Rac 1 signaling in microglia is essential for synaptic proteome plasticity and 1 experience-dependent cognitive performance 2

27 28 Microglial homeostatic functions are fundamental to regulate the central nervous system 29 microenvironment. We use conditional cell-specific gene targeting, RNA-seq profiling, high30 throughput proteomics, phosphoproteomics, systems biology, and animal behavior to report 31 a critical role for the RhoGTPase Rac1 in regulating adult microglia physiology. Ablation of 32 Rac1 in adult microglia impaired their ability to sense and interpret the brain 33 microenvironment and affected their capacity to communicate with synapses to drive 34 cognitive performance, both at the steady-state and during experience-dependent plasticity. 35 Overall, our results reveal a novel and central role for Rac1 as a regulator of microglia 36 homeostasis and a molecular driver of the microglia-synapse crosstalk required for context37 dependent sociability and learning related to memory. 38 39 Introduction 40 Microglia, the largest myeloid cell population in the CNS, are best known for their 41 protective roles in the brain. Not only are microglia involved in physiological synaptic 42 pruning, but they also secrete a plethora of chemical mediators (including neurotransmitters 43 and growth factors) that affects brain synapses . Targeted gene ablation experiments 44 indicate that microglia-secreted factors are putative modulators of synaptic activity and 45 plasticity . Even though the microglial secretome conceivably regulates many aspects of 46 microglia-synapse crosstalk, little is known about the magnitude and specificity of the 47 alterations in synaptic signaling and remodeling elicited by the chemical mediators secreted 48 by microglia . 49 Remodeling of the synaptic structure and signaling network, bona fide synaptic 50 plasticity modalities, occurs continuously throughout life and underlies high-order cognitive 51 functions necessary for an organism to adapt to environmental inputs . Thus, deciphering 52 how the brain encodes and stores experiences triggered by different environmental contexts 53 requires in-depth knowledge of the cellular and molecular mechanisms governing synaptic 54 remodeling, including those in which microglia participate. 55 The GTPases of the Rho family, including the most well-characterized members 56 Rac1, RhoA, and Cdc42, are critical orchestrators of process extension required for cell-cell 57 communication and intracellular trafficking involved in secretion of chemical mediators , 58 making them likely players to govern microglial homeostatic functions. Studies with myeloid 59 cells from outside the CNS have identified a central role for Rac signaling in executing the 60 inflammatory program of the innate immune system . There are three Rac isoforms, but 61 only Rac1 and 2 are significantly expressed in myeloid cells . Gene-targeting approaches 62 revealed essential modulatory roles for Rac1 in the CNS, including axonal growth and 63 stability , oligodendrocyte myelination , and astrocyte morphology . Yet, no information 64 is currently available on Rac1 regulation of microglial homeostatic functions in vivo. 65 Here, by combining conditional cell-specific gene ablation, RNA-seq profiling, high66 throughput proteomics, single-cell live imaging, systems biology, and animal behavior, we 67 revealed a multiscale role for microglial Rac1 in the adult brain. Ablation of Rac1 specifically 68 suppressed the ability of microglia to sense the brain microenvironment, severing the 69 capacity of microglia to communicate with synapses properly. Loss of homeostatic microglia70 to-synapse communication primarily disrupted the remodeling of the proteome network of 71 synapses required for experience-dependent plasticity, impairing context-dependent 72 learning, memory, and social interaction. 73 74 75