A. Kimberly McAllister - US grants

The mammalian visual system requires the proper formation of exquisitely precise circuits to function correctly. These neuronal circuits are assembled during development by the formation of synaptic connections between thousandsof differentiating neurons. Although the formationof glutamatergic synapses is critical for the proper function of the visual system, little is knownabout how these synapses are formed. Recent work has begun to identify some of the early cellular events in synapse formation as well as the molecular signals that initiatethis process. Despite the wealth of information published on this topic in the past few years, some of the most fundamental questions about synapse formation remain unanswered. In particular, the basic mechanisms of transport of synaptic proteins in axons and dendrites before synapse formation havejust begun to be identified. How these mechanisms are altered and regulated during the accumulation of synaptic protein at new mammaliansynapses remains a mystery. Definingthese basic mechanisms of transport and their regulation is critical to understand how synaptogenic signals might alter and direct transport of synaptic proteins to new synapses. The central goal of this proposal is to investigate the molecular mechanisms of the transport and recruitment of synaptic proteins to new synapses between visual cortical neurons. We propose to address these issues directly by combiningtechniques that allow us to visualize and focally manipulatethe transport and recruitment of fluorescently-tagged proteins during synapse formation between dissociated, cultured visual cortical neurons. The specific aims of this proposal are: (1) to identify intracellular signaling pathways that regulate the transport of synaptic vesicle precursors, (2) to identify intracellular signaling pathways that regulate the transport of NMDA receptors, and (3) to determine how transport of synaptic proteins is altered in response to synaptogenic signals. Results from these experiments will be essential for a comprehensive understanding of the cellular and molecular mechanisms underlying the development of the visual cortex. These results will also provide insight into the mechanisms responsible for amblyopia, as well as possible approaches to therapy. More generally, defects in synapse formation are likely to cause many neurodevelopmentaldisorders[unreadable]frommental retardation, to autism, to schizophrenia. Understanding the cellular and molecular mechanismsof synapse formation could revolutionize our ability to identify,prevent, and treat these developmental disorders.

DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) is a complex disease that appears to be caused by a combination of genetic changes and an environmental insult during very early development. Increasing evidence suggests that several such environmental insults work through mechanisms that converge to chronically activate cytokines during fetal and early postnatal brain development. The development of a mouse model for maternal infection has added strong support for a link between maternal immune activation (MIA) and some of the pathology characteristic of ASD. The most compelling MIA mouse model-PolyI:C injection-produces abnormalities in gene expression, neuroanatomy, neurochemistry, and behavioral changes that are similar to those in autistic individuals. Current evidence indicates that these effects of MIA on fetal brain development are mediated by cytokines that cross the placenta. Although little is known about how maternal cytokines alter fetal brain development, the most likely possibility that we will test in this proposal is that MIA leads to chronic neuroinflammation that alters brain development and behavior and may contribute to the symptoms and pathology of ASD. Although cytokines are most often thought of as mediating an immune response, these small proteins and their receptors are found in the healthy, developing CNS where they play important roles in neural development and plasticity. Since cytokines mediate the immune response, are present in the developing cortex, and alter cortical plasticity and connectivity, factors that alter cytokines in the developing brain may lead to changes in synaptic connectivity that contribute to ASD. The goals of this proposal are to define the full complement of cytokines that are changed in specific regions of the CNS of offspring at various postnatal ages following MIA and to determine the effects of those cytokines on cortical neuron connectivity and function. Using the PolyI:C mouse model of MIA, Luminex multiplex cytokine arrays, immunocyto- and histochemistry, cell and slice culture, and whole-cell patch-clamp recording, we will address the following Aims. (1) Define the nature and time-course of changes in cytokines n four distinct brain regions of offspring following MIA. (2) Test combinations of altered cytokines on synaptic connectivity and function during early postnatal development. Results from this proposal will provide insight into the mechanism underlying the effects of MIA on brain development and function and will potentially identify novel targets for therapeutic intervention in ASD. PUBLIC HEALTH RELEVANCE: Although a wide range of environmental stimuli have been proposed to play a role in the pathogenesis of autism spectrum disorders, many of these stimuli have in common the ability to alter immune function. Since cytokines mediate the immune response, are present in the developing brain, and regulate cortical plasticity and connectivity, factors that alter cytokines during development may lead to changes in synaptic connectivity that contribute to ASD. The goals of this proposal are to define the full complement of cytokines that are changed in specific regions of the CNS of offspring at various postnatal ages following MIA and to determine the effects of those cytokines on connectivity and function in several cortical regions at the relevant postnatal ages.

DESCRIPTION (provided by applicant): Although there has been evidence for cross-talk between the immune and nervous systems for years, the idea that immune molecules have non-immune functions in the normal developing nervous system has only recently gained credence. This idea is based on observations that the primary mediators of the adaptive immune response, major histocompatibility complex class I (MHCI) molecules, are expressed in the brain where they mediate activity-dependent refinement of connections. MHCI molecules are also attractive candidates for molecular mediators of the effects of an abnormal or strong immune response on the developing brain, which has been implicated in the pathogenesis of several neurodevelopmental disorders, including autism and schizophrenia. The central goal of this proposal is to determine the function of MHCI during the initial establishment of cortical connections, a comparable developmental stage to the time of the immune challenge that may predispose humans toward neurodevelopmental disorders. Using immunocyto- and histochemistry, electron microscopy, cell and slice culture and transfection, a novel long-term imaging assay, and whole-cell patch-clamp recording, we will address the following Aims. (1) To determine the localization of MHCI molecules in vivo and in vitro during cortical development. (2) To test the hypothesis that MHCI molecules regulate the initial establishment and function of cortical connections in vivo and in vitro. (3) To determine whether MHCI molecules negatively regulate the establishment of cortical connections by inhibiting synapse formation, increasing synapse elimination, or both. (4) To test the hypothesis that MHCI molecules act through natural killer cell receptors to negatively regulate the establishment of cortical connections. Results from this proposal should reveal novel functions of MHCI in the typically developing brain, as well as potential mechanisms for how they might contribute to neurodevelopmental disorders.

SUMMARY: PROJECT 1 Despite its prevalence and enormous cost to society, current treatments for schizophrenia (SZ) are not effective in all individuals and do little to treat the disabling cognitive and negative symptoms. Thus, there is a pressing need to identify new molecular pathways to target in developing new compounds and tools for earlier diagnosis and treatment of SZ. Our Center is focused on testing the hypothesis that immune molecules may constitute such a target pathway. Immune genes located within the major histocompatibility complex (MHC) have been reliability identified in recent genome-wide association studies, while maternal infections are among the best-established environmental risk factors for the illness. The development of mouse and non-human primate (NHP) models of maternal infection has also strengthened the link between maternal immune activation (MIA) and SZ because these models recapitulate many SZ related phenotypes at both the neurobiological and behavioral levels. Although our work is providing increasingly compelling validation for their efficacy in mimicking core phenotypes of SZ, little is known about how MIA alters brain development after birth to cause SZ-related phenotypes in offspring. This project will determine how and when MIA alters neural connectivity during postnatal development and whether those changes depend on MIA-induced changes in MHCI levels in the brains of offspring. To accomplish these objectives, this project will undertake three specific aims to determine: 1) if MIA alters synapse density and type in the brains of offspring of the mouse and NHP models throughout postnatal development and in postmortem brain tissue from individuals with SZ using approaches that include an innovative technique called array tomography; 2) if MIA alters microglial activation and levels of immune molecules, including MHCI, in the brains of offspring in both animal models throughout development as well as in postmortem brain tissue from individuals with SZ; 3) if changes in immune molecules in the brains of offspring from the mouse model mediate the effects of MIA by assessing whether restoring levels of neuronal MHCI back to normal prevents MIA-induced changes in synaptic connectivity and SZ-like behaviors in offspring. By accomplishing these aims, Project 1 will directly address the central hypothesis of the Center in a mechanistic manner. Moreover, performing these experiments across scales will identify which MIA-induced changes in synaptic connectivity are relevant to human disease through their conservation in the NHP model. Results from this project are essential to the success of the other projects in the Center in providing a phenotypic read-out for changes in transcriptional networks (Project 2) and in structural and functional connectivity (Project 3), as well as critical information about whether microglial activation mediates changes in neural inflammation (Project 4) in MIA models and humans with SZ. If successful, results from this project will identify novel targets for developing more effective diagnostic tools and therapies for SZ and other psychiatric illnesses with a neural-immune basis.

Project Summary/Abstract. This is a new application to support advanced predoctoral training in Learning, Memory, and Plasticity (LaMP) at UC Davis. LaMP research at UC Davis is both broad and deep, spanning 4 schools and colleges and all of the major levels of research?cognitive, systems, cellular/molecular, and disease. The 31 Trainers are highly collaborative and value interdisciplinary approaches to their research. We have designed an innovative Program to train the next generation of neuroscientists to bridge the gaps between the major levels of LaMP research?cognitive, systems, cellular/molecular?in order to promote our understanding of LaMP disorders and reduce their tremendous burden on families, society, and public health. Our program proposes to support 6 advanced predoctoral Trainees per year, a size essential for creating a group that crosses traditional disciplinary boundaries, spans levels of analysis, and addresses both basic and translational research. Trainees from 6 participating graduate groups will be eligible to apply to the LaMP Training Program in Year 2 (and Year 3, under exceptional circumstances) of their graduate training. The proposed 2-year Training Program has 6 components. (1) Trainees will conduct research in at least one of the LaMP faculty labs and will be co-mentored by another trainer from a distinct area of LaMP research. (2) Trainees will be exposed to the essential and emerging concepts in each of the fields of LaMP through a 2-quarter core course consisting of interactive lectures as well as lab-based immersion to give trainees the tools to understand the pros and cons of both the concepts and methods used at each level of analysis. (3) Trainees will also be exposed to LaMP disorders through a course consisting of didactic and interview/video sessions that span clinical, cognitive, systems and molecular aspects of each disease. (4) Trainees will receive targeted career development training through a meeting every other week with Program Trainers?emphasizing critical and cross-disciplinary thinking, presentation skills, formal discussion of research-related careers outside of academia, and pressing issues in the field such as data analysis, ethics, and rigor and reproducibility. (5) Trainees will receive extensive training in grant writing, followed by a mock study section prior to submission of their NRSA in the first quarter of their second year of funding. (6) Trainees will participate in an annual program retreat and attend an invited LaMP speaker series. Through these elements, this new Training Program will produce a new generation of scientists who truly think across levels and scales and who have the skills, drive, and motivation to work collaboratively to tackle the most important issues in learning, memory, and plasticity in order to improve human health.

The Planning Grants for Engineering Research Centers competition was run as a pilot solicitation within the ERC program. Planning grants are not required as part of the full ERC competition, but intended to build capacity among teams to plan for convergent, center-scale engineering research.

San Jose State University and University of California, Davis are partnering with Lawrence Livermore National Labs to plan for a potential Engineering Research Center in Cognitive Neuroengineering. Cognitive NeuroEngineering is the next step in the evolution of human interactions with machines. Today, machines are poised to make this important leap: from interfacing with our peripheral nervous system using sight, touch, and sound to interfacing directly with our central nervous system to usher in a world in which everyone will command computers, household machines and other assistive devices simply by thinking about what they want to do. It is in our nation?s interest to be at the forefront of this new engineering discipline and to addresses the cognitive implications of directly interfacing machines with the central nervous system. To promote the progress of science and ensure the nation's health, prosperity and welfare we will develop a diverse team that can effectively address the mission of the proposed ERC and create a Center that has the greatest opportunity for socioeconomic impact. To accomplish this, this project will engage a broad range of stakeholders, from scientist and engineers to clinicians and end-users to improve opportunities for better understanding and trust between these communities. In addition, the specialized knowledge that each community brings will be leveraged to create synergistic partnerships that advances the scientific, educational, and ethical aspects of this new field. Recommendations for national workforce development and any changes to our regulatory environment will be made. There are three major expected benefits of this proposal. The first is to optimize diversity of the stakeholder community to maximize the potential societal benefit of this new field of Cogntive Neuroengineering. The second is improved team dynamics to empower stakeholders to contribute regardless of status and power differences. The third is more effective leadership by utilizing evidence based research in team science.

Just as Human Factors Engineering has grown into Cognitive Engineering, Cognitive NeuroEngineering goes beyond current neuroprosthetic applications and addresses the cognitive implications of directly interfacing machines with the central nervous system. This potential ERC represents one of the most important new challenges of the future of work at the human-technology frontier. Using team-based science, this ERC will embrace convergent research and address the complexity of interfacing with the central nervous system and its impact on cognition. Novel data analytic approaches will be developed, a greater understanding of human cognition will be achieved and innovative neurotechnologies will be created that are expected to impact society beyond the goals of the proposed ERC. To develop the proposed ERC in Cognitive Neuroengineering, this planning grant will be used to 1) enable a series of planning meetings that culminate in three workshops, 2) travel between the partner institutions and to relevant stakeholder communities for first hand exchanges of partner resources and 3) engage a profession facilitator in strategic planning as well as two experts, one in convergent science and the other in the team science. The three workshops revolve around our four Objectives. The first Objective is to identify strengths and weaknesses of our ERC. The second Objective is to identify opportunities for deployment of our proposed engineering system and opportunities for diversity. The third Objective is to fully develop the team and stakeholder community using the latest techniques of team science, including improving our ability to articulate the societal impacts. The fourth Objective is to write the ERC proposal. This planning grant will have the greatest impact on the formulation of a set of strategic objectives and metrics for our ERC. This will allow us to consider carefully and more deeply understand the societal impact of our ERC. Since groups are more likely to engage when they identify with the societal impact of a project, this will improve stakeholder engagement. In addition, the activities are expected to develop new ways of thinking to ease the significant communication and philosophical difference among stakeholders and facilitate the transdisciplinary nature of the ERC whose constituents have significant communication and philosophical differences.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

SUMMARY A lack of understanding of causal etiology is a major barrier to diagnosis and treatment of neurodevelopmental disorders (NDD) such as autism and schizophrenia. This is especially true for the connection between maternal infection and brain disorders, where epidemiological studies have shown that a variety of immune challenges during pregnancy are associated with increased rates of NDD in exposed offspring. Rodent models of maternal infection recapitulate behavioral abnormalities, enabling investigation of the mechanisms underlying pathology. Studies using compounds such as poly(I:C), which mimics viral dsRNA, have shown that maternal immune activation (MIA) alone can produce parallel behavioral, anatomical, and synaptic pathology in exposed offspring even without pathogen exposure. These findings suggest that maternal immune signaling factors directly or indirectly impact fetal brain development. Identifying the root causal pathways activated by MIA and the downstream impact of these acute changes on early brain development will provide insight into prevention or intervention of root causes to ameliorate downstream pathology and lifelong decreases in quality of life. We hypothesized that these root changes would be evident in the transcriptomes of the fetal cerebral cortex after MIA exposure. In preliminary studies, we generated a time course map of neurodevelopmental gene expression changes in a mouse model of poly(I:C) challenge in mid-gestation. These preliminary systems-level transcriptomics data reveal a sequence of neurodevelopmental pathology following MIA, including novel findings indicating that an initial stress response mediates changes in neural progenitor cell behavior and brain structure. We identified a strong acute signature present by 6hrs after MIA that persists over the initial days after exposure. These acute changes are followed by changes in genes associated with proliferative dynamics and neuronal differentiation and, finally, with changes in the level of known cell-identity markers. Here we propose to validate and define how the observed gene expression signatures are associated with anatomical, structural, and cellular changes in the developing and postnatal mouse brain after MIA exposure. We will use the same poly(I:C) model and examine the same time points profiled via transcriptomics, using a combination of histology, functional and structural assays, and cell-specific analysis towards the objectives of 1) identifying the cell-specific and anatomical roots of the acute response to MIA, 2) characterizing changes in identity and proliferative behavior in neuronal progenitors and differentiating neurons, and 3) linking developmental expression signatures to neuroanatomical changes in postnatal brain. These experiments will generate significant insights into generalized pathological mechanisms of MIA and will provide the basis for future in-depth study of the specific causal pathways activated in MIA- exposed fetal brain that we will validate in this work. The results from our studies will additionally seed future translational work focused on developing novel targeted pharmacological interventions.