Samuel Pleasure - US grants

The overall goal of this research plan is to elucidate how the hippocampal and dentate neuroepithelium is converted from a simple epithelial layer to the complex adult structure. The development of the dentate gyrus is particularly unique because it involves the formation, during development, of an ectopic germinative zone that persists and continues to generate new neurons throughout adulthood in rodents and primates (including humans). There is increasing evidence that the dentate gyrus is dysgenic in a subset of patients with temporal lobe epilepsy. Thus, the study of the normal mechanisms of dentate gyrus development, and the relationship of these mechanisms to dysgenic anomalies, will be critically important to our understanding and management of patients in this group. While the neuroanatomic outline of dentate gyrus development is fairly well understood the mechanistic underpinnings of this process are virtually unknown. The primary hypothesis of the proposed research is that development of the dentate neuroepithelium and the dentate gyrus is regulated by a complex cascade of bHLH transcription factors that act as intrinsic cues controlling differentiation of dentate granule cells from their most primitive precursors. The secondary hypothesis is that wnts produced by the cortical hem serve as extrinsic cues that regulate dentate gyrus and granule cell development by acting through cell-cell signaling mechanisms. The specific aims of the proposed research are: l) to characterize the function of the bHLH protein Mash1 in the production of dentate gyrus precursor cells and the development of dentate granule cells; 2) to identify the function of the normal cascade of bHLH proteins in the terminal differentiation of dentate granule cells and the organization of the dentate gyrus;.3) to determine the function of wnts in formation of the dentate gyrus. Experimental design and methods to accomplish the first two of these aims include the use of genetically modified mouse lines, in utero intraventricular retroviral injections and histologic analysis to characterize the function of Mash1 and other bHLH proteins in dentate gyrus development. The final aim consists of expression analysis of wnts, wnt receptors and wnt-inhibitory proteins and the use of cortical explants to understand the function of wnts in hippocampal development. The experiments described in the research plan should allow a detailed account of some intrinsic and extrinsic cues that control the cell fate of dentate granule cells and, hopefully, on the developmental basis of dentate gyrus dysgenesis in humans.

DESCRIPTION (provided by applicant): Structural hippocampal and neocortical abnormalities underlie many cases of pediatric and adult epilepsy and these structures are required for learning and memory. Subtle defects of hippocampal development are also associated with mental retardation, autism, spectrum disorders and schizophrenia. To understand these disorders it is necessary to improve our basic understanding of the regulation of cortical development. Recent studies show that members of the Wnt family of secreted factors are important in the development of this hippocampus. The hippocampus forms from the neuroepithelium adjacent to a midline organizing structure called the cortical hem. The cortical hem secretes many putative morphogenic factors, including a variety of Wnts. Wnt signaling has been demonstrated to regulate hippocampal development, as several lines of mutant mice with defects in Wnt signaling have severe defects in hippocampal development. My primary hypothesis is that Wnt signaling components, including Wnt receptors and effectors control, the morphogenesis of the dentate gyrus by controlling proliferation and cell fate of granule cell neurons. My secondary hypothesis is that these signaling components also regulate a variety of developmental processes in other regions of the hippocampus and neocortex. These hypotheses will be addressed by three specific aims - (1) Regulation of dentate granule cell production by Wnt/beta-catenin signaling; (2) Wnt signaling functions in neocortical development; (3) Determine the functions of Frizzled9 and 10 in the developing cortex. The methodologies used to pursue these aims include the analysis of LRP6 mutant mice, the production and study of LRP6/Lefl double mutant mice, the production of transgenic mice misexpressing dominant-negative LRP6 throughout the cortex, targeting of two Wnt receptors expressed in the early hippocampus to produce mutant mice for Frizzled 9 and 10 and the generation of transgenic mice misexpressing Frizzled9 throughout the cortex. These experiments should produce a more full understanding of the processes regulated by Wnts in the developing hippocampus and neocortex.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): Structural hippocampal and neocortical abnormalities underlie many cases of epilepsy and these structures are required for learning and memory. More subtle defects of hippocampal development are also associated with mental retardation, autism spectrum disorders and schizophrenia and most recently in affective disorders. To understand these disorders it is necessary to improve our basic understanding of the regulation of cortical development. Recent studies show that members of the Wnt family of secreted factors are important in the development of the hippocampus. The hippocampus forms from the neuroepithelium adjacent to a midline organizing structure called the cortical hem. The cortical hem secretes many putative morphogenic factors including a variety of Wnts. Wnt signaling has been demonstrated to regulate hippocampal development, as several lines of mutant mice with defects in Wnt signaling have severe defects in hippocampal development. My primary hypothesis is that Wnt signaling components, including Wnt receptors and effectors control the morphogenesis of the dentate gyrus by controlling proliferation and cell fate of granule cell neurons. My secondary hypothesis is that these signaling components also regulate a variety of developmental processes in other regions of the hippocampus and neocortex. These hypotheses will be addressed by 3 specific aims - 1) Regulation of dentate granule cell production by Wnt/B-catenin signaling; 2) Wnt signaling functions in neocortical development; 3) Determine the functions of Frizzled9 and 10 in the developing cortex. This proposal represents a continuation of Career Development as an independent scientist. Salary support for these activities is sought in conjunction with an already funded award for these studies but this salary support will cover a large portion of the applicant's salary providing the freedom to pursue the scientific goals of the proposal without excess clinical burden. A detailed career development plan is included as a part of the proposal, as is departmental assurance of support in these activities.

DESCRIPTION (provided by applicant): During the entire course of cortical development neuroepithelial stem cells and later the RG maintain constant cellular contact with meningeal cells lining the surface of the developing cortex. Meningeal fibroblasts generate the basement membrane (BM) covering the cortex, and it has long been known that destruction of the meninges or the BM has substantial consequences for cortical development. We have uncovered evidence that defects in the meningeal developmental program caused by mutation of a transcriptional regulator of meningeal development have major consequences for cortical development by disrupting neuronal progenitor proliferation. Our identification of major consequences for cortical neurogenesis due to meningeal defects has led us to develop the main hypothesis of this proposal - that the meninges are a signaling center producing soluble ligands regulating the onset of neocortical neurogenesis. I will address this idea in two aims designed first to characterize the signaling roles of the meninges and second to examine the complex development of the meninges: Aim 1: Examine the signaling role of the meninges on neocortical neurogenesis. One of the most exciting phenotypes in the Foxc1 mutant mice is the apparent failure of neuroepithelial progenitors to properly regulate cell cycle exit and switch to producing neurons. These findings are consistent with the idea that the meninges control the onset and progression of neocortical neurogenesis. We have also now identified the probable meningeally produced factor responsible for the neurogenic switch, and I propose experiments to further establish this finding. I will also proceed with our plan of identifying additional secreted meningeally produced factors that might be responsible additional signaling roles of the meninges. Aim 1a: Characterize the developmental anatomic basis of the Foxc1 mutant neurogenic phenotype. Aim 1b: Determine whether Retinoic Acid (RA), produced by the cortical meninges, regulates the onset and progression of cortical neurogenesis by acting as a diffusible signal. Aim 1c: Use a candidate approach and expression profiling to identify additional meningeally produced factors that regulate cortical neurogenesis. Aim 2: Characterize the differentiation program of the embryonic cortical meninges. Our identification of novel signaling roles for the meninges suggests that further examination of the details how its signaling capacity appears would be of importance. The cortical meninges are formed by a migration of cranial neural crest cells to cover the cortex at about E10. Previous studies have not characterized the developmental events that control this complex migration, or even the details of embryonic meningeal cell phenotype. We have now identified at least four distinct cell types that contribute to the cortical meninges at E12.5, at least two of which are responsible for producing specific secreted signals controlling cortical development (Cxcl12 and RA). This aim will characterize the cellular development of the meninges and will determine whether, as previously proposed, all layers of the cortical meninges develop from a primitive group of meningeal fibroblasts, or whether distinct subsets of these cells migrate in separate streams from cranial neural crest. In addition, we will characterize the developmental function of Foxc1 in the meninges in regulating these events. PUBLIC HEALTH RELEVANCE: Understanding the mechanisms governing the development of the cerebral cortex is one of the central goals of developmental neurobiology and it is abundantly clear that there are a host of human conditions that are impacted by defects in cortical development, including epilepsy, mental retardation, schizophrenia and autism. In addition, in recent years it has become apparent that cortical development and function are also related to many of the things that make us most human - emotion, drive, impulsivity, etc. Despite the fundamental importance of cortical development there remain major questions about the key regulatory events that shape the formation of the brain. In this proposal we explore a novel hypothesis strongly based in our preliminary data. We test the proposition that the meningeal coverings of the brain play a fundamental role in the development of the cortex, controlling many aspects of cortical progenitor behavior by the release of soluble factors that signal to the developing cortex.

[unreadable] DESCRIPTION (provided by applicant): The dentate gyrus and subventricular zone are permissive areas for neurogenesis in the mammalian brain, while most other areas are not. Much emphasis is being placed on finding the set of cues in these environments that differ between permissive and non-permissive settings. As these factors are elucidated it is becoming clear that many signaling molecules active in neurogenic zones are also present in non-permissive areas, particularly after injury. Why then do neural precursor cells respond by making neurons when exposed to some regions while not others? While there are a number of intracellular pathways that are known to modulate the efficiency and ability of neural precursors to produce neurons, there has been little consideration as to whether manipulation of any of these pathways are able to overcome nonpermissive environments in vitro or in vivo. In this proposal we will address the hypothesis that neural precursor cells can overcome non- permissive environments by manipulation of the basic pathways that control cellular fate specification. Specific Aim 1: Define the mechanism(s) underlying directed differentiation of neural precursor cells into neurons or oligodendrocytes in stem cell growth conditions in vitro. Forced expression of Neurogenins (Ngn) or Mash1 drive neuronal differentiation and Sox10 drives oligodendrocyte differentiation within 24 hours without any change in growth conditions. Our preliminary data supports the hypothesis that these manipulations drive multipotential NPC to become fate-restricted precursors. In this aim we will test the mechanistic basis of this process through several sets of experiments: 1) Does directed differentiation with transcription factors change the rate or extent of differentiation when infected cells are exposed to differentiating conditions? 2) Does expression of Ngn, Mash1 or Sox10 instruct differentiation toward the appropriate fate (neurons for Ngn and Mash1 and oligodendrocytes for Sox10) and restrict differentiation into alternative fates? 3) Are the effects of expression of these factors reversible? 4) Is there synergism between neurogenic factors and Pax6? Between Sox10 and Oligs? 5) What is the basis for morphologic differences seen in neurons produced by forced expression of Mash1 compared to Ngn? These experiments are designed to acquire a further understanding behind the mechanism of forced differentiation of neural precursor cells. Specific Aim 2: Determine if directed differentiation overcomes non-permissive conditions in vivo. We will engineer precursors to express Ngn1, Ngn2, Mash1 or Sox10 and transplant them into two conditions. First, we will inject them intraventricularly into the late embryonic mouse brain to test the behavior of these cells in a normally permissive environment. Second, we will inject the cells into the adult mouse striatum to test the behavior of these cells in less permissive conditions for neuronal or oligodendrocyte differentiation. Neural precursor cells present in the already formed brain present an exciting opportunity to promote regenerative repair of the nervous system either by harnessing the potential of endogenous precursors to expand and produce neurons or oligodendrocytes or by transplantation of dividing precursors that then respond to the injured environment to effect repair. If we are able to harness the regenerative capacity of these cells it will be possible to consider stem cell based treatments for multiple sclerosis, neurotrauma or neurodegenerative diseases. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cajal-Retzius (CR) cells and GABAergic interneurons are involved in cortical organization during development and brain function postnatally. Both are born in remote germinative zones and migrate tangentially to adopt their cortical locations. CR cells and many interneurons migrate to cover the cortex in the most superficial layer of the cortex - the Marginal Zone (MZ), adjacent to the meninges. The other primary migratory route of interneurons is in the deeper Intermediate Zone (IZ). The molecular cues that regulate this migratory organization are beginning to be elucidated but there is evidence that important regulators remain to be found. We have found that SDF1 has crucial roles both in organizing the laminar organization of tangential migration and in retaining MZ position of CR cells and interneurons during corticogenesis. This proposal will examine the role of SDF1 and other ligands coupled to the same signaling pathway in three aims. 1) Assess how SDF1 regulates distribution of tangentially migrating neurons in the cortex. 2) Evaluate the postnatal consequences of prenatal disruption of SDF1 signaling. 3) Determine the role of Gi-coupled intracellular signaling in tangential neuronal migration. Relevance to Public Health Many patients with epilepsy, mental retardation and autism have evidence their clinical dysfunction results in part from developmental cortical disorganization. In fact, it is estimated that 15% of refractory epilepsy patients have developmental defects as the cause of their syndrome. This proposal will elucidate mechanisms important in understanding the molecular control of cell migration during cortical development and will establish novel animal models of cortical disorganization affecting groups of cells known to be involved in human cortical malformation pathology.

DESCRIPTION (provided by applicant): We've developed extensive preliminary data showing that Sonic Hedgehog (Shh), produced by the hair follicles dorsal to the cortex, diffuses from the cells of origin to act within the brain to regulate the development of the hippocampal dentate gyrus and the migration of oligodendrocyte progenitors within the cortex at late embryonic and early postnatal stages. Using antibodies to extracellular Shh we are able to show that the amount of Shh reaching the developing cortex is reduced in mice with conditional ablation of Shh in the hair follicles and skin and that there is reduced signaling in transgenic signaling reporter mice in the brain. This indicates that the functional amounts of ligand reaching the cortex are reduced in these mice. These studies suggest the possibility that other protein ligands, if produced in the skin and hair follicles at high enough levels, would have access to the later embryonic and early postnatal developing brain, allowing us to circumvent some of the hurdles inherent in gene therapy or cell transplantation targeted to the cortex. In this proposal I will test this hypothesis and begin to lay the groundwork for determining the viability of this approach in humans. There are several difficult to treat disorders manifesting themselves around birth that may ultimately be amenable to this route of treatment. One particularly important syndrome is perinatal hypoxic-ischemic brain injury. Newborns with this syndrome have decreased oxygen supply due to events surrounding traumatic birth leading to significant injury including death of immature neurons and glial precursors. Recent studies have identified promising parenteral therapies in animals that are moving into human trials, but these face the possibility of causing increased off-target effects and morbidities because of the need to give these protein based agents intravenously. If they could be delivered locally and diffuse from the hair follicles and this might allow more targeted treatment with reduced morbidity. In addition, there are a host of genetic metabolic diseases that present with catastrophic consequences soon after birth due to enzyme deficiencies or other genetic lesions. Thus, I have developed this proposal to test the prospects for this approach.

DESCRIPTION (provided by applicant): The discovery of neural stem/progenitor cells and persistent neurogenesis in restricted brain regions has ignited an unprecedented interest in developing cell restoration therapies, which are designed to repair the malfunctional cells and/or replace the defunct cells in the nervous system in the case of diseases or injuries. Conceivably, we can achieve these either by coaching endogenous stem/progenitor cells for self-repair/self-replenishment, or by transplanting exogenous stem/progenitor cells to differentiate in a defined manner to restore defective or lost cells. However, the successful utilization of neural stem/progenitor cells in either case is contingent on their ability to behave and function in vivo n a biologically meaningful way without causing adverse effects. Many studies in recent years indicate that the fate and behavior of stem/progenitor cells are governed by the local microenvironment, termed the niche. Therefore, a better understanding of the interactions between the stem/progenitor cells and their niche is a critical step toward effective stem cell therapies. We hope to elucidate the molecular cues mediating interactions between stem/progenitor cells and their niche during development and to encourage use of this knowledge to develop novel approaches for treating neural injuries and neurodegenerative diseases through the following two aims. Aim #1: Examine the contribution of the ventral hippocampal ventricular zone to the production of subgranular NSCs throughout the hippocampus. Aim #2: Characterize the roles of various sources of Shh in SGZ development.

? DESCRIPTION (provided by applicant): In preliminary studies we found that mice lacking Sufu in the cortex after E10.5 have an almost complete failure to generate superficial cortical projection neurons but more intact production of deep layer cortical neurons. Preliminary analysis of these mutants demonstrates that progenitors for superficial neurons that appear during corticogenesis progressively lose their appropriate phenotype and instead take on the characteristics of ventral forebrain progenitors and oligodendrocyte precursors. Remarkably, deletion of Sufu at a slightly later age (E13.5) has essentially no effect on production of superficial neurons indicating a temporally sharp role for Sufu in controlling progenitor diversification and cell fate of neurons. Interestingly, when we examined mice with loss of Sufu at E13.5 there was a marked acceleration of the production of oligodendrocyte precursor cells at late embryonic and early postnatal stages in the cortex. These preliminary results have led us to propose a novel primary hypothesis, that controlled restriction of Shh signaling in the early cortical ventricular zone (between E10.5 and E13.5) is required for diversification of neocortical neurons and allows orderly progression from neurogenesis to oligogenesis. Further, our secondary hypothesis is that regulation of Shh signaling in the cortex also has profound effects on the production of oligodendrocyte precursors during cortical development. The aims below will test these hypotheses and examine the cellular and molecular mechanisms governing these phenotypes. Aim 1: Determine how Shh signaling blocks production of superficial cortical neurons. Aim 2: Identify roles of Shh signaling in controlling the postnatal neurogenesis and the production of oligodendrocyte precursors in the cortex. The studies proposed here will provide major new insights into two important questions - 1) What is the role of Shh signaling in the developing forebrain and 2) How is the orderly progression of production of deep layer neurons, then superficial layer neurons then glial cells controlled. This is of great importance to the fiel and also of major relevance to understanding the developmental underpinning of neuropsychiatric disease.

? DESCRIPTION (provided by applicant): In preliminary studies we found that mice lacking Sufu in the cortex after E10.5 have an almost complete failure to generate superficial cortical projection neurons but more intact production of deep layer cortical neurons. Preliminary analysis of these mutants demonstrates that progenitors for superficial neurons that appear during corticogenesis progressively lose their appropriate phenotype and instead take on the characteristics of ventral forebrain progenitors and oligodendrocyte precursors. Remarkably, deletion of Sufu at a slightly later age (E13.5) has essentially no effect on production of superficial neurons indicating a temporally sharp role for Sufu in controlling progenitor diversification and cell fate of neurons. Interestingly, when we examined mice with loss of Sufu at E13.5 there was a marked acceleration of the production of oligodendrocyte precursor cells at late embryonic and early postnatal stages in the cortex. These preliminary results have led us to propose a novel primary hypothesis, that controlled restriction of Shh signaling in the early cortical ventricular zone (between E10.5 and E13.5) is required for diversification of neocortical neurons and allows orderly progression from neurogenesis to oligogenesis. Further, our secondary hypothesis is that regulation of Shh signaling in the cortex also has profound effects on the production of oligodendrocyte precursors during cortical development. The aims below will test these hypotheses and examine the cellular and molecular mechanisms governing these phenotypes. Aim 1: Determine how Shh signaling blocks production of superficial cortical neurons. Aim 2: Identify roles of Shh signaling in controlling the postnatal neurogenesis and the production of oligodendrocyte precursors in the cortex. The studies proposed here will provide major new insights into two important questions - 1) What is the role of Shh signaling in the developing forebrain and 2) How is the orderly progression of production of deep layer neurons, then superficial layer neurons then glial cells controlled. This is of great importance to the fiel and also of major relevance to understanding the developmental underpinning of neuropsychiatric disease.

The corpus callosum (CC) is the largest commissural projection connecting homotopic regions of the hemispheres in mammals. The connectivity of the CC is essential for coordinated motor-sensory function and for many higher cognitive processes. The CC is also a valuable model system for understanding circuit development as well as possible mechanisms underlying disorders of circuit form and function such as psychosis, autism and epilepsy. We have become interested in the role of NMDA receptors (the chief excitatory neurotransmitter receptor in the cortex) in cortical circuit development and maintenance. NMDAR are located at synapses and are known to be critical for circuit plasticity in many contexts and are also known to interact with guidance cues, such as the Eph Receptors (EphR) during development. Also, dysfunction of NMDAR caused by anti-NMDAR antibodies has achieved recent prominence as a model for psychosis. Patients with anti- NMDAR antibody encephalitis present with psychosis, abnormal movements and seizures. The clinical features of this syndrome, including the subacute onset and slow recovery after treatment suggest functional disruption at the level of circuit integrity rather than simple pharmacologic antagonism. We are examining the role of NMDAR in development of somatosensory projections of the CC. We find that Emx1cre/+; NR1fl/fl mice, lacking NMDAR specifically in the cortex after E10.5, have callosal defects in primary somatosensory cortex (S1) and that this is associated with alterations in EphB2 expression. Our analysis shows defects of both initial innervation and subsequent refinement. Also, intraventricular injections of anti-NMDAR antibodies, similar to pathogenic patient antibodies, cause similar defects in the innervation of S1. We propose, that the function of the NMDAR is necessary for homotopic callosal projections, that NMDAR have specific roles in shaping the callosal circuitry by affecting axon pathfinding and/or axon pruning in the developing cortex and that continued NMDAR function is critical for morphologic maintenance of the circuit. The aims below will test these propositions and examine potential molecular mechanisms. Aim1: Determine the anatomic and temporal roles of NMDAR in callosal development. Aim2: Characterize the role of NR2A and NR2B subunits in callosal development. Aim3: Identify interactions between NMDAR and EphrinB/EphB signaling in callosal development.

Project Summary/Abstract This is an application to support a successful training program for students admitted to the UCSF Neuroscience Graduate Program. The program, which dates back to 1977, is designed to provide a highly rigorous training in both the theory and practice of experimental neuroscience, statistical methodologies, quantitative skills development, data analysis and management, and scientific communication. Accomplishing this mission requires an organizational framework dedicated to addressing the challenges inherent in modern neuroscience training. To this end, we have recently overhauled our training program to adapt to our rapidly evolving field. The first year curriculum is devoted to a core course focused on the fundamentals of modern neuroscience, followed by a course in the responsible conduct of science. In addition, students perform 3 laboratory rotations and meet regularly with their graduate advisor to discuss career goals, culminating with entry into a thesis lab at the end of the first year. In year two, students begin their research, take advanced courses, including courses covering scientific writing, experimental design, and data analysis. They also prepare for qualifying examinations. Years three and beyond are focused on developing, conducting, completing, and publishing an independent research project in their thesis laboratory. Students meet 2 times a year with their thesis committee and determine, along with their PI, when enough progress has been made to graduate. Students then prepare and submit a written thesis and give a public exit seminar with all committee members present, with the PhD degree being granted upon the unanimous approval of the thesis committee. The Neuroscience Program currently has 62 training faculty across 14 departments, centers, and affiliated institutes. Program membership is periodically reviewed, and is restricted to faculty who commit to mentorship and teaching, participate in program activities, and maintain a rigorous research program. Virtually all areas of neuroscience are encompassed by the research interests of our faculty. The trainees attend a weekly seminar series and weekly student-faculty research-in-progress seminars, as well as an annual program retreat. In the past, the T32 training grant has been the single most important pillar underlying our program?s success over the past 40 years; it was used to support students during their first and second years of study before they have advanced to candidacy and initiated full-time Ph.D. thesis research. We enroll approximately 15-18 students per year, of whom approximately half would be supported by this training grant during their first two years. Our primary objective is to train a diverse student body in the concepts and methods of modern neuroscience by imparting the skills, knowledge, and leadership skills needed to succeed across a range of neuroscience-related careers. NIH support is essential for us to continue to provide graduate students with the education they will need to become future leaders in their field.

The dentate gyrus (DG) is one of two brain regions acknowledged to sustain neural stem cells (NSCs) continuously producing neurons (termed ?neurogenesis?) beyond development. Newborn neurons produced in the DG are involved in hippocampal-dependent learning and memory. Thus, factors regulating establishment of the NSC pool during development and their life-long maintenance are crucial for hippocampal function. The fate of NSCs is governed by local microenvironmental factors, including neural circuit activity. Hyperexcitation in the hippocampus caused by epileptic seizures aberrantly increases neurogenesis in the adult DG, leading to consumption of many NSCs and results in exhaustion of the NSC pool. Despite accumulating evidence that neuronal activity regulates NSCs dynamics, still little is known about the responsible niche cells and signaling molecules connecting neural activity and NSC dynamics. Similar to the adult DG, NSCs in the developing DG may also be influenced by activity, but whether NSCs are regulated by early neural activity during circuit establishment in the developing DG has not been directly addressed. In the previous funding cycle for this grant, we found that Shh is a key niche signal for the initial production of specialized NSCs populating the DG and for their postnatal expansion to establish the size of the NSC pool for later adult neurogenesis. In more recent preliminary data we have found that Shh signaling is upregulated after seizures in the adult DG and that seizure-induced aberrant neurogenesis is attenuated in Shh deficient mice. We previously showed that Shh is produced from excitatory neurons in the dentate hilus (mossy cells) but have now extended this to show that mossy cell activity enhances neurogenesis. On this basis we have formulated the hypothesis (for Aim 1) that Shh derived from mossy cells is crucial for neuronal activity induced DG neurogenesis. We have also investigated the neuronal circuit in the developing DG and found that the entorhinal cortex projection to the developing DG is established by the first postnatal week coinciding with the appearance of quiescent NSCs in the DG. We have also found that NSCs receive direct inputs from local neurons in the developing DG in this same period. Based on these preliminary results we formulated a second hypothesis (for Aim 2) that development of cortex-dentate-NSCs circuits and their activity control the proliferation state and transition to quiescence of NSCs in the developing DG.

Inflammatory and infectious causes of encephalitis affect 20,000 people a year in the US. More than 50% of patients have inflammatory disorders requiring prolonged treatment with expensive and dangerous biological or immunosuppressive agents. The most common identifiable causes of these syndromes are paraneoplastic, post-infectious or associated with other underlying autoimmune diseases; however, the majority of patients are still classified as idiopathic. In the last 1-2 decades many of these syndromes have been shown to be associated with anti-neural autoantibodies that either cause the pathophysiology or serve as critical biomarkers. The most widely cited examples of these are anti-Hu antibodies, which are highly correlated with an underlying cancer, and anti-NMDA receptor antibodies, which cause psychosis, seizures and memory disturbances. Over the past 5 years, a unique interdisciplinary team of neurologists and basic scientists at UCSF was formed to develop and deploy an integrated approach to rapidly identify anti-neural antibodies associated with encephalitis, with the explicit intent to discover and validate clinically actionable biomarkers in addition to uncovering the fundamental mechanisms of disease pathogenesis underlying these syndromes. The centerpiece of these efforts is a patient cohort being collected at UCSF called the NID (Neuroinflammatory Disease) cohort, consisting of patients with suspected infectious or inflammatory encephalitis. This cohort is now >1,200 patients referred by clinicians at UCSF and from other centers around the world. Already, this cohort has yielded a new paraneoplastic autoimmune syndrome with important implications for men with seminoma. More importantly, we have reason to believe this to be just the tip of the iceberg. Our preliminary data indicates that at least 20% of these patients have associated high titer antibodies in their CSF reactive to neural antigens in mouse brain. Here, we propose to pursue the hypothesis that a plurality of undiscovered autoimmune targets underlie a significant fraction of idiopathic encephalitis cases. Using our unique clinical and molecular approach and our world class patient collection, we will investigate this hypothesis through the following specific aims: Aim 1: To stratify and characterize the UCSF 1,200 patient NID cohort for the presence of anti-neural antibodies in CSF. Aim 2: High-throughput phage display screen to identify and validate auto antigens. Aim 3: Produce recombinant human antibodies from patients with autoantibody syndromes and develop animal models to test autoantibody pathogenicity.

Project Summary/Abstract Project Summary for diversity supplement PA-21-071 is described in the research plan.

PROJECT ABSTRACT COVID-19 is associated with a growing number of peripheral and central nervous system complications. It has become clear that a subset of these syndromes, including acute necrotizing encephalopathy, steroid- responsive encephalitis and Guillain-Barré syndrome, are likely due autoimmunity triggered by SARS-CoV-2 infection. There is an urgent need to prospectively investigate the acute and chronic neurologic complications of COVID-19 and determine which syndromes are neuroinflammatory in origin ? particularly those caused by para- infectious autoimmunity. While anti-viral therapeutics are still being developed for SARS-CoV-2, autoimmune CNS conditions can be very responsive to immunosuppression. Thus, identifying biomarkers for a subset of COVID-19 patients with autoimmune CNS syndromes could immediately impact clinical management. Over the past 7 years, a unique interdisciplinary team of neurologists and basic scientists at UCSF was formed to develop and deploy an integrated approach to rapidly anti-neural antibodies associated with encephalitis, with the explicit intent to discover and validate clinically actionable biomarkers in addition to uncovering the fundamental mechanisms of disease pathogenesis underlying these syndromes. The centerpiece of these efforts is an ongoing patient cohort called the NID (Neuroinflammatory Disease) cohort, consisting of patients with suspected infectious or inflammatory encephalitis. This cohort is now >1,500 patients referred by clinicians at UCSF and from other centers around the world. Already, this cohort has spurred the development of the first ever clinically validated cerebrospinal fluid metagenomic next-generation sequencing assay, the identification of a novel paraneoplastic autoimmune syndrome with important implications for men with seminoma and the identification of enteroviral CSF antibodies in children with acute flaccid myelitis. Here, we propose to adapt this existing clinical research and laboratory infrastructure to enroll and investigate the urgent question whether COVID-19 patients with ongoing neurologic sequelae have CNS inflammation. We will perform this work in collaboration with colleagues at the NIH, Yale University as well as at UCSF Medical Center, Zuckerberg San Francisco General Hospital and UCSF Benioff Children's Hospital. We seek emergency funding to accelerate our work on identifying anti- neural autoantibodies associated with SARS-CoV-2 infection in patients with neurologic syndromes in 1 immediate aim. Aim 1: Identify autoantibodies in the CSF of COVID-19 patients with neurologic syndromes.