DESCRIPTION (provided by applicant): The overarching goal of this application is to develop a model system to evaluate the impact of candidate drugs on human neurons in vivo. Thus, we propose to transplant CDKL5-mutant iPSC-derived neurons into a mouse brain and expose the animal with candidate drugs to evaluate their impact on fully mature human neurons. Taking advantage of our expertise to develop chimeric mouse brains, this model will allow us to perform follow up studies in vivo, to accommodate both molecular and cellular studies at different time-points and to facilitate new drug screening methods. The approach proposed here can also be used to study neuronal networks and, mainly, the effects of brain microenvironment on human neurons. We will strategically use CDKL5-mutant iPSC-derived neurons as a proof of concept considering the patients' early disease onset and its clear morphological phenotype in vitro. Mutations in the CDKL5 gene have been connected to X-linked neurological developmental disorders, including severe mental retardation and early-onset seizures most often seen in the first few months of age. In vitro studies showed that CDKL5 is required for correct genesis/maintenance of dendritic spines and also for synapse formation. Transplanting CDKL5-mutant iPSC-derived NPCs into a mouse brain will allow us to understand the neural formation/maturation of human neurons, investigate their behavior in vivo, and give unparalleled insight into disease progression. More broadly, using this chimeric brain strategy we will further evaluate the effect of lead compounds on the compromised neurons in an environment closer to that one found in patients. Human chimeric mice brains could ultimately serve as a more predictive pre- clinical translational model for studying disease mechanisms. Thus, our specific Aims are: Aim 1 - To transplant human CDKL5-mutant NPCs into a mouse brain and characterize differentiated neurons in vivo; and Aim 2 - To determine the impact of candidate drugs on human CDKL5-mutant neurons in the chimeric brain model. Our main goal is to develop a model to study the development of human neurons for long periods of time in an in vivo setting. The strategy also allows us to evaluate how candidate drugs can affect live human neurons integrated in a mouse brain. Here we propose a new approach to study human neurons in vivo. Such strategy will serve as a proof-of-principle for testing new therapeutic drugs in a more holistic and reliable differentiation system. We truly believe that our efforts will deepy impact and push the field to the next-level.

PUBLIC HEALTH RELEVANCE: The overarching goal of this application is to develop a model system to evaluate the impact of candidate drugs on human neurons in vivo. The approach proposed here can also be used to study neuronal networks and the effects of candidate therapeutical drugs on human neurons.