Eric F. Schmidt - US grants

DESCRIPTION (provided by applicant): Understanding how axons respond to guidance cues to navigate through the developing embryo to connect with the proper target is essential to combat developmental nervous system disorders and regeneration. Sema3A is a common axon repellent which causes growth cone collapse and axon paralysis in numerous types of neurons. To date, not much is known about the intracellular mechanisms that cause cytoskeletal rearrangements necessary for collapse. The collapsing-response-mediator-protein (CRMP) family of cytosolic phosphoproteins is required for Sema3A-induced growth cone collapse. This proposal aims to determine the mechanism of CRMP function at molecular, biochemical, and functional levels of analysis. First, the solved crystal of CRMP1 will be used to generate a series of targeted mutant proteins. These proteins will be used to map functional domains on CRMP1 surface. Second, a constitutively active (CA) mutant CRMP1 that we developed will be used to identify novel CRMP1-binding proteins. Interactions between CRMP1 and putative interacting proteins will be characterized and relevance to Sema3A signaling will be determined. Finally, gain-of-function (via overexpression of CA CRMP1) and loss-of function (RNAi) experiments will be performed to assess the function of CRMP proteins in intact chick embryos. The proposed plan of research will elucidate the mechanism of CRMP action in Sema3A signal transduction at molecular, biochemical, and functional levels of analysis.

This INSPIRE award brings together research areas traditionally supported in the Division of Integrative Organismal Systems in the Directorate for Biological Sciences, in the Division of Chemical, Bioengineering,Environmental, and Transport Systems in the Directorate for Engineering, and in the Division of Behavior and Cognitive Sciences in the Directorate for Social, Behavioral and Economic Sciences. Cognition arises from the activity within complex brain circuits. These brain circuits are laid out according to a species' genetic blueprint. Past and current successes in uncovering the biological basis of cognition include the discovery of areas in the human brain supporting specific cognitive functions, the determination of the roles that specific cell types play in the behavior of animals like the mouse, and the increasingly detailed understanding of the impact specific genes and their alterations can have on cognitive functions in health and disease. The goal of this interdisciplinary project, conducted at The Rockefeller University, is to directly determine the genetic specificity of brain circuit elements that are critically important for high-level cognitive function. This project will be significant by 1) elucidating the complexity of biological organization from the level of genes, through cell types, brain areas, and neural circuits to behavior, 2) developing new technology that will allow researchers to dissect brain circuits underlying cognition with the precision and specificity of model organisms, and 3) improving the understanding of how genetic alterations impact cognition. The interdisciplinary project at the interface of cognitive neuroscience, neural systems, and neurotechnology, is expected to have broader impacts on society by providing insights into some of the deepest questions about the human mind and by offering unique educational and outreach opportunities to improve public understanding of the organization and function of the brain.

The project will investigate the genetic specificity of a multi-node brain circuit that supports cognitive function. The circuit will be localized with functional and structural magnetic brain imaging. Genetic expression patterns of projection neurons within multiple circuit nodes will then be determined using cutting edge molecular techniques. The functions of the projection neurons linking the nodes will then be determined through advanced and custom-designed optogenetic and electrophysiological techniques. The same optogenetic approach will then be used for causal interrogation of projection neurons in cognitive and emotional behaviors. Combining the gene expression and functional data, predictions for how specific polymorphisms in human genes may alter cognitive-emotional abilities will be generated. These predictions will be tested through functional brain imaging and behavioral testing of genotyped subjects. Together, these investigations will provide deep insights into the brain circuits and genetic underpinnings that make possible the cognitive functions of the human mind.

? DESCRIPTION (provided by applicant): Over 30,000 Americans currently suffer from amyotrophic lateral sclerosis (ALS) which is characterized by progressive paralysis due to the degeneration of nerve cells in the brain and spinal cord that control muscles. Almost all cases of ALS are eventually fatal and the rapid progression of the disease makes it particularly terrible, with over 80% of patients dying within five years of diagnosis. No cure exists for ALS and the only available treatment slows disease progression by merely a few months. Therefore a great need exists for more effective and specific therapies that can stop or even reverse neurodegeneration. Innovation for such therapies will only arise from a better understanding of the molecular mechanisms underlying the pathological process. The proposed study aims to identify molecules and pathways dysregulated during disease progression in specific cell populations in the cerebral cortex, including the vulnerable upper motor neurons (UMNs). Such an analysis has never been done before due to the complexity of cortical architecture hampering the ability to distinguish between cell populations. Genetic studies have linked a number of genes to ALS pathology, including SOD1, TDP43, and FUS, yet all of these genes are widely expressed in many cell types throughout the body while ALS afflicts only certain cells in the CNS. This project will utilize the novel translating ribosome affinity purification (TRAP) methodology to overcome these limitations by allowing for the examination of protein translation from genetically defined cell types. Engineered mice harboring the TRAP transgene (bacTRAP mice) in four cortical cell types (two populations of vulnerable UMNs, a non-vulnerable neuronal population, and astrocytes) will be crossed to three mouse models of ALS that utilize disease-linked mutations in the SOD1 (G93A), TDP43 (M337V), and FUS (P525L) genes. These models recapitulate the neurodegeneration seen in human patients and will enable a comprehensive assessment of cell-type specific molecular changes during ALS pathology. Changes in gene expression during disease progression will be determined by analyzing TRAP translational profiles at three time points (early, pre-symptomatic, and late) within each model. While this is a pre-clinical basic research project, efforts will be focused on identifying candidate genes that wil have the strongest and most immediate clinical impact. Particular emphasis will be placed on changes that occur at early and pre-symptomatic stages since earlier intervention will likely have an increased rate of success. These studies aim to improve upon the success rate of therapies arising from animal models by probing genes altered specifically in vulnerable cells across multiple models. Results from the proposed study will provide the field with a valuable resource of novel genes and signaling pathways to serve as candidate targets for more specific and innovative therapeutics to treat ALS.

SUMMARY There is clear evidence that local translation of proteins at distal regions of neurons is an important solution to the highly compartmentalized nature of these cells. Protein synthesis is well documented in dendrites where it serves to facilitate synaptogenesis and neuroplasticity at postsynaptic sites. However, less is known about local translation in axons. A growing body of work has shown that axonal protein synthesis is required for the development, maintenance, and plasticity of axons and presynaptic terminals and may underlie a number of neurodegenerative conditions. However, much of these studies have relied on cultured neuronal systems since axonal ribosomes are not abundant and the lack of reliable methods for isolating axonal compartments have made in vivo studies extremely challenging. The proposed study aims to develop a novel method that overcomes many of these obstacles to examine locally translated axonal mRNAs in distinct cell populations in vivo. This method is based on the translating ribosome affinity purification (TRAP) technique and will utilize viral vectors to deliver tagged ribosome proteins to the axons of genetically defined cell populations, allowing for the isolation of ribosome- associated transcripts. This viral strategy will then be used to perform a comprehensive comparative analysis of axonal translation across four distinct classes of projection neurons with distinct neurotransmitter profiles. Bioinformatics analysis will, for the first time, establish shared and cell type specific properties and regulation of axonal translated mRNAs. The development of this viral approach will provide the field with a simple and versatile method for profiling locally translated transcripts by axons in vivo and offer a strategy for measuring changes in axonal gene expression during development, plasticity, or neurodegenerative disorders.

The autism spectrum disorders (ASDs) are characterized by impairments of social and communicative behavior. The different, yet specific behavioral phenotypes of autism suggest impairments of specific neural circuits of the social brain. Yet, as genetic studies of autism implicate several hundred gene variants, it remains unclear how these genetic variants cause the behavioral phenotypes of autism. Several studies have implicated dysregulation of gene expression in the cerebral cortex in the pathophysiology of ASD. However, they do not address the specificity of cell types involved, how genetic changes alter brain function, or the involvement of functionally specific brain areas. Thus, we do not know whether and how they are altering social brain function selectively or what it is about social brain function that makes it particularly vulnerable in autism. In order to understand autism and its causes, we need to understand how genetic alterations cause the specific changes in the brain circuits that mediate the social and communicative behaviors altered in the condition. The current proposal aims to establish a new approach and a new model system to answer these questions. Using an animal model close to humans, gene expression patterns in functionally defined circuits of the social brain will be characterized. As in human functional magnetic resonance imaging (fMRI) studies, functionally specific regions of the social brain will be localized. This pilot proposal will focus on face-selective brain regions, but the overall approach, once established, will easily translate to other systems. The functional characterizations of the social brain will be complemented by the determination of the connectome of face areas through diffusion-weighted brain imaging. With this knowledge, long-range projection neurons within this functionally defined network will be labeled through a retrograde adeno-associated virus and cell-type specific gene expression patterns will be measured using the Translating Ribosome Affinity Purification (TRAP). The approach will allow for the determination of these expression patterns in glutamatergic cortical projection neurons located in the supra- and infra-granular cortical layers. These are the exact neurons which two recent studies have found to be highly correlated with ASD risk genes. Gene expression patterns of projection neurons will be compared in functionally defined social brain areas to known catalogs of autism-associated gene variations and pathways. The main expected outcome of this study will be the first determination of autism-risk gene expression patterns of functionally identified nodes of the social brain. The rationale of this study is that it will allow us to link autism risk genes to social brain circuits, advance the development of etiological models of autism, and provide crucial information for the generation of transgenic non-human primate autism models. In doing so, critical new links will be forged between the genetic analysis of ASD and functional imaging of brain function in ASD.