Microglia complement signaling promotes neuronal elimination and normal brain functional connectivity.

Complement signaling is thought to serve as an opsonization signal to promote the phagocytosis of synapses by microglia. However, while its role in synaptic remodeling has been demonstrated in the retino-thalamic system, it remains unclear whether complement signaling mediates synaptic pruning in the brain more generally. Here we found that mice lacking the Complement receptor 3, the major microglia complement receptor, failed to show a deficit in either synaptic pruning or axon elimination in the developing mouse cortex. Instead, mice lacking Complement receptor 3 exhibited a deficit in the perinatal elimination of neurons in the cortex, a deficit that is associated with increased cortical thickness and enhanced functional connectivity in these regions in adulthood. These data demonstrate a role for complement in promoting neuronal elimination in the developing cortex.

Phospholipid scramblase Xkr8 is required for developmental axon pruning via phosphatidylserine exposure.

The mature mammalian brain connectome emerges during development via the extension and pruning of neuronal connections. Glial cells have been identified as key players in the phagocytic elimination of neuronal synapses and projections. Recently, phosphatidylserine has been identified as neuronal "eat-me" signal that guides elimination of unnecessary input sources, but the associated transduction systems involved in such pruning are yet to be described. Here, we identified Xk-related protein 8 (Xkr8), a phospholipid scramblase, as a key factor for the pruning of axons in the developing mammalian brain. We found that mouse Xkr8 is highly expressed immediately after birth and required for phosphatidylserine exposure in the hippocampus. Mice lacking Xkr8 showed excess excitatory nerve terminals, increased density of cortico-cortical and cortico-spinal projections, aberrant electrophysiological profiles of hippocampal neurons, and global brain hyperconnectivity. These data identify phospholipid scrambling by Xkr8 as a central process in the labeling and discrimination of developing neuronal projections for pruning in the mammalian brain.

Large neutral amino acid levels tune perinatal neuronal excitability and survival.

Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.

What microglia depletion approaches tell us about the role of microglia on synaptic function and behavior.

Microglia are dynamic cells, constantly surveying their surroundings and interacting with neurons and synapses. Indeed, a wealth of knowledge has revealed a critical role of microglia in modulating synaptic transmission and plasticity in the developing brain. In the past decade, novel pharmacological and genetic strategies have allowed the acute removal of microglia, opening the possibility to explore and understand the role of microglia also in the adult brain. In this review, we summarized and discussed the contribution of microglia depletion strategies to the current understanding of the role of microglia on synaptic function, learning and memory, and behavior both in physiological and pathological conditions. We first described the available microglia depletion methods highlighting their main strengths and weaknesses. We then reviewed the impact of microglia depletion on structural and functional synaptic plasticity. Next, we focused our analysis on the effects of microglia depletion on behavior, including general locomotor activity, sensory perception, motor function, sociability, learning and memory both in healthy animals and animal models of disease. Finally, we integrated the findings from the reviewed studies and discussed the emerging roles of microglia on the maintenance of synaptic function, learning, memory strength and forgetfulness, and the implications of microglia depletion in models of brain disease.

Substrate stiffness effect on molecular crosstalk of epithelial-mesenchymal transition mediators of human glioblastoma cells.

The complexity of the microenvironment effects on cell response, show accumulating evidence that glioblastoma (GBM) migration and invasiveness are influenced by the mechanical rigidity of their surroundings. The epithelial-mesenchymal transition (EMT) is a well-recognized driving force of the invasive behavior of cancer. However, the primary mechanisms of EMT initiation and progression remain unclear. We have previously showed that certain substrate stiffness can selectively stimulate human GBM U251-MG and GL15 glioblastoma cell lines motility. The present study unifies several known EMT mediators to uncover the reason of the regulation and response to these stiffnesses. Our results revealed that changing the rigidity of the mechanical environment tuned the response of both cell lines through change in morphological features, epithelial-mesenchymal markers (E-, N-Cadherin), EGFR and ROS expressions in an interrelated manner. Specifically, a stiffer microenvironment induced a mesenchymal cell shape, a more fragmented morphology, higher intracellular cytosolic ROS expression and lower mitochondrial ROS. Finally, we observed that cells more motile showed a more depolarized mitochondrial membrane potential. Unravelling the process that regulates GBM cells' infiltrative behavior could provide new opportunities for identification of new targets and less invasive approaches for treatment.

Microglia control glutamatergic synapses in the adult mouse hippocampus.

Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX-induced synaptic changes were absent in Cx3cr1-/- mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses.

Antibiotics Treatment Modulates Microglia-Synapses Interaction.

'Dysbiosis' of the adult gut microbiota, in response to challenges such as infection, altered diet, stress, and antibiotics treatment has been recently linked to pathological alteration of brain function and behavior. Moreover, gut microbiota composition constantly controls microglia maturation, as revealed by morphological observations and gene expression analysis. However, it is unclear whether microglia functional properties and crosstalk with neurons, known to shape and modulate synaptic development and function, are influenced by the gut microbiota. Here, we investigated how antibiotic-mediated alteration of the gut microbiota influences microglial and neuronal functions in adult mice hippocampus. Hippocampal microglia from adult mice treated with oral antibiotics exhibited increased microglia density, altered basal patrolling activity, and impaired process rearrangement in response to damage. Patch clamp recordings at CA3-CA1 synapses revealed that antibiotics treatment alters neuronal functions, reducing spontaneous postsynaptic glutamatergic currents and decreasing synaptic connectivity, without reducing dendritic spines density. Antibiotics treatment was unable to modulate synaptic function in CX3CR1-deficient mice, pointing to an involvement of microglia-neuron crosstalk through the CX3CL1/CX3CR1 axis in the effect of dysbiosis on neuronal functions. Together, our findings show that antibiotic alteration of gut microbiota impairs synaptic efficacy, suggesting that CX3CL1/CX3CR1 signaling supporting microglia is a major player in in the gut-brain axis, and in particular in the gut microbiota-to-neuron communication pathway.

Microglial-glucocorticoid receptor depletion alters the response of hippocampal microglia and neurons in a chronic unpredictable mild stress paradigm in female mice.

Chronic psychological stress is one of the most important triggers and environmental risk factors for neuropsychiatric disorders. Chronic stress can influence all organs via the secretion of stress hormones, including glucocorticoids by the adrenal glands, which coordinate the stress response across the body. In the brain, glucocorticoid receptors (GR) are expressed by various cell types including microglia, which are its resident immune cells regulating stress-induced inflammatory processes. To study the roles of microglial GR under normal homeostatic conditions and following chronic stress, we generated a mouse model in which the GR gene is depleted in microglia specifically at adulthood to prevent developmental confounds. We first confirmed that microglia were depleted in GR in our model in males and females among the cingulate cortex and the hippocampus, both stress-sensitive brain regions. Then, cohorts of microglial-GR depleted and wild-type (WT) adult female mice were housed for 3 weeks in a standard or stressful condition, using a chronic unpredictable mild stress (CUMS) paradigm. CUMS induced stress-related behavior in both microglial-GR depleted and WT animals as demonstrated by a decrease of both saccharine preference and progressive ratio breakpoint. Nevertheless, the hippocampal microglial and neural mechanisms underlying the adaptation to stress occurred differently between the two genotypes. Upon CUMS exposure, microglial morphology was altered in the WT controls, without any apparent effect in microglial-GR depleted mice. Furthermore, in the standard environment condition, GR depleted-microglia showed increased expression of pro-inflammatory genes, and genes involved in microglial homeostatic functions (such as Trem2, Cx3cr1 and Mertk). On the contrary, in CUMS condition, GR depleted-microglia showed reduced expression levels of pro-inflammatory genes and increased neuroprotective as well as anti-inflammatory genes compared to WT-microglia. Moreover, in microglial-GR depleted mice, but not in WT mice, CUMS led to a significant reduction of CA1 long-term potentiation and paired-pulse ratio. Lastly, differences in adult hippocampal neurogenesis were observed between the genotypes during normal homeostatic conditions, with microglial-GR deficiency increasing the formation of newborn neurons in the dentate gyrus subgranular zone independently from stress exposure. Together, these findings indicate that, although the deletion of microglial GR did not prevent the animal's ability to respond to stress, it contributed to modulating hippocampal functions in both standard and stressful conditions, notably by shaping the microglial response to chronic stress.

Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development.

De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). In mouse, constitutive Cul3 haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs.

Molecular mechanisms for targeted ASD treatments.

The possibility to generate construct valid animal models enabled the development and testing of therapeutic strategies targeting the core features of autism spectrum disorders (ASDs). At the same time, these studies highlighted the necessity of identifying sensitive developmental time windows for successful therapeutic interventions. Animal and human studies also uncovered the possibility to stratify the variety of ASDs in molecularly distinct subgroups, potentially facilitating effective treatment design. Here, we focus on the molecular pathways emerging as commonly affected by mutations in diverse ASD-risk genes, on their role during critical windows of brain development and the potential treatments targeting these biological processes.

Dimethyl Fumarate Reduces Microglia Functional Response to Tissue Damage and Favors Brain Iron Homeostasis.

Dimethyl fumarate (DMF) is the only available approved drug for first line treatment of multiple sclerosis (MS), a lethal condition impairing central nervous system (CNS). To date, however, little is known of its mechanisms of action. Only recently, it has been suggested that DMF exerts neuroprotective effects acting as an immunomodulator and that it may alter the activation state of microglia cells, crucial in MS pathogenesis. However, DMF effects on microglia functions are still not well determined. Here, we examine the effects of DMF treatment on microglia functional activities, as phenotype, morphology, processes motility and rearrangement, migration, ATP response and iron uptake in mouse primary microglia culture and acute hippocampal slices. We found that DMF treatment reduces microglia motility, downregulating functional response to ATP, increases ferritin uptake and pushes microglia towards an anti-inflammatory phenotype, thus reducing its proinflammatory reactivity in response to tissue damage. These results highlight the effects of this compound on microglia functions and provide new insights on the mechanism of action of DMF in MS treatment.

Microglia-neuron crosstalk: Signaling mechanism and control of synaptic transmission.

The continuous crosstalk between microglia and neurons is required for microglia housekeeping functions and contributes to brain homeostasis. Through these exchanges, microglia take part in crucial brain functions, including development and plasticity. The alteration of neuron-microglia communication contributes to brain disease states with consequences, ranging from synaptic function to neuronal survival. This review focuses on the signaling pathways responsible for neuron-microglia crosstalk, highlighting their physiological roles and their alteration or specific involvement in disease. In particular, we discuss studies, establishing how these signaling allow microglial cells to control relevant physiological functions during brain development, including synaptic formation and circuit refinement. In addition, we highlight how microglia and neurons interact functionally to regulate highly dynamical synaptic functions. Microglia are able to release several signaling molecules involved in the regulation of synaptic activity and plasticity. On the other side, molecules of neuronal origin control microglial processes motility in an activity-dependent manner. Indeed, the continuous crosstalk between microglia and neurons is required for the sensing and housekeeping functions of microglia and contributes to the maintenance of brain homeostasis and, particularly, to the sculpting of neuronal connections during development. These interactions lay on the delicate edge between physiological processes and homeostasis alteration in pathology and are themselves altered during neuroinflammation. The full description of these processes could be fundamental for understanding brain functioning in health and disease.

Time-lapse Whole-field Fluorescence Imaging of Microglia ProcessesMotility in Acute Mouse Hippocampal Slices and Analysis.

Microglia are the resident immune cells of the central nervous system (CNS). In the last year, the improvements in the transgenic mouse technologies and imaging techniques have shed light on microglia functions under physiological conditions. Microglia continuously scan the brain parenchyma with their highly motile processes, maintaining tissue homeostasis and participating in neuronal circuits refinement. Here, we describe a protocol that enables us to perform time-lapse imaging of microglial cells in acute hippocampal slices, making image acquisition possible on an electrophysiology rig equipped with a standard imaging system. Using this ex vivo approach, we investigated microglial processes scanning abilities under physiological condition in hippocampus.

Microglia shape presynaptic properties at developing glutamatergic synapses.

Deficient neuron-microglia signaling during brain development is associated with abnormal synaptic maturation. However, the precise impact of deficient microglia function on synaptic maturation and the mechanisms involved remain poorly defined. Here we report that mice defective in neuron-to-microglia signaling via the fractalkine receptor (Cx3cr1 KO) show reduced microglial branching and altered motility and develop widespread deficits in glutamatergic neurotransmission. We characterized the functional properties of CA3-CA1 synapses in hippocampal slices from these mice and found that they display altered glutamatergic release probability, maintaining immature properties also at late developmental stages. In particular, CA1 synapses of Cx3cr1 KO show (i) immature AMPA/NMDA ratio across developmental time, displaying a normal NMDA component and a defective AMPA component of EPSC; (ii) defective functional connectivity, as demonstrated by reduced current amplitudes in the input/output curve; and (iii) greater facilitation in the paired pulse ratio (PPR), suggesting decreased release probability. In addition, minimal stimulation experiments revealed that excitatory synapses have normal potency, but an increased number of failures, confirming a deficit in presynaptic release. Consistently, KO mice were characterized by higher number of silent synapses in comparison to WT. The presynaptic deficits were corrected by performing experiments in conditions of high release probability (Ca2+ /Mg2+ ratio 8), where excitatory synapses showed normal synaptic multiplicity, AMPA/NMDA ratio, and proportion of silent synapses. These results establish that neuron-microglia interactions profoundly influence the functional maturation of excitatory presynaptic function.

Environmental stimuli shape microglial plasticity in glioma.

In glioma, microglia and infiltrating macrophages are exposed to factors that force them to produce cytokines and chemokines, which contribute to tumor growth and to maintaining a pro-tumorigenic, immunosuppressed microenvironment. We demonstrate that housing glioma-bearing mice in enriched environment (EE) reverts the immunosuppressive phenotype of infiltrating myeloid cells, by modulating inflammatory gene expression. Under these conditions, the branching and patrolling activity of myeloid cells is increased, and their phagocytic activity is promoted. Modulation of gene expression depends on interferon-(IFN)-γ produced by natural killer (NK) cells. This modulation disappears in mice depleted of NK cells or lacking IFN-γ, and was mimicked by exogenous interleukin-15 (IL-15). Further, we describe a key role for brain-derived neurotrophic factor (BDNF) that is produced in the brain of mice housed in EE, in mediating the expression of IL-15 in CD11b+ cells. These data define novel mechanisms linking environmental cues to the acquisition of a pro-inflammatory, anti-tumor microenvironment in mouse brain.