Project Summary/Abstract Cell replacement therapies are one of the best options for multifactorial diseases of the retina like aged related macular degeneration (AMD), which normally cannot be treated by correcting mutations in individual genes. The retina is also highly surgically accessible, allowing stem cell derived neurons to be precisely transplanted and tracked post-operatively. While transplant therapies into the retina are ongoing, including Phase I human trials, success has been limited due to the inability of photoreceptors (PRs) to integrate properly into complex existing circuits. One potential means to improve PR integration is to transplant genetically modified PRs that have enhanced ability to extend processes and form synapses. We recently found that stem cell derived PRs have a limited time they are capable of autonomous axon extension, which correlated with a loss of an organized actin cytoskeleton within their terminals and an upregulation of synaptic proteins (Rempel et al., 2022). This result suggests that PRs become immobile due to their decreased ability to polymerize and organize actin, which is essential for cell and growth cone motility. Therefore, identifying age- dependent molecular changes that account for the loss of actin filaments and decreased PR terminal motility would provide potential targets to potentiate intrinsic PR terminal motility. Transplanting more motile older PRs that also express synaptic proteins could greatly improve circuit integration. It also should be noted that even young human PRs only extend axons an average of 20 um in isolation, so methods to improve terminal motility at any age could help restore circuits in the adult retina. Transplantation of “motility enhanced” PRs could greatly improve their integration into existing circuits. Here we propose a series of innovative experiments to first test likely upstream modulators that may be lost as PRs mature within retinal organoids (ROs). Next, we will conduct an unbiased proteomic screen of protein expression changes within PRs across relevant time points. Candidates will be tested to determine if PR actin dynamics, terminal motility and axon extension can be enhanced by increasing the activity of key proteins that show age-dependent loss of expression.