Basic Biology IV
$1 338 400
The project aims at investigating the contribution of MeCP2 to synaptogenesis. MeCP2 is a transcriptional regulator, attracted to methylated DNA. Mutations and duplications of MeCP2 are observed in a large spectrum of neurological disorders, including Rett syndrome (RTT), X-linked mental retardation, severe neonatal encephalopathy, schizophrenia and autism. We recently developed an induced pluripotent stem cell (iPSC) system for RTT and showed that MeCP2 is directly involved on the regulation of glutamatergic synapses in human neurons. The aims and experiments of this proposal were designed to understand the function of MeCP2 during development. Using an array of controls and MeCP2 mutant iPSCs lines, we will investigate how MeCP2 levels can contribute to the formation glutamatergic synapses. We will use genetic tools to allow the visualization and quantification of newly synthesized synapses. MeCP2 targets will be tested for causal effects based on a series of gene expression and proteomics experiments. Finally, we will measure the impact of non-neuronal cell types on the synaptic defects observed in RTT neurons. The data generated here will be useful to design novel therapeutic targets for several neurological disorders. The results of this research will have broad implication in ASDs, with direct consequences for the understanding of early stages of synaptic development in human neurons.
Statement of Benefit to California:
Mutations in the MeCP2 gene were linked to several mental disorders, including Rett syndrome (RTT), X-linked mental retardation, severe neonatal encephalopathy, schizophrenia and autism, affecting many Californian children. Because MeCP2 plays an important role in the pathogenesis of multiple disorders, the investigation of MeCP2 function and regulatory pathways in the cell may show promise for developing broad-spectrum therapies. In the absence of a functionally effective cure or early diagnostic tool, the cost of caring for patients with such diseases is high, in addition to a major personal and family impact since childhood. Our major goal is to understand the regulatory network of MeCP2 molecular interactions in human neurons. Our preliminary data strongly suggest that MeCP2 is an important factor for the formation of excitatory synapses during development. The proposed experiments will bring novel insights on the identification of new therapeutic targets, potential diagnostics for early detection of several diseases risk, or ability to predict severity of particular symptoms. In addition, the development of this type of validation approach in California will serve as an important proof of principle and stimulate the formation of businesses that seek to develop novel types of therapies in California with consequent economic benefit.
This application describes a proposal to investigate the contribution of the transcriptional regulator MeCP2 to synaptogenesis, the process by which neurons form new synaptic connections. Mutations and duplications of MeCP2 are observed in a large spectrum of neurological disorders, including Rett syndrome (RTT), X-linked mental retardation, severe neonatal encephalopathy, schizophrenia and autism. The applicant has recently developed an induced pluripotent stem cell (iPSC) system for RTT and has shown that MeCP2 is directly involved on the regulation of glutamatergic synapses in human neurons. The aims and experiments described in this proposal have been designed to elucidate the function of MeCP2 during development. In Aim 1, the applicant will investigate how MeCP2 levels can contribute to the formation glutamatergic synapses and uses genetic tools to allow the visualization and quantification of newly formed synapses. In Aim 2, MeCP2 targets will be tested for causal effects based on a series of gene expression and proteomics experiments. Finally, in Aim 3, the applicant will measure the impact of non-neuronal cell types on the synaptic defects observed with RTT neurons. Significance and Innovation - Rett syndrome is not a classic autistic disorder, but it offers a clean genetic system to model an autistic spectrum disorder, and the study may provide insight into other neurodevelopmental disorders. - Although a number of innovative approaches are proposed, not all of them are presented with sufficient preliminary data to judge their feasibility, and the rationale for using particular approaches was frequently unclear. - Reviewers were not convinced that the proposed approaches would significantly advance an understanding of mechanisms underlying synaptogenesis. Feasibility and Experimental Design - Reviewers expressed concern that the relatively poor control of neuronal differentiation and cell fate using the proposed protocols would yield a highly heterogeneous population of neurons that would severely limit the project’s feasibility. - Preliminary data did not convincing demonstrate the applicant’s ability to produce large numbers of layer-defined cortical neurons. - Reviewers viewed aim 1 as feasible and appreciated the inclusion of appropriate alternative approaches. - Aim 2 was not well focused and experiments were not adequately developed or described. Reviewers further questioned the quality and significance of the preliminary data supporting this aim. - Reviewers found Aim 3 confusing, with unclear and inadequately developed experimental design. Furthermore, this aim was not sufficiently supported by preliminary data, and issues of engraftment and immunosuppression were not adequately addressed. Principal Investigator (PI) and Research Team - The PI is an outstanding young investigator with expertise in iPSC-based disease modeling. - The PI has an outstanding funding and publication record, although he/she has yet to publish many papers as an independent investigator. Responsiveness to the RFA -This proposal is responsive to the RFA as it is focused on iPSC-based disease modeling and the analysis of basic cellular and molecular mechanisms.
- PROGRAMMATIC DISCUSSION
- A motion was made to move this application into Tier 1, Recommended for Funding. GWG members cited the need to expand research into autism spectrum disorders. The proposal’s scientific strengths and weaknesses were reiterated. The motion failed.