Anna V. Molofsky, M.D/Ph.D. - US grants

DESCRIPTION (provided by applicant) The existence of stem cells in the adult mammalian central nervous system (CNS) suggests that the brain is capable of regeneration and repair, yet the mechanisms that regulate stem cell self-renewal and the generation of new neurons are poorly understood. The oncogene bmi1 promotes the self-renewal of CNS stem cells in part by repressing the cell cycle regulator p16ink4a. Mice lacking Bmi1 experience progressive gait disturbances and postnatal depletion of forebrain stem cells. To better understand the role of Bmil in neurogenesis and stem cell self-renewal three specific aims are proposed: First, to characterize the effects of Bmi1 deficiency on adult olfactory bulb neurogenesis by quantifying neurogenesis in Bmi1 deficient mice. Secondly, to determine if the depletion of postnatal stem cells in Bmi1 deficient mice is mediated by the tumor suppressor p16ink4a. Finally, to overexpress Bmi1 in forebrain stem cells in order to determine whether Bmi1 is sufficient to promote stem cell self-renewal. In sum, the goal of these aims is explore a model system in which stem cells are depleted in order to understand the normal role of stem cells in building and regenerating the adult mammalian brain.

DESCRIPTION (provided by applicant): Astrocytes (AS) are the most numerous cells in the central nervous system and are increasingly recognized as key players in the pathology of neurodevelopmental and neurodegenerative diseases including the autism- associated disorders Rett Syndrome and Fragile X syndrome, Amyotrophic Lateral Sclerosis (ALS), and Huntington's Disease. The goal of this research program is to define the role of AS in guiding the formation of functional neural circuits. The applicant for this K08 Mentored Clinical Scientist Research Career Development Award is a Psychiatrist and Clinical Instructor in the Department of Psychiatry, and a postdoctoral fellow with Dr. David Rowitch at the University of California San Francisco. This proposal outlines a 4-year career development plan and research strategy that includes mentorship by Dr. Rowitch, Dr. John Rubenstein and Dr. Erik Ullian to accomplish the research aims described below. Together, these will enable the applicant to fulfill a career goal of running an independent laboratory spanning the interface of glial biology and neuropsychiatric disease. Preliminary data that forms the basis for this proposal demonstrates that the guidance molecules Sema3a and EphA5 are uniquely expressed by ventral, but not dorsal spinal cord AS. AS-encoded Sema3a secreted from ventral AS has local effects on motor neuron position, postnatal survival, and synaptogenesis, demonstrating molecularly defined AS heterogeneity for the first time. The research strategy expands these findings in mechanistic and functional directions to determine how these heterogeneous AS modulate the formation of a sensorimotor circuit. Aim one will investigate the mechanism by which AS-encoded Sema3a protein polarizes motor neurons and its effects on motor neuron dendrite outgrowth. Aim two will study the effects of AS- encoded Sema3a in motor neuron circuit function, via additional training in slice electrophysiology. Aim three will determine the role of AS in guiding incoming sensory afferents that synapse on motor neurons through function of AS-encoded positional molecule EphA5. Together, these aims will greatly expand our understanding of the role of AS on the maturation of a defined monosynaptic CNS circuit. The training and mentorship proposed will prepare the candidate for an independent investigator position addressing the role of AS in neural circuit formation throughout the CNS. Ultimately, this strategy and training plan will open new avenues of research on glial-based treatments for neuropsychiatric disorders.

Project Summary/Abstract Remodeling of neural circuits is essential for development, learning, and normal brain function, and intimately dependent on astrocytes and microglia -- the glial support cells of the brain. The goal of this proposal is to identify novel glial-encoded genes that promote neural circuit remodeling and to design glial-based tools to track and manipulate brain plasticity in vivo. The central premise of this proposal is that microglia, the resident phagocytes of the brain, respond to cues from local astrocytes, stromal cells that are intimately associated with neuronal synapses. Together these cells coordinate a tissue remodeling response, physically clearing space to enable new synapse formation. We predict that this coordinate regulation between the stromal and immune cells of the brain will co-opt innate immune pathways that are increasingly implicated in tissue remodeling elsewhere in the body. This model will be tested in the mouse barrel cortex in which whisker removal early in postnatal life leads to plasticity and topographic rearrangement of neuronal inputs. This is an inherently heterogeneous response that requires local detection of altered sensory inputs, removal of superfluous synapses, and formation of new synapses. Our experimental design will proceed in three phases. First, we will perform single cell RNA sequencing of astrocytes and microglia during barrel cortex structural remodeling to identify coordinate regulation modules: ligand-receptor pairs that are upregulated or alternately spliced in astrocytes and microglia respectively during barrel cortex remodeling. Our top candidates will be misexpressed or deleted during barrel cortex plasticity via adeno-associated virus (AAV) delivery of transgenes or CRISPR/Cas9 genome editing constructs. We predict that the top candidate pathways will be heterogeneously expressed in remodeling barrel cortex in situ and will impact cortical plasticity when misexpressed in vivo. Our ultimate goal is to design new glial-based tools to both report and regulate neural circuit plasticity that can be used to study less experimentally accessible circuits throughout the nervous system, particularly those involved in cognition and behavior. We anticipate that prospective identification of glial-encoded plasticity pathways will have broad ranging applications for understanding and modulating neural circuits in the context of development, injury, and neuropsychiatric diseases.

PROJECT SUMMARY/ ABSTRACT Regulation of synapse homeostasis is essential for normal brain development and function, and intimately dependent on astrocytes and microglia -- the support cells of the brain. Astrocytes contact thousands of synapses and promote developmental synapse formation. Microglia are brain-resident immune cells increasingly implicated in both synapse formation and pruning. We have discovered a novel signaling circuit between astrocytes and microglia that promotes synapse elimination in the developing central nervous system (CNS). We found that astrocytes express the immune signal Interleukin-33 (IL-33) whereas microglia express the IL-33 receptor (IL1RL1.) We subsequently showed that in the spinal cord, eliminating IL-33 from developing astrocytes leads to excess synapses, and that IL-33 signals to microglia to drive synapse engulfment and lead to synapse depletion. Our central hypothesis is that astrocytes express and release IL-33 in response to neuron-derived signals, and that IL-33 in turn drives microglial synapse elimination. We will test this hypothesis in three distinct but interrelated aims, focusing on a well-defined and experimentally accessible circuit in the ventrobasal sensory thalamus (VB), where IL-33 is highly expressed during synapse refinement. In Aim One we will determine how astrocytic IL-33 regulates thalamic synapse subtypes and circuit function. We previously showed that global deletion of IL-33 lead to hyperexcitability of the VB circuit and excess synapses. Here we will conditionally delete IL-33 from astrocytes and explore these phenotypes in more detail, quantifying subtypes of afferent excitatory and inhibitory synapses to understand how different components of the circuit are altered. In Aim Two, we will determine the molecular mechanisms regulating microglial synapse engulfment. We previously found that IL-33 promotes engulfment of postsynaptic proteins by microglia. Here, using both standard and high resolution techniques (expansion microscopy), we will quantify engulfment of both pre- and postsynaptic excitatory elements, as well as inhibitory synapses. We will test the requirement for direct signaling to microglia via conditional deletion of its receptor. In Aim 3, we will identify neuronal molecules that induce astrocyte expression and release of IL-33. Our preliminary data demonstrates that norepinephrine is a neuron-derived cue that promotes expression of IL-33 in gray matter astrocytes. Here we will further test which noradrenergic receptors on astrocytes mediate this effect in vivo. We will also test the hypothesis that extracellular release of IL-33 is dependent on neuronal synaptic activity, by modulating activity in vitro and in vivo. Together, these three aims explore the role of a novel glial-neuronal circuit mediating synapse homeostasis. We predict that a broader understanding of how glia communicate via immune molecules to regulate synapses will fundamentally impact our understanding of how neural circuits change in learning and development as well as in neurodevelopmental diseases.

PROJECT SUMMARY Structural remodeling of synapses and circuits is essential to experience-dependent plasticity, as occurs during the consolidation of learned experiences into long-term memory. Immune dysfunction has been implicated in numerous cognitive disorders, including schizophrenia and neurodegenerative diseases, leading to increasing interest in the function of brain-resident macrophages known as microglia, which are the dominant immune cell in the brain parenchyma. We previously published that the IL-1 family cytokine Interleukin-33, which is made by astrocytes during development, signals to its obligate receptor IL1RL1 expressed on microglia to promote microglial activation and phagocytic function. In preliminary data that forms the basis for this proposal, we identify a novel population of IL-33 expressing neurons in two adult brain regions: hippocampus and frontal cortex. In detailed structural analyses in the hippocampus, we find that neuron-specific deletion of IL-33 or microglial- specific deletion of IL-33 R leads to fewer dendritic spines, diminished markers of spine plasticity known as spine head filopodia, and impaired neurogenesis. Furthermore, loss of this signaling pathway leads to deficits in contextual fear conditioning: mice are able to normally learn to recognize a fear context but have a progressive decrease in their ability to discriminate the fear context from a neutral context emerging at 15-30 days post training. Mechanistically, we find that extracellular matrix proteins (the chondroitin sulfate proteoglycans brevican and aggrecan) accumulate in the hippocampus of IL-33 deficient animals. We find that microglia engulf aggrecan, and that loss of IL-33 signaling diminishes this engulfment. We also developed a neuronal gain of function construct that constitutively secretes IL-33. We find this is sufficient to increase hippocampal spine numbers, microglial ECM engulfment, and to clear ECM around dendritic spines. Based on these preliminary data, this proposal will test the central hypothesis that neuron-derived IL-33 drives microglial remodeling of ECM to promote circuit plasticity in support of memory consolidation. Aim One will explore the molecular regulation of IL-33 release from neurons and its activity dependence, and test two candidate proteases mediating microglial remodeling in response to IL-33. Aim Two will test the impact of this neuron-microglia signaling on the ECM composition of the frontal cortex with a focus on perineuronal nets and determine its impact on connectivity of the cortical microcircuit. Aim 3 will use calcium imaging in a contextual fear conditioning task to test how neuronal activity patterns shift during the transition from recent to remote memories, addressing key questions regarding the structural changes that underlie memory consolidation. Together, these studies will systematically dissect the role of a novel cellular circuit regulating plasticity in the healthy brain. The outcome of this work has implications for understanding cognitive dysfunction in numerous diseases linked to microglia and the immune system , including schizophrenia and neurodegenerative conditions such as Alzheimer's Disease.