Alain Bessis

The Role of Microglia in the Healthy Brain

Microglia were recently shown to play unexpected roles in normal brain development and adult physiology. This has begun to dramatically change our view of these resident “immune” cells. Here, we briefly review topics covered in our 2011 Society for Neuroscience minisymposium “The Role of Microglia in the Healthy Brain.” This summary is not meant to be a comprehensive review of microglia physiology, but rather to share new results and stimulate further research into the cellular and molecular mechanisms by which microglia influence postnatal development, adult neuronal plasticity, and circuit function.

A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression

A growing number of long nuclear‐retained non‐coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis‐associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II‐dependent transcription is active. Knock‐down studies revealed that Malat1 modulates the recruitment of SR family pre‐mRNA‐splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1‐depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock‐down of Malat1 decreases synaptic density, whereas its over‐expression results in a cell‐autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.

Microglial control of neuronal death and synaptic properties

Microglia have long been characterized by their immune function in the nervous system and are still mainly considered in a beneficial versus detrimental dialectic. However a review of literature enables to shed novel lights on microglial function under physiological conditions. It is now relevant to position these cells as full time partners of neuronal function and more specifically of synaptogenesis and developmental apoptosis. Indeed, microglia can actively control neuronal death. It has actually been shown in retina that microglial nerve growth factor (NGF) is necessary for the developmental apoptosis to occur. Similarly, in cerebellum, microglia induces developmental Purkinje cells death through respiratory burst. Furthermore, in spinal cord, microglial TNFα commits motoneurons to a neurotrophic dependent developmental apoptosis. Microglia can also control synaptogenesis. This is suggested by the fact that a mutation in KARAP/DAP12, a key protein of microglial activation impacts synaptic functions in hippocampus, and synapses protein content. In addition it has been now demonstrated that microglial brain‐derived neurotrophin factor (BDNF) directly regulates synaptic properties in spinal cord. In conclusion, microglia can control neuronal function under physiological conditions and it is known that neuronal activity reciprocally controls microglial activation. We will discuss the importance of this cross‐talk which allows microglia to orchestrate the balance between synaptogenesis and neuronal death occurring during development or injuries.

Microglia activation triggers astrocyte-mediated modulation of excitatory neurotransmission

Fine control of neuronal activity is crucial to rapidly adjust to subtle changes of the environment. This fine tuning was thought to be purely neuronal until the discovery that astrocytes are active players of synaptic transmission. In the adult hippocampus, microglia are the other major glial cell type. Microglia are highly dynamic and closely associated with neurons and astrocytes. They react rapidly to modifications of their environment and are able to release molecules known to control neuronal function and synaptic transmission. Therefore, microglia display functional features of synaptic partners, but their involvement in the regulation of synaptic transmission has not yet been addressed. We have used a combination of pharmacological approaches with electrophysiological analysis on acute hippocampal slices and ATP assays in purified cell cultures to show that activation of microglia induces a rapid increase of spontaneous excitatory postsynaptic currents. We found that this modulation is mediated by binding of ATP to P2Y1R located on astrocytes and is independent of TNFα or NOS2. Our data indicate that, on activation, microglia cells rapidly release small amounts of ATP, and astrocytes, in turn, amplified this release. Finally, P2Y1 stimulation of astrocytes increased excitatory postsynaptic current frequency through a metabotropic glutamate receptor 5-dependent mechanism. These results indicate that microglia are genuine regulators of neurotransmission and place microglia as upstream partners of astrocytes. Because pathological activation of microglia and alteration of neurotransmission are two early symptoms of most brain diseases, our work also provides a basis for understanding synaptic dysfunction in neuronal diseases.

Developmental neuronal death in hippocampus requires the microglial CD11b integrin and DAP12 immunoreceptor.

In several brain regions, microglia actively promote neuronal apoptosis during development. However, molecular actors leading microglia to trigger death remain mostly unknown. Here, we show that, in the developing hippocampus, apoptotic neurons are contacted by microglia expressing both the integrin CD11b and the immunoreceptor DAP12. We demonstrate that developmental apoptosis decreases in mice deficient for CD11b or DAP12. In addition, function-blocking antibodies directed against CD11b decrease neuronal death when injected into wild-type neonates, but have no effect when injected into DAP12-deficient littermates. This demonstrates that DAP12 and CD11b act in converging pathways to induce neuronal death. Finally, we show that DAP12 and CD11b control the production of microglial superoxide ions, which kill the neurons. Thus, our data show that the process of developmental neuronal death triggered by microglia is similar to the elimination of pathogenic cells by the innate immune cells.

Microglia Modulate Wiring of the Embryonic Forebrain

Dysfunction of microglia, the tissue macrophages of the brain, has been associated with the etiology of several neuropsychiatric disorders. Consistently, microglia have been shown to regulate neurogenesis and synaptic maturation at perinatal and postnatal stages. However, microglia invade the brain during mid-embryogenesis and thus could play an earlier prenatal role. Here, we show that embryonic microglia, which display a transiently uneven distribution, regulate the wiring of forebrain circuits. Using multiple mouse models, including cell-depletion approaches and cx3cr1(-/-), CR3(-/-), and DAP12(-/-) mutants, we find that perturbing microglial activity affects the outgrowth of dopaminergic axons in the forebrain and the laminar positioning of subsets of neocortical interneurons. Since defects in both dopamine innervation and cortical networks have been linked to neuropsychiatric diseases, our study provides insights into how microglial dysfunction can impact forebrain connectivity and reveals roles for immune cells during normal assembly of brain circuits.

Impaired Synaptic Function in the Microglial KARAP/DAP12-Deficient Mouse

Several proteins are expressed in both immune and nervous systems. However, their putative nonimmune functions in the brain remain poorly understood. KARAP/DAP12 is a transmembrane polypeptide associated with cell-surface receptors in hematopoeitic cells. Its mutation in humans induces Nasu-Hakola disease, characterized by presenile dementia and demyelinization. However, alteration of white matter occurs months after the onset of neuropsychiatric symptoms, suggesting that other neuronal alterations occur in the early phases of the disease. We hypothesized that KARAP/DAP12 may impact synaptic function. In mice deficient for KARAP/DAP12 function, long-term potentiation was enhanced and was partly NMDA receptor (NMDAR) independent. This effect was accompanied by changes in synaptic glutamate receptor content, as detected by the increased rectification of AMPA receptor EPSCs and increased sensitivity of NMDAR EPSCs to ifenprodil. Biochemical analysis of synaptic proteins confirmed these electrophysiological data. In mutants, the AMPA receptor GluR2 subunit expression was decreased only in the postsynaptic densities but not in the whole membrane fraction, demonstrating specific impairment of synaptic receptor accumulation. Alteration of the BNDF-tyrosine kinase receptor B (TrkB) signaling in the mutant was demonstrated by the dramatic decrease of synaptic TrkB with no change in other regulatory or scaffolding proteins. Finally, KARAP/DAP12 was detected only in microglia but not in neurons, astrocytes, or oligodendrocytes. KARAP/DAP12 may thus alter microglial physiology and subsequently synaptic function and plasticity through a novel microglia-neuron interaction.

Prenatal Activation of Microglia Induces Delayed Impairment of Glutamatergic Synaptic Function

Background Epidemiological studies have linked maternal infection during pregnancy to later development of neuropsychiatric disorders in the offspring. In mice, experimental inflammation during embryonic development impairs behavioral and cognitive performances in adulthood. Synaptic dysfunctions may be at the origin of cognitive impairments, however the link between prenatal inflammation and synaptic defects remains to be established. Methodology/Principal Findings In this study, we show that prenatal alteration of microglial function, including inflammation, induces delayed synaptic dysfunction in the adult. DAP12 is a microglial signaling protein expressed around birth, mutations of which in the human induces the Nasu-Hakola disease, characterized by early dementia. We presently report that synaptic excitatory currents in mice bearing a loss-of-function mutation in the DAP12 gene (DAP12KI mice) display enhanced relative contribution of AMPA. Furthermore, neurons from DAP12KI P0 pups cultured without microglia develop similar synaptic alterations, suggesting that a prenatal dysfunction of microglia may impact synaptic function in the adult. As we observed that DAP12KI microglia overexpress genes for IL1β, IL6 and NOS2, which are inflammatory proteins, we analyzed the impact of a pharmacologically-induced prenatal inflammation on synaptic function. Maternal injection of lipopolysaccharides induced activation of microglia at birth and alteration of glutamatergic synapses in the adult offspring. Finally, neurons cultured from neonates born to inflamed mothers and cultured without microglia also displayed altered neuronal activity. Conclusion/Significance Our results demonstrate that prenatal inflammation is sufficient to induce synaptic alterations with delay. We propose that these alterations triggered by prenatal activation of microglia provide a cellular basis for the neuropsychiatric defects induced by prenatal inflammation.

Morphologically identified glycinergic synapses in the hippocampus

Inhibitory transmission in the hippocampus is predominantly GABAergic, but electrophysiological data evidenced strychnine-sensitive glycine-induced currents. However, synaptic currents have not been reported. Here, we describe, for the first time, the presence of GlyR clusters in several areas of the hippocampus as well as in cultured hippocampal neurons. In contrast with spinal cord, hippocampal GlyRs contain alpha2 but no alpha1 subunit. Optical and electron microscopy indicates that GlyRs can be synaptic as well as extrasynaptic. Synaptic GlyRs were apposed to glycinergic boutons characterized by the expression of the vesicular and the plasma membrane transporters of glycine (VIAAT and GlyT2, respectively). Double labeling with calcium-binding proteins showed that GlyT2 could be detected in boutons innervating both excitatory cells (soma and dendrites) and interneurons. Finally, GlyR clusters could be detected at synaptic sites with the GABAA receptor gamma2 subunit and gephyrin, suggesting that mixed GABA/glycine synapses might exist in the hippocampus.

Association of gephyrin with synaptic and extrasynaptic GABAA receptors varies during development in cultured hippocampal neurons.

Several studies have reported extrasynaptic clusters of GABAA receptors in hippocampal neurons. Yet their functional relevance as well as their evolution in relation with gephyrin during synaptogenesis remain unknown. We have analyzed the expression pattern of the main proteins of the GABAergic synapses during synaptogenesis in cultured hippocampal neurons. We found that GABAergic terminals, characterized by VIAAT and GAD-65 expression, differentiated 3 to 7 days after the glutamatergic endings. At the postsynaptic side, the GABAAR- beta3 subunit was first diffuse and then clustered when GABAergic terminals differentiated and gephyrin formed large clusters. Colocalization of these proteins was high and increased with development. At later stages, GABAAR beta3 clusters colocalized with gephyrin at synaptic but also at extrasynaptic sites. GABAAR gamma2 subunits were directly expressed as clusters which were first extrasynaptic and not associated with gephyrin. Subsequently, the GABAAR gamma2 subunits associated with gephyrin at synaptic and/or extrasynaptic sites. Our data indicate that formation of GABAAR gamma2 subunit clusters is gephyrin independent.