Identifying and leveraging strategies of inherently resilient retinal neurons to treat degeneration

摘要 视网膜神经节细胞（RGC）是眼睛和大脑之间的唯一联系。它们特别 易变性，它们的损伤和死亡导致视力丧失，如青光眼，糖尿病 视网膜病、视神经胶质瘤和视神经炎。这些疾病的大多数治疗方法并不关注 特别是拯救RGC，但缓解疾病进展的明显驱动因素。例如电流 青光眼治疗集中在降低升高的眼内压（IOP），但在大多数情况下无效 病人。此外，许多青光眼患者也出现RGC变性，但没有IOP升高。因此，新 在退行性疾病中保留RGC的治疗代表了重要的未满足的临床需求。虽然 RGC细胞死亡导致视力丧失，退行性疾病中的RGC死亡是不完全的，即使在严重的 受影响的患者和强大的动物模型。了解一些RGC如何在退行性变中天然坚持 条件可以告知新的治疗策略的发展。为了确定本地应对策略，我们将 在变性之前和过程中直接观察单个RGC的细胞特征，重点是 细胞内稳态我们已经建立了纵向，在体内，2光子成像的基因编码的 RGC中的生物传感器，以重复单个RGC分辨率直接观察能量和Ca 2+稳态 在很长一段时间内。这种方法允许通常需要两端的测量 点样本采集，合并多个视网膜的RGC，或两者兼而有之;模糊人群的局限性 异质性和个体细胞动力学。我们将描述能量和Ca 2+的基线异质性 内稳态，沿着轴突损伤后动力学，并将这些测量与RGC直接相关 生存或死亡内稳态机制与一系列退行性疾病高度相关， 在RGC变性模型中还有待于彻底研究。我们的初步数据显示， 天然存活的视神经损伤的RGC具有显著的能量和Ca 2+稳态特征， 在诱导退化之前，应与研资局的整体人口区分开来。这些结果强烈 表明在严重变性模型中，稳态设定点影响RGC存活结果。此外，本发明还 我们将进行实验，通过操纵这些通路， 使用基因过表达或抑制干预来模拟弹性RGC的特性。我们这样做 可以验证我们的观察是相关的还是因果的。因此，我们建议的目标是：更多 彻底定义存活良好的RGC的稳态指纹;确定轴突切断术如何诱导 退化影响了存活良好与存活不良的RGC的稳态;并将其转化为 将信息转化为干预措施，以保护否则会退化的RGC。综合我们的 实验将确定和验证保护RGC的新方法。

ABSTRACT Retinal ganglion cells (RGCs) are the sole connection between the eye and the brain. They are particularly susceptible to degeneration, and their damage and death leads to vision loss in conditions like glaucoma, diabetic retinopathy, optic nerve glioma, and optic neuritis. Most treatments for these diseases are not focused on specifically rescuing RGCs, but on relieving apparent drivers of disease progression. For example, current glaucoma treatments focus on reducing elevated intraocular pressure (IOP), but are not effective in the majority of patients. Further, many glaucoma patients also have RGC degeneration without IOP elevations. Thus, new treatments to preserve RGCs in degenerative diseases represent an important unmet clinical need. Although RGC cell death leads to vision loss, RGC death in degenerative conditions is incomplete even in severely affected patients and robust animal models. Understanding how some RGCs natively persist in degenerative conditions can inform the development of new treatment strategies. To identify native coping strategies, we will directly observe cellular traits of individual RGCs prior to and during the course of degeneration, focusing on cellular homeostasis. We have established longitudinal, in vivo, 2-photon imaging of genetically encoded biosensors in RGCs to directly observe energetic and Ca2+ homeostasis at single RGC resolution repeatedly over a protracted period of time. This approach allows for measurements that would normally require either end point sample collection, pooling of RGCs from multiple retinae, or both; limitations that obscure population heterogeneity and individual cell dynamics. We will characterize baseline heterogeneity of energetic and Ca2+ homeostasis, along with dynamics following axon injury and directly relate these measurements with RGC survival or death. Mechanisms of homeostasis are highly relevant to a range of degenerative diseases but have yet to be thoroughly investigated in models of RGC degeneration. Our preliminary data indicate that mouse RGCs that natively survive optic nerve crush have salient features of energetic and Ca2+ homeostasis that can be distinguished from the RGC population as a whole prior to induction of degeneration. These results strongly suggest that homeostatic set-points influence RGC survival outcomes in a severe degeneration model. Further, we will conduct experiments to preserve RGCs in optic nerve crush models by manipulating these pathways to mimic the properties of resilient RGCs using both gene overexpression or repression interventions. Doing so we can validate which of our observations are correlative or causative. The goals of our proposal are thus to: more thoroughly define the homeostatic fingerprint of well surviving RGCs; determine how axotomy induced degeneration impinges on homeostasis of well-surviving versus poorly-surviving RGCs; and translate this information into interventions that preserve RGCs that would otherwise degenerate. Taken together our experiments will identify and validate new approaches towards protection of RGCs.