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

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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.