progression of AMD, there are genetic variations that alter the transcriptional landscape in the retina. Transcriptional analysis of 460 human postmortem retinas (age range: 19-101 yr; mean=68.1), including males and females, identified 1370 genes associated with aging and 1272 sex-biased genes. We used our analysis to develop a transcriptomic aging clock that accurately predicts chronological age based on retinal gene expression profiles. These retinal aging signatures correlated to signatures identified in brain and adipose tissues. Moreover, cross-species analysis identified 39 genes with conserved aging trajectories. Our future work aims to establish high-resolution retinal eQTL and mQTL maps in our aging cohort to determine how genetic polymorphisms affect DNA methylation and gene expression, QTLs contribute to genetic age, and how aging affects the interplay between genetic and epigenetic-mediated changes in transcription. We aim to understand how unique genetic variants contribute to healthy retinal aging and compare these changes in AMD cohorts to define unique molecular subtypes, separate inherited vs acquired disruptions contributing to pathogenesis, and compare sex-specific regulatory effects in AMD progression. Epigenetic Clocks for AMD, Glaucoma, and IRD Coding and non-coding genetic variations work in concert to modify gene expression. Genome-wide association studies of AMD and glaucoma cohorts has identified retinopathy-associated genes that could be potential candidates for further characterization. Our previous endeavors identified PILRB as a likely AMD gene and used a mouse model to mimic AMD pathogenesis. To examine these multifactorial diseases, we must implement various strategies to elucidate the various mechanisms that promote disease progression. We analyzed DNA methylation profiles of 160 post-mortem controls and AMD retinal samples to generate epigenetic clocks that may predict biological age of retinal tissue and focus on loci that contribute to progression of retinal diseases, such as AMD and glaucoma. Whole exome and whole genome sequencing of age-related macular degeneration (AMD) families WGS of 2394 AMD patients and 2393 controls identified single variant association signals at 4 loci and independent gene-based signals in CFH, C2, C3, and NRTN. We also found an enrichment of predicted rare loss-of-function variants in CFH, CFI, and an unreported gene ORMDL2. WES from 107 families (480 individuals) and WGS data from 20 AMD families (98 individuals) demonstrated clustering of advanced AMD across generations. The premise is that the genetic burden of these rare variants would be high and that coding variants will be easier to link to their role in pathogenesis of AMD. Our analysis relies on our existing and tested pipeline for WES and WGS using exome-wide association study (ExWAS), segregation, and case control collapsing analysis to identify rare variants and 304 genes associated with AMD. Segregating variants in 10 genes, identified in 18 families, are associated with two pathways that have not been linked to AMD. We have replicated, in an independent cohort, 192 genes involved in multiple signaling pathways. AMD signalome includes MAP Kinase, HIPPO, mTOR, VEGF and Complement pathways. Drug-gene interaction analysis reveals 17 of the 45 AMD signalome genes that could be potential drug targets. We hope to submit this manuscript in September. Genome topology of human trabecular meshwork (HTM) Intraocular pressure (IOP) increase is a common factor in primary open angle glaucoma (POAG). Dexamethasone (dex) increases IOP, therefore treatment of HTM cells with dex was useful in elucidating the molecular mechanisms in POAG progression. GWAS have identified over 300 POAG/IOP associated risk loci; however, causal genes and variants are poorly understood. With a goal of gene prioritization, we generated the first comprehensive genome topology maps of control and dex-treated HTM cells by integrating promoter capture HiC, ATAC-seq, Cut&Run for key histone marks, and total RNA-seq data. We have identified tissue-specific enhancers and promoter-centered chromatin contacts that are enriched for critical HTM functions including extracellular matrix organization, actomyosin organization, and cell adhesion. Dex-induced transcriptional shifts are concordant with changes in histone marks, compartment and accessibility, and looping. Our results implicate TNF signaling and transcription factors such as NFKB, AP-1 and CEBPD as critical hubs orchestrating steroid-induced alterations in TM. We also uncover direct physical contact between glaucoma GWAS variants and 73 novel genes, providing a framework for deciphering mechanisms underlying IOP and POAG. Splicing in Human Retina We have collaborated with the Segrè lab (Harvard) to generate splicing QTL (sQTL) map by integrating RNA-seq data from >400 human retina and 188 macula samples with genotype data and uncovered association of common cis DNA variants (±1Mb around the TSS) with differential splicing of expressed genes. After correcting for age, sex, AMD grade, population stratification, and hidden expression covariates, we have identified 2,850 genes with at least one significant sQTL in retina and 1,390 genes with one sQTL in macula. These genes are enriched in retinal biological processes, including phototransduction, cilia transport, and metabolism of xenobiotics. About 7% of sQTLs are specific for the retina. We are evaluating splicing differences between peripheral retina and macula and their relationship to AMD. RNA editing in Human Retina In collaboration with Bahlo lab (Australia), we have completed a study on genetic regulation of RNA editing using our postmortem human retina (451) and macula (185) transcriptome datasets and identified 153,118 and 104,968 high-confidence RNA editing sites, respectively. We then identified 1,525 retina and 644 macula RNA editing QTLs (edQTLs) and integrated these with AMD-GWAS using SMR and COLOC to AMD-GWAS. Notably, 14 RNA edited amino acid substitutions could be uncovered in publicly available retina proteomics dataset. Using targeted MS with C13/N15 synthetic peptides as internal controls, we have validated the expression of several edited amino acid changes in control and AMD retina. miRNAome of Human Retina We generated microRNA expression dataset (miRNAome) from 136 postmortem human retinas (from Ferrington). Banfi lab is now doing the analyses. This study could lend insight into the role of noncoding RNAs in retinal aging and disease. Protein QTLs for Human Retina Protein expression levels do not always correlate with transcription, and inter-individual variations are common. How genetic variants control protein levels in human retina has not been explored. We will generate a protein quantitative trait loci (pQTL) landscape of >100 human retina samples from Dr. Ferrington. We will use MS-based platforms to identify inter-individual variation of proteins across controls and AMD retinas, integrate this with respective genotypes, and then with mQTL and AMD-GWAS data. These studies should provide new directions for functional studies. Molecular mechanism(s) and gene networks underlying retinal degeneration Extensive genetic and epigenetic variety, as well as environmental factors contribute to disease progression and severity. These unique alterations have posed a challenge in generating therapeutic strategies for IRDs and other retinopathies. We proposed a convergence of molecular pathways resulting in the observed common phenotype, photoreceptor death that can be used to develop gene-independent interventions. Following these common pathways, we can also determine mechanisms active or inactive before onset of photoreceptor degeneration. Our studies have now shown significant differences in transcriptome, proteome and metabolome preceding rod degeneration in rd1 retinas and implicated metabolic and mitochondria pathways. Using deep transcriptomic profiling of flow-sorted rod photoreceptors from wildtype and retinopathy mouse models, including rd1, rd10, rd16, rds, Aipl1-/- and Rpgrip1-/-, identified common (termed core) pathways in at least 4 out of 6 models; these include mitochondrial metabolism, signaling (P53, MAPK and PI3K-AKT) and proteasomal processes. Pathways specific to 1-2 models are grouped as signature pathways and include amino acid metabolism, TGF-beta and cGMP-PKG signaling. Gene therapy using LCA patient-derived retinal organoids We established in vitro models of CRX-LCA and NPHP5-LCA using patient-derived human iPSCs, validated the phenotype by histological and gene expression profiling, and rescued the phenotypes by AAV-mediated gene augmentation. NEI has filed patent applications for these promising therapies. Gene-independent drug discovery using retinal organoids Due to the genetic heterogeneity that mediates IRD progression and severity, we aim to develop cost-effective, gene-independent therapies that can target neurodegenerative molecular pathways in various retinal and macular diseases. Not all human diseases can be mimicked using mouse models, therefore we utilize human iPSC-derived retinal organoids to develop efficient, high-throughput platforms to test pharmaceutical candidates. Our group in collaboration with National Center for Advancing Translational Sciences (NCATS) developed a scalable, phenotype-based platform for high-throughput untargeted small molecule drug screening using GFP-labeled rod photoreceptors of iPSC-derived retinal organoids from rd16 mouse model of CEP290-LCA. Of the 5 identified molecules (out of >6000) that prolonged rod survival, reserpine (a FDA approved antihypertensive drug) was validated for efficacy in maintaining photoreceptors in human CEP290-LCA patient-derived retinal organoids as well as in rd16 mouse retina in vivo, likely by resolving proteostasis imbalance. We then looked at other drugs resembling reserpine’s structure, quinoline derivatives of antimalarials, and other antimalarials with similar function but different function. We identified and confirmed that the drug halofantrine was able to restore normal protein expression and cellular organization in iPSC-derived retinal organoids from CEP290-LCA patients. Halofantrine was also able to restore tissue architecture and function in rd10 mice and P23H rats. We are currently working to develop eye drops to apply halofantrine for treating IRDs. We are also working to determine the efficacy of halofantrine in RPE degenerative models and AMD.