The role of Tetraspanin-10 in the retinal pigment epithelium and its influence in age-related macular degeneration

Abstract

Age related macular degeneration (AMD) is the leading cause of vision loss in the Western World [1], accounting for 5.6% of global blindness [1][2, 3]. Irreversible visual impairment arises due to the gradual loss of photoreceptors and their supportive cells: the retinal pigment epithelium (RPE). Currently therapeutics are only available to treat 50% of patients with AMD and in the long-term are ineffective. This is due to an incomplete understanding of the pathophysiology. Genetic risk is a key element of AMD susceptibility and multiple risk loci have been identified including a novel single nucleotide polymorphism (SNP) at Tetraspanin 10 (TSPAN10) [4-7]. The purpose of this work was to elucidate the role of TSPAN10 within the RPE and subsequently understand how it contributes to AMD.
CRISPR/Cas-9 gene editing was utilised to produce two TSPAN10 knock-out (k.o) clones in human embryonic stem cells (hESCs) that were then differentiated into RPE. Three separate differentiations for both TSPAN10 k.o and WT cells were produced for robustness, and their genomic DNA, transcripts and protein characterised. TSPAN10 has a significant influence on hESC-RPE pigmentation. TSPAN10 k.o cells were less pigmented than WT hESC-RPE, evidenced both macroscopically in culture, microscopically using transmission electron microscopy (TEM), and through measurement of tyrosinase production and function.
Transcriptomics suggested TSPAN10 k.o has a significant influence on oxidative stress in the RPE. Catalase (CAT) is a key enzyme protecting the cell from oxidative stress, CAT mRNA expression was significantly reduced in TSPAN10 k.o clones (padj= 1.39 x 10-20). Functional correlation revealed increased oxidative stress in k.o cells at baseline and reduced catalase production. A functional assay also assessed the effect of TSPAN10 k.o on RPE phagocytic function, revealing a similar response to oxidatively stressed WT RPE.
Finally, a cohort study in human beings analysing the impact of AMD genetic risk loci on outer retinal thickness, revealed the AMD risk SNP at TSPAN10 to be significantly associated with thinning of the RPE-Bruch’s membrane and photoreceptor complexes.
The work presented within this thesis demonstrates the multiple plausible roles that TSPAN10 may have in the RPE and gives insight into its role in AMD. Aligning this with further functional work is a key next step

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Identifiers

ORCID for Rebecca Kaye: orcid.org/0000-0002-1504-3201

ORCID for Andrew Lotery: orcid.org/0000-0001-5541-4305

ORCID for Jorn Lakowski: orcid.org/0000-0003-4214-7580

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