Ricardo E. Dolmetsch - US grants

DESCRIPTION (provided by applicant): Voltage-gated calcium (Ca2+) channels play a central role in neuronal function and are essential for converting electrical activity into biochemical events. The main goal of this proposal is to identify the molecular mechanisms by which voltage-gated calcium channels activate signaling cascades that mediate gene expression and promote neuronal survival. Neurons and muscle cells express at least ten different kinds of voltage-gated calcium channels that vary in their subcellular localization and biophysical properties. L-type channels (LTCs) are particularly effective at activating transcriptional pathways and at promoting neuronal survival. The transcription factors CREB and MEF-2 are two important targets of LTC signaling that regulate differentiation and plasticity in the nervous system. The biophysical and biochemical features that allow LTCs to activate gene expression and suppress apoptosis are not well understood. To address the question of how LTCs are linked to signaling pathways the following specific aims are proposed: 1) To determine the structural and biophysical features that allow L-type calcium channels to activate transcription. 2) To determine whether specific L-type channel interacting proteins link the channel to signaling pathways that lead to the activation of transcription. 3) To determine what features of L-type calcium channels allow them to inhibit neuronal apoptosis and promote survival. Biochemical, cell biological and electrophysiological techniques will be used to develop these specific aims. We have recently developed a method of using dihydropyridine insensitive LTCs to investigate LTC signaling in primary neurons and we plan to use this approach for these studies. We have also identified several LTC-interacting proteins that may be important for channel regulation and signaling and we plan to investigate their importance for signaling to the nucleus. The results of these experiments will provide critical insights into how voltage-gated channels activate the signaling pathways that regulate the structure and function of the nervous system.

Psychiatric diseases are devastating and poorly understood. The goal of this project is to [unreadable] identify the cell biological phenotypes that underlie autism and other psychiatric[unreadable] disorders, taking advantage of new developments in human stem cell technology. I[unreadable] propose to harvest skin fibroblasts from patients with autism, to reprogram these cells to[unreadable] generate induced pluripotent stem (iPS) cells, and to differentiate the iPS cells into[unreadable] neurons. I will then use a set of semi-automated in vitro assays to develop a phenotypic[unreadable] fingerprint for each cell line focusing on the developmental and functional phenotypes[unreadable] that are likely to lead to autism. We will use automated microscopes, cell sorters and[unreadable] semi-automated patch clamps to measure the differentiation, migration, survival,[unreadable] morphology, and excitability of neurons derived from patients. Finally, we will take[unreadable] advantage of the genetic information available for some autistic patients to determine[unreadable] whether deletion, duplication, or mutation of specific genes leads the phenotypes[unreadable] observed in the neurons from that patient. This approach has the potential to[unreadable] revolutionize our study of autism and other psychiatric disorders. It will allow us to link[unreadable] the phenotype and genotype of patients with the cell biological defects that give rise to[unreadable] disease. In addition, it will allow us to develop cell-based assays to both investigate the[unreadable] etiology of the disease and to find new treatments for these untreatable disorders.

DESCRIPTION (provided by applicant): Autism spectrum disorders (ASD) are highly heritable complex neurodevelopmental disorders of the brain, which cannot be explained by mutation or mutations in any single gene. In the last couple of years linkage and association studies have led to the identification of several mutations that confer susceptibility to ASDs. Studying the functional effects of these mutations offers a unique window to a better understanding of the underlying neurobiology. One of the major obstacles is the difficulty in obtaining neurons and glial cells from patients with an ASD. The goal of this project is to develop the methods to convert skin cells from patients with ASDs into neurons and to characterize these neurons using high content screens. To achieve this goal we will convert fibroblasts into pluripotent progenitor (iPS) cells. In the next step we will differentiate these iPS cells into neurons in vitro. Finally we will study the specific cell- intrinsic aspects of neuronal function that are likely to be disrupted in ASDs including synapse formation, axonal and dendritic morphology and calcium signaling. We have already established all of these techniques in our laboratory. Before we can apply these techniques on a larger scale we need to first address some of their limitations. The focus of R21 phase of the proposal is to improve and standardize the methodology. We will first generate and characterize iPS cells from human fibroblasts harvested from healthy controls and ASD patients with mutations in the CACNA1C and SHANK3 gene, mutations known to affect neuronal development, and optimize and characterize the differentiation of iPS cells (Specific Aim 1). We will develop standardized protocols for differentiating iPS cells into mixed populations of cortical, dopaminergic, and inhibitory neurons (Specific Aim 2). We will then characterize the cellular phenotypes of neurons from ASD and from controls, focusing on calcium signaling, dendritic arborization, and cell survival (Specific Aim 3). In the R33 phase of the project we will target a larger number of individuals with ASD a known to have a mutation in others gene/s affecting neuronal development (Specific Aim 4 and 5). PUBLIC HEALTH RELEVANCE: Autism is considered to be among the most common of the serious developmental disabilities, second only to mental retardation. The lifetime per capita incremental societal cost is estimated at $3.2 million. Because the deficits in autism affect human- specific social behaviors, the mechanisms underlying autism will need to be studied in human patients and in cells. The goal of this project is to develop the methods to convert skin cells from patients with autism into neurons and to characterize these neurons using high content screens. These experiments will allow researchers to study the neurons of individuals diagnosed with autism and will lead to a better understanding of the development and differentiation of neurons.