Beth Stevens, Ph.D. - US grants

DESCRIPTION (provided by applicant): The formation of mature neural circuits requires selective pruning of inappropriate synapses and strengthening of appropriate synaptic connections. A longstanding question in neurobiology is what determines which synapses will be eliminated? While the role of spontaneous and experience-driven synaptic activity in developmental synaptic pruning is well established, surprisingly little is known about the molecules and mechanisms that link neural activity with the physical elimination of specific synapses. Our recent studies reveal that glial cells-microglia and astrocytes--are key players in developmental synaptic pruning. We discovered that C1q, the initiating protein of the classical complement cascade, is significantly upregulated in developing neurons by immature astrocytes. Both C1q and downstream C3 are moreover localized to synapses and are required for developmental synapse elimination in the visual system; however the mechanisms by which complement mediates synaptic pruning are completely unknown. The primary role of the complement cascade in the innate immune system is to opsonize or tag unwanted cells or debris for removal by phagocytic macrophages via specific complement receptors. Our preliminary studies support a model in which inappropriate synapses in the developing brain are similarly tagged by complement and then eliminated by microglia, the primary phagocytic cells in CNS. Given the importance of neural activity in synaptic pruning, a major goal of the proposed research is to determine whether and how the complement system cooperates with neuronal activity to give rise to precise visual circuit wiring. Activity-dependent competition between neighboring axons is thought to drive the elimination of weak synaptic inputs; however, the molecular mechanisms remain elusive. We propose a model in which activity and astrocyte-derived factor(s) act cooperatively to upregulate C1q in developing neurons which leads to local activation of the complement cascade at neighboring weak synapses and the elimination of complement (C3) tagged synapses by microglia. We will use the mouse retinogeniculate system as a model to manipulate neural activity at specific synapses in vivo to examine the role of microglia (Aim 1), C1q and C3 (Aim 2) and astrocytes (Aim 3) in activity-dependent synapse elimination. Specifically, we will ask: 1) Does complement specifically tag weak synapses for elimination? 2) Do microglia actively prune synapses or engulf synapses already undergoing elimination? 3) Does neuronal activity regulate the complement cascade, and if so, how? The answers to these questions will add to our understanding of the role of microglia and the complement cascade in developmental synapse elimination, and may ultimately provide new insight into how CNS synapses are eliminated during normal brain wiring, and possibly in diseases involving aberrant synapse loss and synaptic connectivity, such as epilepsy and autism.

Understanding how learning and memory take place in the brain is one of the major questions in neuroscience, with important benefits to health and disease, and practical applications in devising better ways of teaching that are science-based. Despite decades of research this remains one of the most challenging questions in brain science. Until recently, all research on learning and memory at the cellular level was focused on neurons, even though the majority of cells in the brain are not neurons. These non-neuronal cells, glia, do not generate electrical impulses, but new research reveals that glia can communicate chemically and that they can sense and control communication between neurons in several different ways. Glia are therefore likely to participate in learning and memory and to provide unique capabilities to information processing that may resolve some long-standing questions about cognition and learning that have been elusive. The workshop would synthesize the latest information on glia in learning and cognition and bring together researchers from traditionally separate disciplines from across a broad scope that includes cellular, systems-level, behavioral, and computational neuroscience, to begin to understand how glia may participate in learning. The two-and-a-half-day workshop would seek to outline future directions for research on glia in learning, with an emphasis on identifying how glia may contribute a new direction for research to solve long-standing problems in the field of learning.

The objectives of this workshop are at the cutting edge of several scientific disciplines, including cellular neuroscience and the rapidly expanding research on neuron-glia interactions, human brain imaging during learning, and forging new directions in neuronal modeling. Broader impacts include: The inclusion of women scientists and scientists from many nationalities will provide the opportunity for significant cross-fertilization of ideas between fields and pioneer new directions of research to explore novel solutions to aspects of learning that have been difficult to solve from an exclusively neuronal perspective. The plans for dissemination will make these new findings and new ideas available to a wide scientific audience through a review article published in a major scientific journal, a special issue of a journal and a book.

? DESCRIPTION (provided by applicant): Microglia, the brain's resident immune cells and phagocytes, are emerging as critical regulators of developing synaptic circuits in the healthy brain. Recent studies from our lab and others indicate that microglia engulf synapses in the developing brain; however, how microglia know which synapses to target remains a major open question. Our previous work demonstrates that microglia-mediated pruning underlies developmental synaptic refinement, an essential process required for the formation of mature circuits in which weak or inappropriate synapses are eliminated and remaining connections are maintained and strengthened. We found that microglial engulfment of presynaptic inputs is activity-dependent and driven by complement molecules C1q and C3, and microglial complement receptor CR3. These molecules are innate immune eat me signals known for promoting macrophage phagocytosis of apoptotic cells or debris, and mice lacking these signals exhibit reduced microglial engulfment of synaptic inputs and impaired refinement. This suggests that microglia-mediated pruning may be analogous to the removal of non-self material by phagocytes in the immune system. However, we do not yet know how microglia precisely determine which inputs to engulf and which to avoid, an important decision regarding the specificity needed to sculpt precise, mature connections. We propose that protective don't eat me signals are required to prevent inappropriate microglial engulfment of necessary connections during synaptic refinement, just as they prevent inappropriate engulfment of healthy self-cells by phagocytes during an immune response. Our preliminary data support this hypothesis, as don't eat me signals CD47 and SIRP? are present in the developing brain and required to prevent excess microglial engulfment of synaptic inputs. We will investigate the anatomical, functional, and behavioral abnormalities in mice lacking CD47 and SIRP? to better understand the consequences of excess microglial engulfment. We will also investigate whether and how these don't eat me signals are regulated by activity to determine if they direct microglia to engulf specific synapses in an activity-dependent manner. Finally, as don't eat me signals are known to be downregulated in the brains of patients with neurodegenerative diseases, we will examine whether these molecules are dysregulated in mouse models of Huntington's disease (HD) and could thereby underlie synapse loss caused by aberrant microglial engulfment. This study would be the first to demonstrate that synaptic protection is required to prevent inappropriate microglial engulfment of necessary connections during development. This research program will provide insight not only into the mechanisms regulating microglial engulfment of specific synapses, but also into possible mechanisms underlying synapse loss in CNS neurodegenerative diseases.

ABSTRACT It is becoming clear that autism spectrum disorder (ASD) likely occurs due to dysfunction of developing synapses and synaptic remodeling. Tuberous sclerosis complex (TSC) is a monogenetic disease with a high incidence of ASD. To obtain a deeper understanding of the underlying pathogenic mechanisms of ASD, we propose to take advantage of a TSC mouse model, which is missing the Tsc1 gene only in the cerebellar Purkinje cells (PCs). These conditional TSC mutant mice exhibit the common core characteristics of ASD: lack of interest in socializing, repetitive behaviors, and cognitive inflexibility. Importantly, Tsc1-deficient PCs display increased spine density, a phenotype previously reported in patients with neurodevelopmental disorders; however the neuronal and non- neuronal mechanisms that contribute this process remain elusive. In this project, we will investigate two complimentary mechanisms that contribute to the synaptic and behavioral phenotypes in this newly developed TSC mouse model of ASD. In Aim 1, we will test the hypothesis that impaired autophagy, driven by excess mTOR signaling, prevents normal synaptic remodeling and leads to the increased dendritic spine density on PCs, which contribute to the behavioral abnormalities found in the PC-Tsc1 CKO mice. We will characterize the rate of autophagy including autophagy of mitochondria (mitophagy), and modulate autophagy pharmacologically to test whether we can improve the spine and behavioral phenotypes. In Aim 2, we turn to cell-extrinsic mechanisms and ask whether the interaction between mutant PCs and microglia, resident immune cells and key mediators of synaptic remodeling, contributes to the spine and ASD-like phenotypes. We hypothesize that Tsc1-deficient Purkinje cells lead to early disruption in microglia development and function, including their ability to prune and signal to synapses. Moreover, our preliminary findings suggest that microglia activation and inflammatory signaling further contribute to synaptic and ASD like phenotypes. We are uniquely positioned to explore the spatio-temporal relationship of microglia changes relative to Tsc1-null PCs using a combination of novel transcriptional profiling (single cell Drop-Seq), and functional assays. We will perform the first detailed transcriptional analysis of microglia and neurons from TSC patients and compare these data with mouse models. Finally, we will determine whether specific manipulation of autophagy and microglia dysfunction in PC-TSC cKO mice rescue synaptic and specific ASD- relevant behaviors. We will leverage four IDDRC cores (Cellular Imaging, Molecular Genetics, Neurodevelopmental Behavior and Clinical Translational Cores) and complimentary expertise of co-PIs, Sahin and Stevens and IDDRC collaborators. Together, these experiments will shed light on the cell intrinsic and extrinsic mechanisms mediating synaptic modeling and may inform new therapeutic targets and biomarkers for TSC and related neurodevelopmental disorders.

The Administrative Core will act as an enabling-hub for the highly collaborative, synergistic scientific project cores, act to support deep, multidisciplinary training opportunities for our future scientists and to facilitate efficient, and effective dissemination of our work and resources. The core will provide the administrative and organizational resources to foster synergy both between the project cores and with existing departmental initiatives, support training and outreach, and planning and evaluation activities. The director of the center will be Beth Stevens and she work closely with CoPIs, McCarroll and Carroll to ensure the tasks of the center are efficiently delegated and executed in a coherent manner. The team will include a part time administrative assistant, who will assist with operational tasks and an Research Administrator who will be responsible for creating and updating website content, organizing and corresponding with SAB members and symposium speakers. In addition to augmenting interdisciplinary training, the administrative core will coordinate dissemination and outreach work to both to the scientific community and the public, including establishing and maintaining a website, a biannual interdisciplinary scientific symposium (spanning genetics, immunology, and developmental neuroscience) and a biannual meeting for educators and clinicians on youth mental health. The latter will bring together middle school and high-school teachers with care providers including school counselors, mental health social workers, nurses, child & adolescent psychiatrists, and students of these disciplines for a series of talks, workshops and discussion forums on brain development and psychiatric disorder. Finally, the core will spearhead the creation and operation of our platform for resource sharing.

Brain imaging in schizophrenia patients reveals excessive loss of gray matter, already visible in young adults at the first psychotic episode. Post mortem brains from schizophrenia (SCZ) patients have decreased numbers of synapses in the prefrontal cortex (PFC), a region involved in executive function and working memory. However, it is not known whether synapse loss results from excessive developmental pruning or why schizophrenia often becomes clinically apparent during adolescence. Dendritic spine density on layer 3 pyramidal neurons is dramatically reduced in SCZ patients, suggesting a synapse and circuit- specific mechanism of vulnerability. As the PFC integrates information from multiple brain regions, defects in pruning could significantly impact synaptic connectivity, cognitive function, and behavior. Our goal is to map the development and refinement of PFC synapses (Aim 1) and to interrogate the functional and behavioral consequences of local and global defects in synaptic pruning (Aims 2?3). We have identified C4A and the classical complement cascade as key mediators of synaptic pruning in the mouse postnatal visual system (Sekar et al., 2016)(Project 2); however, it is unknown if it is also necessary in the cortex and circuits relevant to SCZ and if aberrant pruning affects anatomical and functional connectivity and the behaviors dependent on it. In Aim 2, we will test the hypothesis that over-activation of the complement cascade enhances pruning in the PFC, perturbing anatomical and functional connectivity as well as behavior. We will use global complement KO mice (C1q, C4KO) and novel hC4A-overexpressing mice (Project 2) to ask if early postnatal pruning defects impact cortical connectivity and function later in life. We will also use viral strategies to test if circuit-specific and temporally restricted activation and inhibition of the complement cascade is sufficient for circuit-specific phenotypes. In Aim 3, we will seek to understand how second hits (genetic or environmental) on a background of genetic risk (increased copy number of C4A) combine to impact neural circuit development and behavior. We hypothesize that a second genetic hit (e.g., loss of CSMD1) or environmental hit (immune challenge) might worsen synaptic and behavioral phenotypes in mice over-expressing the human C4A risk allele. To link the central and peripheral effects of C4 expression explored in Projects 1 and 2, we will investigate the potential role of the choroid plexus, a major source of cerebral spinal fluid (CSF), in the regulation of complement and cytokine levels in the brain in Aim 3. This will lead to a better understanding of the cellular and molecular players linking peripheral immune dysregulation to brain dysfunction and may identify novel biomarkers.

The pathophysiological processes underlying neuropsychiatric disorders have been unknown; as a result, these disorders have lacked innovative medical therapies with new mechanisms of action. We recently identified the alleles underlying the human genome's largest population-level influence on risk of schizophrenia ? a series of structural alleles of the complement C4A and C4B genes, each of which appears to affect schizophrenia risk in proportion to the amount of C4A expression it generates in the brain. We also found that C4 shapes synaptic refinement in a mouse model of postnatal activity-dependent synapse elimination. These findings may help explain known features of schizophrenia, including reduced numbers of synapses in key cortical regions and an adolescent age of onset that corresponds with developmentally timed waves of synaptic pruning in these regions. The goal of the work we envision for a Conte Center is to develop our understanding of neural-immune interactions and synapses while also generating novel scientific resources that can be used to evaluate current and future hypotheses about schizophrenia-implicated genes, neural-immune interactions, and critical periods for synaptic refinement. Our proposed work arises from close, successful collaboration of scientists with expertise in genomics, immunology, and neuroscience. We aim to accomplish our Center's missions through scientific projects and cores. Project 1 will seek to understand how CNS cells regulate the expression of complement and reprogram gene expression as they traverse critical periods in the maturation of their circuits. Project 2 will create mice that carry human C4 genes and alleles; examining how human C4 allelic diversity and expression levels affect microglia-mediated synaptic pruning and other processes. Project 3 will reveal the functional consequences of complement-cascade dysregulation ? both over- and under-pruning ? on circuit function and behavior. A Computational and Statistical Analysis Core will contribute to research in all three projects by facilitating analyses of genome-wide expression data and genome sequence data. An administrative core will coordinate biweekly lab meetings and outward-facing activities, including an annual symposium on emerging research at the interface of neuroscience, immunology and genomics. We hope to advance the search for molecular understanding of schizophrenia while advancing the understanding of brain development, the interacting influences of genes and environment on brain and behavior, and possibly general principles that could be applicable to the mechanisms and pathways that go awry in other mental illnesses.