Project description:Unilateral whisker lesioning at age P4 leads to removal of thalamocortical synapses in layer IV of the barrel cortex over the course of the next 6 days. Synapse removal is mediated by microglia and induced by CX3CL1-CX3CR1 signaling between neurons and microglia. Downstream of CX3CL1-CX3CR1 activation, microglia induce astrocyte morphological changes. To identify molecular crosstalk between astrocytes and microglia, we performed translating ribosome affinty purification followed by RNA sequencing (TRAP-Seq).
Project description:Methamphetamine (Meth) is a powerful illicit psychostimulant, widely used for recreational purposes. Besides disrupting the monoaminergic system and promoting oxidative brain damage, Meth also causes neuroinflammation, contributing to synaptic dysfunction and behavioral deficits. Aberrant activation of microglia, the largest myeloid cell population in the brain, is a common feature in neurological disorders triggered by neuroinflammation. In this study, we investigated the mechanisms underlying the aberrant activation of microglia elicited by Meth in the adult mouse brain. We found that binge Meth exposure caused microgliosis and disrupted risk assessment behavior (a feature that usually occurs in individuals who abuse Meth), both of which required astrocyte-to-microglia crosstalk. Mechanistically, Meth triggered a detrimental increase of glutamate exocytosis from astrocytes (in a process dependent on TNF production and calcium mobilization), promoting microglial expansion and reactivity. Ablating TNF production, or suppressing astrocytic calcium mobilization, prevented microglia reactivity and abolished the behavioral phenotype elicited by Meth in the elevated plus maze (EPM). Overall, our data indicate that glial crosstalk is critical to relay alterations caused by acute Meth exposure.
Project description:This study defines a molecular interaction between neurons and microglia that drives experience-dependent synapse remodeling in the hippocampus.
Project description:Genetic studies implicate Clusterin (CLU) in the pathogenesis of Alzheimer’s disease (AD), yet its precise molecular impact remains unclear. Through unbiased proteomic profiling and functional validation in CLU-deficient astrocytes, we identify increased NfkB-dependent signaling and complement C3 secretion. Reduction of astrocyte CLU induced microglia dependent modulation of extracellular APOE and phosphorylated tau, as well as increased microglial phagocytosis and reduced synapse numbers. By integrating mouse and human cellular models with comprehensive analyses of human plasma and brain tissue, we demonstrate that CLU AD risk alleles are associated with reduced CLU protein and heightened inflammatory profiles. These findings establish a mechanistic link between AD genetic risk factors, astrocyte reactivity, and microglia-mediated effects on synaptic integrity. Collectively, these results support a model in which CLU upregulation in response to neuropathology is associated with maintenance of cognitive function, while diminished astrocyte CLU levels heighten disease susceptibility.
Project description:Genetic studies implicate Clusterin (CLU) in the pathogenesis of Alzheimer’s disease (AD), yet its precise molecular impact remains unclear. Through unbiased proteomic profiling and functional validation in CLU-deficient astrocytes, we identify increased NfkB-dependent signaling and complement C3 secretion. Reduction of astrocyte CLU induced microglia dependent modulation of extracellular APOE and phosphorylated tau, as well as increased microglial phagocytosis and reduced synapse numbers. By integrating mouse and human cellular models with comprehensive analyses of human plasma and brain tissue, we demonstrate that CLU AD risk alleles are associated with reduced CLU protein and heightened inflammatory profiles. These findings establish a mechanistic link between AD genetic risk factors, astrocyte reactivity, and microglia-mediated effects on synaptic integrity. Collectively, these results support a model in which CLU upregulation in response to neuropathology is associated with maintenance of cognitive function, while diminished astrocyte CLU levels heighten disease susceptibility.
Project description:To investigate the influence of Aβ40WT and Aβ42WT fibrils on astrocyte and microglia, we isolated astrocyte and microglia from P2 SD rats and treated glia cells with Aβ40WT and Aβ42WT fibrils for 24 hours. We then performed gene expression profiling analysis using data obtained from RNA-seq of astrocyte and microglia treated with Aβ fibrils.
Project description:Neuronal synapse formation and remodeling is essential to central nervous system (CNS) development and is dysfunctional in neurodevelopmental diseases. Innate immune signals regulate tissue remodeling in the periphery, but how this impacts CNS synapses is largely unknown. Here we show that the IL-1 family cytokine Interleukin-33 (IL-33) is produced by developing astrocytes and is developmentally required for normal synapse numbers and neural circuit function in the spinal cord and thalamus. We find that IL-33 signals primarily to microglia under physiologic conditions, that it promotes microglial synapse engulfment, and that it can drive microglial-dependent synapse depletion in vivo. These data reveal a cytokine-mediated mechanism required to maintain synapse homeostasis during CNS development.
Project description:Neuronal synapse formation and remodeling is essential to central nervous system (CNS) development and is dysfunctional in neurodevelopmental diseases. Innate immune signals regulate tissue remodeling in the periphery, but how this impacts CNS synapses is largely unknown. Here we show that the IL-1 family cytokine Interleukin-33 (IL-33) is produced by developing astrocytes and is developmentally required for normal synapse numbers and neural circuit function in the spinal cord and thalamus. We find that IL-33 signals primarily to microglia under physiologic conditions, that it promotes microglial synapse engulfment, and that it can drive microglial-dependent synapse depletion in vivo. These data reveal a cytokine-mediated mechanism required to maintain synapse homeostasis during CNS development.
Project description:SPEACC-seq is a novel high-throughput method which enables forward genetic screens to identify cell-cell interaction mechanisms that uncovered an astrocyte-microglia regulatory circuit mediated by amphiregulin and IL33-ST2. signaling. Cell-cell interactions in the central nervous system (CNS) play central roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. For example, several factors mediate microglia-astrocyte interactions that promote CNS pathology, but less is known about regulatory interactions that limit tissue pathology. Here we report the development of SPEACC-seq (Stimulation, Perturbation, and Encapsulation of interACting Cells followed by Sequencing), a forward genetic screening platform which combines genome-wide CRISPR/Cas9 perturbations, cell co-culture in picoliter droplets, and microfluidic-based fluorescence activated droplet sorting to identify mechanisms of cell-cell communication. Using SPEACC-seq in combination with an in vivo perturb-seq screen, we identified microglia-produced amphiregulin as a suppressor of disease promoting astrocyte responses in experimental autoimmune encephalomyelitis (EAE), a pre-clinical model of multiple sclerosis (MS). The production of microglial amphiregulin was induced via ST2 signaling by IL-33 released from astrocytes during EAE. Indeed, the genetic inactivation of ST2 or amphiregulin in microglia, or IL-33 or amphiregulin signaling in astrocytes resulted in the worsening of EAE, suggesting that IL-33-induced microglial amphiregulin limits disease-promoting astrocyte responses associated with CNS pathology. This regulatory loop was also detected in human astrocytes and microglia both in vitro and in MS patient CNS samples. In summary, we developed SPEACC-seq, a high-throughput, droplet-based forward genetic screening platform for the identification of cell-cell interaction mechanisms, which identified a novel microglia-astrocyte negative feedback loop that limits CNS pathology.
Project description:Spatially heterogeneous synapse loss is a characteristic of many psychiatric and neurological disorders, but the underlying mechanisms are unclear. Here, we show that spatially-restricted complement activation mediates stress-induced heterogeneous microglia activation and synapse loss localized to the upper layers of the mouse medial prefrontal cortex (mPFC). Single cell RNA sequencing also reveals a stress-associated microglia state marked by high expression of the apolipoprotein E gene (Apoehigh) localized to the upper layers of the mPFC. Mice lacking complement component C3 are protected from stress-induced layer-specific synapse loss, and the Apoehigh microglia population is markedly reduced in the mPFC of these mice. Furthermore, C3 knockout mice are also resilient to stress-induced anhedonia and working memory behavioral deficits. Our findings suggest that region-specific complement and microglia activation can contribute to the disease-specific spatially restricted patterns of synapse loss and clinical symptoms found in many brain diseases.