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

项目摘要 据估计，美国有300万人受到青光眼的影响，而且预期寿命正在增加 加剧了该疾病的社会经济影响。青光眼和其他视神经疾病导致永久性 视神经损伤和视网膜神经节细胞(RGC)丢失。目前还没有治疗方法可用于 减轻不可逆转的视力损失。细胞替代疗法的可行性最近被证明是通过 RGC是从发育中的视网膜分离出来的。此外，我们已经证明，有可能实现稳健和 干细胞来源的视网膜神经节细胞的可重复移植。而我们的供体视网膜节细胞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，结构和功能都很差 整合仍然是成功取代研资局的一项重大挑战。 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 在发育过程中引导体细胞间隔和树枝分枝的残留的嗜同性分子线索。 唐氏综合征细胞黏附分子(DSCAM)被认为是一种关键的分子信号通路 神经元自我回避，以防止在发育过程中视网膜中的束状物和保持马赛克间距。 我们假设这些相同的机制管理着移植的视网膜节细胞的整合，并且 包括DSCAM在内的亲水性分子信号限制了供体RGC向其自然方向的迁移 视网膜内的连接点。因此，这项建议的目的是在研资局的背景下调查DSCAM 移植以了解自我回避机制如何有助于神经回路的发育和 修理。 以RGC移植到视网膜作为模型系统，我们将确定DSCAM是否介导了自身的 回避机制是剂量介导的，依赖于跨细胞的相互作用，并且功能相似，与 神经迁移的结果。为了研究RGC自我回避的跨细胞DSCAM表达的必要性，我们将 使用获得和丧失功能(GOF和LOF)的小鼠进行一系列移植实验。 玻璃体内注射siRNA抑制小鼠干细胞来源RGC中DSCAM的表达 打针。同样，为了调查DSCAM是否独立于模式调节供体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 细胞骨架改变了它们在胞体易位和多极迁移之间的迁移方式。实时成像 并将使用定量免疫组织化学方法评估供体细胞在 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估 与宿主双极细胞形成突触。总之，这种机械化的方法将显著影响 青光眼和其他神经退行性疾病的细胞替代疗法的发展。

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.