Regulating cell-cell interactions to improve donor retinal ganglion cell integration

项目摘要 据估计，美国有300万人患有青光眼， 加剧了疾病的社会经济影响。青光眼和其他视神经病变导致永久性 视神经损伤和视网膜神经节细胞（RGC）损失。目前没有治疗方法可用于 减轻不可逆转的视力丧失。细胞替代疗法的可行性最近得到了证实， 从发育中的视网膜分离的RGC。此外，我们已经表明，有可能实现鲁棒性和 干细胞衍生的RGCs的可重复移植。虽然我们的供体RGC在宿主视网膜中存活， 移植后，细胞存活并不等同于视力的恢复，而且结构和功能较差 要成功取代研资局，整合工作仍是一项重大挑战。 限制供体RGC整合到现有电路中的关键分子特征之一可能是 在发育过程中指导体细胞间隔和树突分支的退化的嗜同性分子线索。 唐氏综合症细胞粘附分子（DSCAM）已被确定为介导 神经元的自我回避，以防止成束，并在发育过程中保持视网膜中的马赛克间距。 我们假设这些相同的机制支配移植的RGCs的整合， 包括DSCAM在内的嗜同性分子线索限制了供体RGC向其天然受体的迁移。 连接视网膜内的点。因此，本建议旨在研资局的背景下研究DSCAM 移植，以了解自我回避机制如何有助于神经回路的发展， 修复. 使用RGC移植到视网膜作为模型系统，我们将确定是否DSCAM介导的自我， 回避机制是剂量介导的，依赖于跨细胞相互作用，并且功能相似， 神经迁移。为了研究跨细胞DSCAM表达对RGC自我回避的需要，我们将 使用功能获得和丧失（GOF和LOF）小鼠进行一系列移植实验。 在玻璃体内注射前用siRNA抑制小鼠干细胞来源的RGC表达DSCAM 注射剂类似地，为了研究DSCAM是否独立于DCAM的模式调节供体RGC间距， 迁移，我们将抑制宿主和供体RGC中的DSCAM，同时暂时破坏供体RGC的稳定 细胞骨架改变其迁移方式之间的染色体易位和多极迁移。实时成像 和定量免疫组织化学将用于评估供体细胞形态和分布， 视网膜。将使用顺行追踪和在多电极阵列上培养的视网膜外植体来评价 与宿主双极细胞形成突触。总而言之，这种机械的方法将大大影响 开发用于青光眼和其他神经退行性疾病的细胞替代疗法。

Project Summary An estimated 3 million people are affected by glaucoma in the United States, and increasing life expectancy exacerbates the disease’s socio-economic impact. Glaucoma and other optic neuropathies lead to permanent damage of the optic nerve and loss of retinal ganglion cells (RGCs). No therapies are currently available to mitigate irreversible vision loss. The feasibility of cell replacement therapy was recently demonstrated using RGCs isolated from a developing retina. Furthermore, we have shown that it is possible to achieve robust and reproducible transplants with stem cell-derived RGCs. While our donor RGCs survived in host retinas following transplantation, cell survival does not equate to the restoration of vision, and poor structural and functional integration remains a significant challenge for successful RGC replacement. One of the key molecular features limiting donor RGC integration into the existing circuitry is likely to be the vestigial homophilic molecular cues that guide somatic spacing and dendritic arborization during development. Down Syndrome Cell Adhesion Molecule (DSCAM) has been identified as a key molecular cue that mediates neuronal self-avoidance to prevent fasciculation and preserve mosaic spacing in the retina during development. We hypothesize that these same mechanisms govern the integration of transplanted RGCs and that homophilic molecular cues, including DSCAM, limit donor RGC migration towards their natural connecting points within the retina. Therefore, this proposal aims to investigate DSCAM in the context of RGC transplantation to understand how self-avoidance mechanisms contribute to neural circuit development and repair. Using RGC transplantation into the retina as a model system, we will determine if DSCAM-mediated self- avoidance mechanisms are dose-mediated, rely on transcellular interactions, and function similarly irrespective of neural migration. To investigate the need for transcellular DSCAM expression for RGC self-avoidance, we will conduct a series of transplantation experiments using gain- and lose-of-function (GOF and LOF) mice. Expression of DSCAM by mouse stem cell-derived RGC will be suppressed with siRNA before intravitreal injections. Similarly, to investigate if DSCAM regulates donor RGC spacing independent of the mode of migration, we will suppress DSCAM in host and donor RGCs while temporarily destabilizing the donor RGC’s cytoskeleton to alter their migratory modality between somal translocation and multipolar migration. Live imaging and quantitative immunohistochemistry will be used to assess donor cell morphology and distribution in the retina. Anterograde tracing and retinal explants cultured on multielectrode arrays will be used to evaluate synapse formation with host bipolar cells. Altogether, this mechanistic approach would significantly impact the development of cell replacement therapy for glaucoma and other neurodegenerative diseases.