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.

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 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.