Ziaka, Kalliopi; (2024) Investigation and manipulation of photoreceptor stress responses in rhodopsin retinitis pigmentosa. Doctoral thesis (Ph.D), UCL (University College London).

Text Ziaka Kalliopi_PhD_thesis.pdf - Accepted Version Access restricted to UCL open access staff until 1 March 2027. Download (43MB)

Abstract

Photoreceptors are sensory neurons with high metabolic demands, as they are responsible for carrying out visual phototransduction, a complex and multistep process that involves a large number of signalling protein components. The viability of photoreceptors relies on mechanisms that ensure a well-balanced and functional proteome that maintains cellular protein homeostasis, or proteostasis. These include mechanisms such as, the heat shock response (HSR), unfolded protein response (UPR), and the mitochondrial associated UPR (UPRmt). Autosomal dominant Retinitis Pigmentosa (adRP), can be caused by mutations in rhodopsin, the visual pigment of rod photoreceptor cells, which can disrupt the proteostasis network. This study explored photoreceptor stress responses in the presence of rhodopsin mutations in new mouse and induced pluripotent stem cell (iPSC)-derived retinal organoid (RO) models of adRP. Initially, phenotypic assessment of mouse knock-in models carrying the rhodopsin mutations M39R, T58R, R135W and P347L revealed patterns of retinal degeneration which represent the retinal phenotype of adRP patients. Transcriptomic analyses of R135W and P347L mouse models, which had the most severe forms of retinal degeneration, showed distinct changes in key markers of HSR, UPR and UPRmt stress mechanisms. Specifically, the R135W model exhibited signs of HSR and UPR induction. HSR was further enhanced in the R135W retinae after treatment with the HSR potentiator ARUK4008447, which led to augmented levels of HSP70, a key player of HSR. Surprisingly, treatment with another HSR potentiator, arimoclomol, led to accelerated retinal degeneration. The P347L mouse model showed induction of HSR and UPRmt related genes, and the presence of prominent extracellular vesicles (EVs). Proteomic analysis on the isolated EVs provided further insights of the phenotype of this model. In addition, patient fibroblasts carrying the rhodopsin mutation P347L were reprogrammed into iPSCs, which were then successfully differentiated into ROs. The P347L ROs were used as a complementary model to the mouse studies. Key UPRmt genes were also found to be upregulated in the P347L ROs, further supporting a potential involvement of mitochondrial stress. To follow up these observations, mitochondrial function was assessed in the P347L ROs, which demonstrated an impaired mitochondrial bioenergetic profile. In summary, this thesis describes evidence of potential involvement of distinct cellular stress pathways and identifies potential mechanisms of disease in these new rhodopsin models of adRP.

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