Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for the routine non-inflammatory clearance of dead brain cells1. Here we show that the TAM receptor tyrosine kinases Mer and Axl2 regulate these microglial functions. We find that mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells (ACs) specifically in neurogenic regions of the adult CNS, and that microglial phagocytosis of the ACs generated during adult neurogenesis3,4 is normally driven by both TAM receptor ligands – Gas6 and Protein S5. Live two-photon imaging demonstrates that the microglial response to brain damage is also TAM-regulated, as TAM-deficient microglia display reduced process motility and delayed convergence to sites of injury. Finally, we show that microglial expression of Axl is prominently up-regulated in the inflammatory environment that develops in a mouse model of Parkinson’s disease6. Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease.

In the nucleus accumbens (NAc), a key structure to the effects of all addictive drugs, presynaptic cannabinoid CB1 receptors (CB1Rs) and postsynaptic metabotropic glutamate 5 receptors (mGluR5s) are the principal effectors of endocannabinoid (eCB)-mediated retrograde long-term depression (LTD) (eCB-LTD) at the prefrontal cortex-NAc synapses. Both CB1R and mGluR5 are involved in cocaine-related behaviors; however, the impact of in vivo cocaine exposure on eCB-mediated retrograde synaptic plasticity remains unknown. Electrophysiological and biochemical approaches were used, and we report that a single in vivo cocaine administration abolishes eCB-LTD. This

The dynamins comprise a large family of mechanoenzymes known to participate in membrane modeling events. All three conventional dynamin genes (Dyn1, Dyn2, Dyn3) are expressed in mammalian brain and produce more than 27 different dynamin proteins as a result of alternative splicing. Past studies have suggested that Dyn1 participates in specialized neuronal functions such as rapid synaptic vesicle recycling, while Dyn2 may mediate the conventional clathrin-mediated uptake of surface receptors. Currently, the distribution, expression, and function of Dyn3 in neurons, or in any other cell type, are completely undefined. Here, we demonstrate that Dyn1 and Dyn3 localize differentially in the synapse. Dyn1 concentrates within the presynaptic compartment, while Dyn3 localizes to dendritic spine tips. Within the postsynaptic density (PSD), we found Dyn3, but not Dyn1, to be part of a biochemically isolated complex comprised of Homer and metabotropic glutamate receptors. Finally, although dominant-negative Dyn3 did not seem to inhibit receptor endocytosis, overexpression of a specific Dyn3 spliced variant in mature neurons caused a marked remodeling of dendritic spines. These data suggest that Dyn3 is a postsynaptic dynamin and, like its binding partner Homer, plays a significant role in dendritic spine morphogenesis and remodeling.

Glutamate receptors are clustered at the membrane through interactions with intracellular scaffolding proteins and cytoskeletal elements but can also be found in intracellular compartments or dispersed in the membrane. This distribution results from an equilibrium between the different pools of receptors whose dynamic is poorly known. The group I metabotropic glutamate receptor 5 (mGluR5) is concentrated in an annulus around the postsynaptic density but also found in large amounts in the extrasynaptic membrane. To analyze the dynamic of stabilization of mGluR5, we used single-particle tracking, force measurements, and fluorescence recovery to measure the mobility of mGluR5. We found that receptor activation increases receptor diffusion, whereas the scaffolding protein Homer favors confinement of receptor movements within clusters of Homer-mGluR5. However, this stabilization is reversible, because even in the presence of Homer, receptors still enter and exit from clusters at fast rates. Furthermore, clusters themselves are highly dynamic both in their movements and in their composition, which can vary within tens of seconds. Thus, exchange of receptors between dispersed and clustered states is fast and regulated during physiological processes. These properties may explain certain fast changes in receptor composition observed at postsynaptic densities.

Omega-3 fatty acids (n-3 PUFAs) are essential for the functional maturation of the brain. Westernization of dietary habits in both developed and developing countries is accompanied by a progressive reduction in dietary intake of n-3 PUFAs. Low maternal intake of n-3 PUFAs has been linked to neurodevelopmental diseases in Humans. However, the n-3 PUFAs deficiency-mediated mechanisms affecting the development of the central nervous system are poorly understood. Active microglial engulfment of synapses regulates brain development. Impaired synaptic pruning is associated with several neurodevelopmental disorders. Here, we identify a molecular mechanism for detrimental effects of low maternal n-3 PUFA intake on hippocampal development in mice. Our results show that maternal dietary n-3 PUFA deficiency increases microglia-mediated phagocytosis of synaptic elements in the rodent developing hippocampus, partly through the activation of 12/15-lipoxygenase (LOX)/12-HETE signaling, altering neuronal morphology and affecting cognitive performance of the offspring. These findings provide a mechanistic insight into neurodevelopmental defects caused by maternal n-3 PUFAs dietary deficiency.

BackgroundATP-gated P2X7 is a non-selective cation channel, which participates in a wide range of cellular functions as well as pathophysiological processes including neuropathic pain, immune response, and neuroinflammation. Despite its abundant expression in microglia, the role of P2X7 in neuroinflammation still remains unclear.MethodsPrimary microglia were isolated from cortices of P0-2 C57BL/6 wild-type or P2X7 knockout (P2X7−/−) mouse pups. Lipopolysaccharide, lipopolysaccharide plus IFNγ, or IL4 plus IL13 were used to polarize microglia to pro-inflammatory or anti-inflammatory states. P2rx7 expression level in resting or activated mouse and human microglia was measured by RNA-sequencing and quantitative real-time PCR. Microglial cell death was measured by cell counting kit-8 and immunocytochemistry, and microglial secretion in wild-type or P2X7−/− microglia was examined by Luminex multiplex assay or ELISA using P2X7 agonist BzATP or P2X7 antagonist A-804598. P2X7 signaling was analyzed by Western blot.ResultsFirst, we confirmed that P2rx7 is constitutively expressed in mouse and human primary microglia. Moreover, P2rx7 mRNA level was downregulated in mouse microglia under both pro- and anti-inflammatory conditions. Second, P2X7 agonist BzATP caused cell death of mouse microglia, while this effect was suppressed either by P2X7 knockout or by A-804598 under both basal and pro-inflammatory conditions, which suggests the mediating role of P2X7 in BzATP-induced microglial cell death. Third, BzATP-induced release of IL1 family cytokines including IL1α, IL1β, and IL18 was blocked in P2X7−/− microglia or by A-804598 in pro-inflammatory microglia, while the release of other cytokines/chemokines was independent of P2X7 activation. These findings support the specific role of P2X7 in IL1 family cytokine release. Finally, P2X7 activation was discovered to be linked to AKT and ERK pathways, which may be the underlying mechanism of P2X7 functions in microglia.ConclusionsThese results reveal that P2X7 mediates BzATP-induced microglial cell death and specific release of IL1 family cytokines, indicating the important role of P2X7 in neuroinflammation and implying the potential of targeting P2X7 for the treatment of neuroinflammatory disorders.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0904-8) contains supplementary material, which is available to authorized users.

SUMMARY Microglia are the intrinsic immune sentinels of the central nervous system. Their activation restricts tissue injury and pathogen spread, but in some settings, including viral infection, this response can contribute to cell death and disease. Identifying mechanisms that control microglial responses is therefore an important objective. Using replication-incompetent adenovirus 5 (Ad5)-based vectors as a model, we investigated the mechanisms through which microglia recognize and respond to viral uptake. Transgenic, immunohistochemical, molecular-genetic, and fluorescence imaging approaches revealed that phosphatidylserine (PtdSer) exposure on the outer leaflet of transduced cells triggers their engulfment by microglia through TAM receptor-dependent mechanisms. We show that inhibition of phospholipid scramblase 1 (PLSCR1) activity reduces intracellular calcium dysregulation, prevents PtdSer externalization, and enables months-long protection of vector-transduced, transgene-expressing cells from microglial phagocytosis. Our study identifies PLSCR1 as a potent target through which the innate immune response to viral vectors, and potentially other stimuli, may be controlled.

Metabotropic glutamate receptors 5 (mGluR5) are members of the growing group C G protein-coupled receptor family. Widely expressed in mammalian brain, they are involved in modulation of the glutamate transmission. By means of transfection of mGluR5 receptors in COS-7 cells and primary hippocampal neurons in culture followed by immunocytochemistry and quantitative image analysis and by a biochemical assay, we have studied the internalization of mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well as receptors in which endogenous activity had been inhibited by an inverse agonist. We have measured a constitutive rate of endocytosis of 11.7%/min for mGluR5a. We report for the first time the endocytosis pathway of mGluR5. Internalization of mGluR5 is not mediated by clathrin-coated pits. Indeed, inhibition of this pathway by Eps15 dominant negative mutants did not disturb their endocytosis. However, the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5 in COS-7. mGluR5 is the first shown member of the group C G-protein coupled receptor family internalized by a nonconventional pathway.Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. It exerts its effects by binding to ionotropic and metabotropic receptors. Metabotropic glutamate receptors (mGluRs) 1 belong to the seven-transmembrane domain G protein-coupled receptor (GPCR) family. Together with calcium-sensing receptors, ␥-aminobutyric acid receptor B (GABA B ) and pheromone, olfactory and taste receptors, mGluRs form the third family of GPCRs (group C) (for reviews, see Refs. 1 and 2). They have little homology with other GPCRs from groups A and B and are characterized by a large N-terminal extracellular domain that binds glutamate. Eight genes code for mGluRs that generate more than 15 proteins by alternative splicing. On the basis of their sequence similarity, pharmacology, and signal transduction mechanisms, mGluRs have been classified into three groups. Group 1 consists of mGluR1 and mGluR5 that are preferentially positively coupled to phospholipase C via G q/11 proteins. mGluRs of group 2 and 3 are negatively coupled to adenylate cyclase. Group 1 mGluRs are expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in several forms of synaptic plasticity.Currently, very little is known about the targeting and trafficking mechanisms of mGluRs in cells. During the last few years, attention has focused mainly on endocytosis of a subtype of ionotropic glutamate receptors: the ␣-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptors. Internalization of AMPA receptors in response to various stimuli is an important process for the rapid attenuation of neurotransmission during synaptic plasticity (3). By different approaches, it has been s...