Farkas, M. H. | bioRxiv Science

bioRxiv2022

Patterns of gene expression and allele-specific expression vary among macular tissues and clinical stages of Age-related Macular Degeneration

Age-related macular degeneration (AMD) is a complex neurodegenerative disease and is the leading cause of blindness in the aging population. Early AMD is characterized by drusen in the macula and causes minimal changes in visual function. The later stages are responsible for the majority of visual impairment and blindness and can be either manifest as geographic atrophy (dry) or neovascular disease (wet). Available medicines are directed against the wet form and do not cure vision loss. Therefore, it is imperative to identify preventive and therapeutic targets. As the mechanism for AMD is unclear, we aim to interrogate the disease-affected tissue - the macular neural retina and macular retina pigment epithelium (RPE)/choroid. We investigated differentially expressed genes expression (DEG) across the clinical stages of AMD in meticulously dissected and phenotyped eyes using a standardized published protocol (Owen et al., 2019). Donor eyes (n=27) were obtained from Caucasian individuals with an age range of 60-94 and 63% were male, and tissue from the macula RPE/choroid and macula neural retina were taken from the same eye. Donor eyes were recovered within 6 hours post mortem interval time to ensure maximal preservation of RNA quality and accuracy of diagnosis. Eyes were then phenotyped by retina experts using multi modal imaging (fundus photos and SD-OCT). Utilizing DESeq2, followed PCA, Benjamini Hochberg adjustment to control for the false discovery rate, and Bonferonni correction for the number of paired comparisons: a total of 26,650 genes were expressed in the macula RPE/choroid and/or macula retina among which significant differential expression was found for 1,204 genes between neovascular AMD and normal eyes, 40 genes between intermediate AMD and normal eyes, and 1,194 genes between intermediate AMD and neovascular AMD. A comparison of intermediate AMD versus normal eyes included TCN2, PON1, IFI6, GPR123, and TIMD4 as being some of the most significant DEGs in the macula RPE/choroid. A comparison of neovascular AMD versus normal eyes included SLC1A2, SLC24A1, SCAMP5, PTPRN, and SEMA7A as being some of the most significant DEGs in the macula RPE/choroid. Top pathways of DEGs in the macular RPE/choroid identified through Ingenuity Pathway Analysis (IPA) for the comparison of intermediate AMD with normal eyes were interferon signaling and Th1 and Th2 activation, while those for the comparison of neovascular AMD with normal eyes were the phototransduction and SNARE signaling pathways. Allele-specific expression (ASE) in coding regions of previously reported AMD risk loci identified by GWAS (Fritsche et al, 2016) revealed significant ASEs for C3 rs2230199 and CFH rs1061170 in the macula RPE/choroid for normal eyes and intermediate AMD, and for CFH rs1061147 in the macula RPE/choroid for normal eyes and intermediate and neovascular AMD. An investigation of the 34 established AMD risk loci revealed that 75% of them were significantly differentially expressed between normal macular RPE/choroid and macular neural retina, with 75% of these loci showing higher expression in the RPE. Similarly, disease state differences for the GWAS loci were only found to be statistically differentially expressed in the macular RPE/choroid. Moreover, the known coding variants in the previously identified GWAS loci including, CFH, C3, CFB, demonstrated ASE across AMD clinical stages in the macular RPE/choroid and not in the neural retina. These data at the bulk level underscore the importance of the RPE/choroid to AMD pathophysiology. While many bulk RNASeq data sets are publicly available, to the best of our knowledge this is one of the first publicly available datasets with both maculae RPE/choroid and macula neural retina from the same well phenotyped donor eye(s) where the macula is separated from the periphery. Our findings also underscore the importance of studying both macular tissue types to gain a full understanding of mechanisms leading to AMD. Our results provide insights into underlying biological mechanisms that may differentiate the disease subtypes and into the tissues affected by the disease.

genomics↗

bioRxiv2022

Exploring the lncRNA localization landscape within the retinal pigment epithelium under normal and stress conditions

Long noncoding RNAs (lncRNAs) are emerging as a class of genes whose importance has yet to be fully realized. It is becoming clear that the primary function of lncRNAs is to regulate gene expression, and they do so through a variety of mechanisms that are critically tied to their subcellular localization. Although most lncRNAs are poorly understood, mapping lncRNA subcellular localization can provide a foundation for understanding these mechanisms. Here, we present an initial step toward uncovering the localization landscape of lncRNAs in the human retinal pigmented epithelium (RPE) using high throughput RNA-Sequencing (RNA-Seq). To do this, we differentiated human induced pluripotent stem cells (iPSCs) into RPE, isolated RNA from nuclear and cytoplasmic fractions, and performed RNA-Seq on both. Furthermore, we investigated lncRNA localization changes that occur in response to oxidative stress. We discovered that, under normal conditions, most lncRNAs (76%) are seen in both the nucleus and the cytoplasm to a similar degree, but of the transcripts that are highly enriched in one compartment, more are nuclear (18.6%) than cytoplasmic (5.6%). Interestingly, under oxidative stress conditions, we observed an increase in lncRNA localization in both nuclear (23.5%) and cytoplasmic (9.7%) fractions. In addition, we found that nuclear localization was partially attributable to the presence of previously described nuclear retention motifs, while adenosine to inosine (A-to-I) RNA editing appeared to play a very minimal role. Our findings regarding lncRNA localization in the RPE provide two avenues for future research: 1) how lncRNAs function in the RPE, and 2) how one environmental factor, in isolation, may play a role in retinal disease pathogenesis.

genetics↗

bioRxiv2021

Oxidative stress alters transcript localization of disease-causing genes in the retinal pigment epithelium

Nuclear retention is a mechanism whereby RNA transcripts are held in the nucleus to maintain a proper nuclear-to-cytoplasmic balance or as a stockpile for use in responding to stimuli. Many mechanisms are employed to determine whether transcripts are retained or exported to the cytoplasm, though the extent to which tissue- or cell-type, stressors, or disease pathogenesis affect this process remains unclear. As the most biochemically active tissue in the body, the retina must mitigate endogenous and exogenous stressors to maintain cell health and tissue function. Oxidative stress, believed to contribute to the pathogenesis, or progression, of age-related macular degeneration (AMD) and inherited retinal dystrophies (IRDs), is produced both internally from biochemical processes, as well as externally from environmental insult. To evaluate the effect of oxidative stress on transcript localization in the retinal pigment epithelium (RPE), we performed poly-A RNA sequencing on nuclear and cytoplasmic fractions from induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells exposed to hydrogen peroxide, as well as untreated controls. Under normal conditions, the number of mRNA transcripts retained in the nucleus exceeded that found in studies of other tissues. Further, the nuclear-to-cytoplasmic ratio of transcripts is altered following oxidative stress, as is the retention of genes associated with AMD, IRDs, and those important for RPE physiology. These results provide a retention catalog of all expressed mRNA in iPSC-RPE under normal conditions and after exposure to hydrogen peroxide, offering insight into one of the potential roles oxidative stress plays in the progression of visual disorders.

genetics↗