Defining the mechanism of photoreceptor cell death in Retinitis Pigmentosa

色素性视网膜炎（RP）是一种多基因、无法治疗的遗传性视网膜营养不良（IRD）。超过100个基因的突变导致视网膜感光细胞死亡，导致夜盲症、视野缩小，最终完全丧失视力。IRDs是儿童和工作成年人视力丧失的主要原因，并与之相关的巨大经济和福祉成本。尽管基因治疗已经成为一种潜在的治疗方法，但由于每个基因只能一次靶向一个，因此改善幅度很小且进展缓慢。据推测，RP中光感受器死亡的机制对所有致病等位基因是共同的。如果是这样，了解这一途径可能会导致一个治疗靶点，将适用于所有形式的疾病。然而，它还没有被完全定义。本项目将采用4D细胞医学方法，以单细胞分辨率确定RP中光感受器从健康到应激再到死亡过程中发生的转录变化。为此，我们将使用两种不同的小鼠RP模型；每个都有一个基因突变，涉及光感受器维持和功能所需的不同途径。Atrd1小鼠携带Pde6b突变；它的蛋白质对视觉周期至关重要。Atrd1小鼠在6周内迅速发生光感受器退化。RPGR似乎参与光感受器外段的维持/更新。在24个月的时间里，rpgr突变的小鼠发生了缓慢的光感受器退化。通过比较两种小鼠共同的转录变化，我们希望能够找到一种靶向途径来挽救RP中的光感受器损失。假设：在多种RP模型中有一个共同的光感受器死亡机制。该项目有3个目标：-充分表征Atrd1小鼠（pde6b介导的RP模型）的视网膜变性。-确定导致theAtrd1小鼠光感受器丧失的细胞死亡的潜在机制。-将Atrd1数据集与进展缓慢的RP RPGR模型的数据集进行功能整合，以识别是否存在共同机制。在这个多学科项目中，将应用实验室和计算技术的结合。Atrd1小鼠将在Roly Megaw博士的监督下，在MRC HGU的眼睛表型组中进行全面表征，以绘制视网膜功能（视网膜电图，光动力鼓）和结构（光学相干断层扫描）随时间的变化。Atrd1是一种视网膜变性的快速模型，光感受器的丧失会在数周内发生。在此之后，将在分离视网膜的早期（P14）和晚期（P28）时间点进行单细胞测序实验。数据集将在Catalina Vallejos博士的监督下进行分析，目的是确定突变光感受器死亡时的路径变化。此外，入侵的胶质细胞和循环的单核细胞将被询问。结果将与rp -疾病（Rpgr）慢变性模型的scSeq数据（12个月和24个月时间点）进行比较。细胞死亡和视网膜入侵的机制将通过聚焦qPCR、RNAscope等空间转录组学方法、蛋白质组学方法和成像实验来证实。确定的途径将随后靶向拯救在我们的突变模型中看到的光感受器变性。这个博士项目的目标是更好地理解光感受器细胞死亡的机制，通过确定可以挽救或减缓所有形式RP的视网膜变性的靶点来开发新的和急需的治疗方案。

Retinitis pigmentosa (RP) is a multigenic, untreatable inherited retinal dystrophy (IRD). Mutations in over 100 genes lead to death of the light sensing photoreceptors in the retina, resulting in night blindness, visual field constriction and eventual total visual loss. The IRDs are the leading cause of visual loss in children and working adults and have immense economic and well-being costs associated with them. Although gene therapy has emerged a potential therapeutic, improvements are minimal and slow-moving, as each gene must be targeted one at a time.It has been postulated that the mechanism through which photoreceptors die in RP is common to all disease-causing alleles. If so, understanding this pathway could lead to a therapeutic target that would be applicable for all forms of disease. It has, however, yet to be fully defined. This project will adopt a 4D cellular medicine approach to determine the transcriptional changes that occur in photoreceptors with single cell resolution as photoreceptors move from health to stress to death in RP. To do so, we will use two different murine models of RP; each with a mutation in a gene involved in a different pathway required for photoreceptor maintenance and function. The Atrd1 mouse harbours a mutation in Pde6b; its protein is crucial for the visual cycle. The Atrd1 mouse develops a fast photoreceptor degeneration over 6 weeks. RPGR appears to be involved in photoreceptor outer segment maintenance / turnover. The Rpgr-mutant mouse develops a slow photoreceptor degeneration over 24 months. By comparing transcriptional changes common to both mice, it is hoped we will identify a pathway to be targeted to rescue photoreceptor loss in RP.Hypothesis: There is a shared common mechanism of photoreceptor death across multiple models of RP.This project has 3 aims:- To fully characterise the retinal degeneration seen in the Atrd1 mouse, a model for Pde6b-mediated RP.- To define the underlying mechanism of cell death that leads to photoreceptor loss in theAtrd1 mouse.- To functionally integrate Atrd1 datasets with those of the slow progressing RP RPGR modelto identify whether common mechanisms exist.In this multidisciplinary project, a combination of both laboratory and computational techniques will be applied. The Atrd1 mouse will comprehensively characterised in the MRC HGU's eye phenotyping suite under the supervision of Dr Roly Megaw, to map changes in retinal function (electroretinography, optokinetic drum) and structure (optical coherence tomography) over time. The Atrd1 is a fast model of retinal degeneration, with photoreceptor loss occurring over weeks.Following this, single cell sequencing experiments will be performed on dissociated retinas at early (P14) and late (P28) timepoints. Datasets will be analysed under the supervision of Dr Catalina Vallejos, with the aim of defining pathway changes in mutant photoreceptors as they die. Further, invading glial and circulating monocytic cells will be interrogated. Results will be compared to scSeq data (12 and 24 month timepoints) of a slow degeneration model of RP-disease (Rpgr). Mechanisms of cell death and retinal invasion will be confirmed with focussed qPCR, spatial transcriptomic methods such as RNAscope, as well as proteomics methods and imaging experiments. Identified pathways will subsequently be targeted to rescue the photoreceptor degeneration seen in our mutant models.The objective of this PhD project is a better understanding of the mechanisms of photoreceptor cell death to develop novel and much needed treatment options by identifying targets that could rescue or slow down retinal degeneration in all forms of RP.