Defining the mechanism of photoreceptor cell death in Retinitis Pigmentosa

定义视网膜色素变性感光细胞死亡的机制

• 批准号: 2605683
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2605683
• 关键词: Defining; mechanism; photoreceptor; cell; death

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.

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 个基因的突变会导致视网膜中的感光感光细胞死亡，从而导致夜盲症、视野收缩并最终导致完全视力丧失。 IRD 是导致儿童和职业成年人视力丧失的主要原因，并带来巨大的经济和福利成本。尽管基因疗法已成为一种潜在的治疗方法，但改进很小且进展缓慢，因为每次必须针对每个基因。 据推测，RP 中光感受器死亡的机制对于所有致病等位基因来说都是常见的。如果是这样，了解这一途径可能会产生适用于所有形式疾病的治疗靶点。然而，它尚未得到完全定义。该项目将采用 4D 细胞医学方法，以单细胞分辨率确定光感受器在 RP 中从健康状态转变为应激状态直至死亡时发生的转录变化。为此，我们将使用两种不同的 RP 小鼠模型；每一种都含有涉及光感受器维持和功能所需的不同途径的基因突变。 Atrd1 小鼠的 Pde6b 发生突变；它的蛋白质对于视觉循环至关重要。 Atrd1 小鼠在 6 周内出现快速光感受器退化。 RPGR 似乎参与感光器外节的维护/周转。 Rpgr 突变小鼠在 24 个月内会出现缓慢的光感受器退化。通过比较两只小鼠共有的转录变化，希望我们能够找到一条有针对性的途径来挽救 RP 中的光感受器丧失。假设：多种 RP 模型之间存在共同的光感受器死亡机制。该项目有 3 个目标：- 全面表征 Atrd1 小鼠（Pde6b 介导的 RP 模型）中观察到的视网膜变性。- 定义潜在机制 导致 Atrd1 小鼠光感受器丧失的细胞死亡。- 将 Atrd1 数据集与缓慢进展的 RP RPGR 模型的数据集进行功能整合，以确定是否存在共同机制。在这个多学科项目中，将应用实验室和计算技术的组合。 Atrd1 小鼠将在 Roly Megaw 博士的监督下在 MRC HGU 的眼表型分析套件中进行全面表征，以绘制视网膜功能（视网膜电图、光动鼓）和结构（光学相干断层扫描）随时间的变化。 Atrd1 是视网膜变性的快速模型，光感受器损失会在数周内发生。随后，将在早期 (P14) 和晚期 (P28) 时间点对分离的视网膜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途径变化。此外，入侵的神经胶质细胞和循环单核细胞将受到询问。结果将与 RP 疾病 (Rpgr) 缓慢退化模型的 scSeq 数据（12 和 24 个月时间点）进行比较。细胞死亡和视网膜侵袭的机制将通过聚焦 qPCR、RNAscope 等空间转录组学方法以及蛋白质组学方法和成像实验来证实。随后将针对已确定的途径来挽救我们突变模型中出现的光感受器变性。这个博士项目的目的是更好地了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的 RP 中视网膜变性的靶点来开发新颖且急需的治疗方案。