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

调节细胞间相互作用以改善供体视网膜神经节细胞整合

• 批准号: 10389799
• 负责人: Jonathan R Soucy
• 金额: $ 6.99万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389799
• 关键词: Affect; Animals; Biological Models; Blindness; CDK5 gene; Cell Adhesion Molecules; Cell Communication; Cell Surface Receptors; Cell Survival; Cell Transplantation; Cells; Cellular Morphology; Cellular biology; Cerebrum; Colcemid; Cues; Cytoskeleton; Development; Disease; Dose; Down Syndrome; Down Syndrome Cell Adhesion Molecule; Electrophysiology (science); Event; Ganglion Cell Layer; Glaucoma; Harvest; Image; Immunohistochemistry; Investigation; Isogenic transplantation; Lead; Life Expectancy; Masks; Mediating; Modality; Molecular; Mosaicism; Mus; Muscle fasciculation; Natural regeneration; Neurodegenerative Disorders; Neurons; Optic Nerve; Outcome; Pattern; Peptide Hydrolases; Persons; Pharmaceutical Preparations; Population; Positioning Attribute; Process; Reproducibility; Research Proposals; Retina; Retinal Degeneration; Retinal Ganglion Cells; Role; Series; Signal Transduction; Small Interfering RNA; Spinal; Surface; System; Transplantation; Trisomy; United States; age related; cell motility; cell replacement therapy; economic impact; experimental study; improved; induced pluripotent stem cell; inhibitor; intravitreal injection; loss of function; loss of function mutation; migration; multi-electrode arrays; neural circuit; neuron loss; neuronal cell body; optic nerve disorder; preservation; prevent; receptor expression; relating to nervous system; repaired; retina transplantation; retinal damage; retinal neuron; retinogenesis; sight restoration; small molecule; socioeconomics; stem cell differentiation; stem cells; synaptogenesis; transcriptome

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.

项目概要 据估计，美国有 300 万人受到青光眼的影响，并且预期寿命不断延长 加剧了该疾病的社会经济影响。青光眼和其他视神经病变会导致永久性的 视神经损伤和视网膜神经节细胞（RGC）损失。目前尚无可用疗法 减轻不可逆的视力丧失。最近使用的方法证明了细胞替代疗法的可行性 从发育中的视网膜中分离出 RGC。此外，我们已经证明，实现稳健且可靠的目标是可能的。 使用干细胞衍生的 RGC 进行可重复的移植。虽然我们的供体 RGC 在宿主视网膜中存活下来，但 移植后，细胞存活不等于视力恢复，结构和功能较差 集成仍然是成功替代 RGC 的一个重大挑战。 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距和树突分枝的残余同质分子线索。 唐氏综合症细胞粘附分子 (DSCAM) 已被确定为介导唐氏综合症细胞粘附分子的关键分子线索 神经元自我回避，以防止发育过程中的束震并保留视网膜中的马赛克间距。 我们假设这些相同的机制控制着移植的 RGC 的整合，并且 同质分子线索，包括 DSCAM，限制供体 RGC 向其自然迁移 视网膜内的连接点。因此，本提案旨在在 RGC 的背景下研究 DSCAM 移植以了解自我回避机制如何促进神经回路的发育和 维修。 使用 RGC 移植到视网膜作为模型系统，我们将确定 DSCAM 是否介导自我调节 回避机制是剂量介导的，依赖于跨细胞相互作用，并且无论何种情况都具有相似的功能 的神经迁移。为了研究跨细胞 DSCAM 表达对 RGC 自我回避的需求，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。 玻璃体内注射前，小鼠干细胞来源的 RGC 的 DSCAM 表达将被 siRNA 抑制 注射。同样，为了研究 DSCAM 是否独立于 RGC 模式调节供体 RGC 间距 迁移时，我们将抑制宿主和供体 RGC 中的 DSCAM，同时暂时破坏供体 RGC 的稳定性 细胞骨架改变其在体细胞易位和多极迁移之间的迁移方式。实时成像 定量免疫组织化学将用于评估供体细胞的形态和分布 视网膜。顺行追踪和在多电极阵列上培养的视网膜外植体将用于评估 与宿主双极细胞形成突触。总而言之，这种机械方法将显着影响 开发针对青光眼和其他神经退行性疾病的细胞替代疗法。