Understanding of retinal degeneration through the lens of high throughput gene expression

Abstract

The retina is comprised of an intricate network of neurons, including the light-sensing photoreceptors, and supporting glia. Retinal glia maintain tissue homeostasis, while the underlying retinal-pigmented epithelium (RPE) and vascular choroid synergise to ensures a constant nutrient supply and waste removal from the highly metabolically active photoreceptors. Photoreceptor degeneration is central to almost all retinal degenerative diseases, including the increasingly prevalent and currently untreatable Age-Related Macular Degeneration (AMD). Oxidative stress and retinal inflammation are established as drivers of degeneration and are instigated by retinal glia and immune cells from circulation and choroid. Although reductionist approaches and histological observations have unravelled some of the molecular players driving photoreceptor degeneration, the application of high-throughput methods is required to penetrate the bewildering molecular complexity of retinal degeneration. To this end, the work presented in this thesis leverages powerful RNA profiling technologies to uncover novel gene expression patterns in mRNA and regulatory microRNA (miRNA) underpinning retinal degeneration. This thesis integrates data from 7 bulk miRNA/mRNA datasets and 3 single-cell RNA sequencing datasets (scRNAseq) to probe the transcriptomes of (1) the whole degenerating retina, as well as (2) effector cells driving degeneration including Muller glia and choroidal melanocytes and (3) extracellular vesicles (EV) as follow: Published results [1] presented in Chapter 3 describe changes in the mRNA and miRNA in the mouse retina following retinal degeneration induced by photo-oxidative damage. miRNA are short, non-coding RNAs working within a protein complex (RNA-induced silencing complex - RISC) to repress the translation of their mRNA targets. This chapter demonstrates that retinal degeneration is underpinned by a shift in miRNA and mRNA expression towards a proinflammatory state. Simultaneously, retinal degeneration alters miRNA binding sites within pro-inflammatory glial mRNAs allowing targeting by RISC-bound, neuronal miRNA. Published results [2] in Chapter 4 explore the role of EVs in retinal degeneration. EVs are membrane-bound vesicles secreted by nearly all cells as a form of intercellular communication between anatomically separated cells. This chapter developed the first published method for EV isolation from mouse retinas. Using this method, retinal EVs were found to be enriched with neuronal miRNA, and retinal degeneration results in a depletion of EV bioavailability. Lastly, this chapter demonstrates that inhibition of EV biogenesis accelerates retinal degeneration and results in impaired miRNA trafficking. Chapter 5 the explores miRNA-mRNA interactions in Muller glia using data integration from miRNA expression and scRNAseq. These results demonstrate that Muller glia activate a transient gene expression programme driving proliferation and pluripotency early in degeneration, then a shift towards a sustained pro-inflammatory programme follows. Published work [3] in Chapter 6 focuses on the response of choroidal melanocytes to inflammation. The choroid is a reservoir of and entry portal for immune cells in retinal degeneration, yet the relevance of the melanocytes (non-immune, highly abundant choroidal cells) in retinal degeneration is unknown. This chapter demonstrates that choroidal melanocytes have extensive immunomodulatory properties that are activated by both inflammatory challenge and retinal degeneration. In summary, this thesis uncovers novel tissue-level and cell-level gene expression patterns and regulatory networks altered in retinal degeneration, potentially providing the impetus for future therapeutics tacking complex retinal degenerations such as AMD. Show more