Jonathan T. Ting, Ph.D. - US grants

DESCRIPTION (provided by applicant): The goal of this project is to determine the role of Synaptotagmin (Syt) IV in the control of mammalian synaptic transmission. The 15 identified Syts comprise a family of calcium (Ca) binding proteins that generally regulate membrane trafficking events. Syts I, II, and IV are found in synaptic vesicle membranes, which suggests that these isoforms are important for neurotransmission. Recent evidence reveals that Syts I and II are primary Ca sensors for neurotransmission, but there is little consensus on the role of Syt IV. The Syt IV protein is strongly elevated following seizures and has diminished Ca binding relative to Syt I and other isoforms. This proposal focuses on two Specific Aims to determine the function of Syt IV. Specific Aim 1 tests the hypothesis that Syt IV is a Ca sensor that is less effective than Syt I in promoting neurotransmitter release. Specific Aim 2 tests the hypothesis that elevation of Syt IV (as occurs following seizure) reduces neurotransmission. The experimental design will employ viral-mediated Syt IV expression and electrophysiological measurement of neurotransmission in cultured mammalian neurons. Evidence in support of these hypotheses would suggest a neuroprotective role for Syt IV following seizures.

PROJECT SUMMARY How the brain performs its computational task is a great unsolved problem in biology, but this answer is vital for us to understand and combat disorders of brain function like autism, schizophrenia, and Alzheimer?s. One appealing strategy towards solving this problem is to deconstruct the brain into the component parts?the cell types?and to determine their respective key features and dissect which physiological functions are sub served by each distinct type. Recently, advances in single cell RNA-sequencing technologies have catalyzed the identification of dozens of transcriptomically distinct cell types in the mammalian neocortex, many of which share homology between human and mouse, and it will likely soon be possible to have a complete transcriptomic taxonomy of neocortical cell types for multiple mammalian species. However, the ability to probe the function of most of these refined cell types is lacking, especially in human. As a result, the defining functional characteristics of human neocortical cell types remain largely unknown. In order to address this shortcoming, the current proposal lays out a comprehensive strategy to generate a first-in-class toolbox of cell type-specific genetic tools. Using recently developed methods to explore chromatin landscapes as well as Allen Institute-generated single-cell transcriptomics datasets, Aim 1 seeks to identify conserved neocortical cell type- and cell class-specific cis-regulatory modules across adult mouse and human. Candidate cis-regulatory modules near cell type- and cell class-specific marker genes identified in Aim 1 will be filtered for sequence conservation and accessibility conservation. In Aim 2, top candidates from Aim 1 will be used to generate cell class-specific viral vector libraries and screened for reporter expression in adult mouse neocortex, followed by secondary verification of individual on-target reporter vectors in adult mouse and human neocortex. In Aim 3 the most promising subset of these tools will be more deeply evaluated and validated using single cell RNA-seq and patch clamp electrophysiology on labeled cell populations in parallel for adult mouse and human neocortex. On the whole this project will generate a toolbox of novel reagents for the interrogation of neocortical cell type function that is compatible with diverse mammalian species, including mouse, monkey, and human. As such, these tools will not only enable direct cross-species functional comparisons of presumed orthologous neocortical cell types, but may also be suitable for human gene therapy applications. If successful, these tools will be of exceptional value and a welcome new resource for the neuroscience community.

Abstract: Many cell types together assemble the functional circuitry of the human brain. For over a century, neuroscientists have categorized brain cell types by their features, including shape, position, physiology, molecules, and function. Single cell transcriptomics studies are now defining molecular cell types at a resolution not previously possible, uncovering a taxonomy of hundreds to thousands of brain cell types. These studies have also revealed dramatic differences in molecular signatures of homologous cell types across species, showing decisively that the difference between mouse and human brain is not simply the total number of neurons. However, the function of each cell class or type in brain circuitry, and dysfunction in disease, is only beginning to be evaluated. To characterize the roles of human brain cell classes in normal function and disease, it is critical that tools be developed to allow genetic access to cell classes in vivo. Such tools would enable precise therapeutic gene delivery to brain cell classes, permitting targeted treatment for class-specific etiologies like some epilepsies. Few genetic tools are available to mark and manipulate cell classes and types in non-genetically tractable species like human and non-human primate (NHP). Viruses including adeno-associated viruses (AAVs), containing cell class and type selective enhancers can be leveraged to gain genetic access to, and drive gene expression in specific brain cell classes in these species. We have initiated a project through the BRAIN Initiative to generate and validate reporter AAVs to mark specific cell classes in the mouse cortex in vivo and in human neocortical tissue ex vivo. Our groups have engineered AAV vectors and optimized capsids to access neurons and express transgenes in many discrete cell classes and types in mouse and primate. New and improved AAV tools promise to fuel human brain scientific discovery and clinical progress, but one impediment has been the costly and time-consuming process of validating new vectors in primates. We present three Aims to translate these promising new AAV vectors into a high-value set of primate-optimized tools that could eventually be used for gene therapies in humans. First, we will develop a platform for screening AAV vectors in NHP ex vivo brain slices, followed by individual validation of promising vectors in NHP in vivo and human ex vivo brain slice cultures. Second, we will identify optimal AAV capsids to: a) support widespread NHP neuronal transduction in vivo when applied intravenously or to cerebrospinal fluid (CSF), two preferred routes of delivery for human CNS gene therapy, and b) support AAV transduction of human primary brain tissue ex vivo. Third, we will perform proof-of-concept experiments using cell class-selective vectors to express a therapeutic transgene in defined classes to treat a severe and intractable form of childhood epilepsy called Dravet syndrome (DS). These experiments represent a significant step towards converting cell class-selective AAVs into first-in-class viral tools optimized for in vivo NHP brain studies and human gene therapy applications.