Catherine Béchade

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.

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.

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.

Expression of Glycine Receptor α Subunits and Gephyrin in Cultured Spinal Neurons

The inhibitory glycine receptor is a pentameric membrane protein composed of α and β subunits. In the postsynaptic membrane, the glycine receptor and the copurifying peripheral membrane protein gephyrin are clustered underneath glycine‐releasing nerve terminals. Here, we describe the expression of gephyrin and the neonatal and adult glycine receptor α subunit isoforms α1 and α2 during in vitro differentiation of rat spinal neurons. Analysis by immunoassays and the reverse transcriptase—polymerase chain reaction showed that gephyrin and α subunit mRNA and protein levels exhibited a marked increase from 1 to 5 days in vitro, i.e. prior to the formation of functional synaptic contacts. Using confocal and standard immunofluorescence, we determined the number of immunoreactive cells and the cellular localization of the α subunits and gephyrin. At 3 days in vitro, glycine receptor immunoreactivity revealed by the monoclonal antibody mAb4a was found in >10% of cells and was mainly localized intracellularly; in contrast, gephyrin was detected in <50% of cells. At 7 days in vitro, gephyrin was essentially localized at the neuronal surface. At this stage, the number of glycine receptor‐positive cells approached that of gephyrin‐containing neurons (50%), and glycine receptor antigen was found both intracellularly and at the periphery of the cells. The antibody mAb2b, which binds exclusively to the α1 subunit, revealed aggregates at the surface of a few neurons. At 10 days in vitro, glycine receptor and gephyrin staining was localized in clusters at the periphery of the soma and the neurites. This quantitative analysis corroborates temporal differences in the cellular distribution of gephyrin and glycine receptor α subunits, the former being accumulated first at the neuronal surface.

Physiological roles of microglia during development

J. Neurochem. (2011) 119, 901–908.

Nerve growth factor (NGF) induces motoneuron apoptosis in rat embryonic spinal cord in vitro.

Recent studies have demonstrated that nerve growth factor (NGF) induces apoptosis of several cell types in the central nervous system through its low‐affinity p75 neurotrophin receptor (p75NTR). To test the effect of NGF on embryonic motoneuron survival, we developed an organotypic culture system which allowed the in vitro development of intact embryonic rat spinal cords. In our system, neural tubes were taken and cultured at E13, just before the onset of physiological motoneuron death. After 2 days in vitro (DIV), motoneurons underwent apoptosis over a time‐course similar to that in vivo. In this system, the addition of NGF (200 ng/mL) for 2 days enhanced the number of apoptotic motoneurons by 37%. This pro‐apoptotic effect was completely reversed by the blocking anti‐p75NTR (REX) antibody which inhibits NGF binding to p75NTR. Other neurotrophins, e.g. brain‐derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5) did not have any effect, while glial cell‐derived neurotrophic factor (GDNF) promoted motoneuron survival. Anti‐BDNF blocking antibodies enhanced motoneuron death indicating that endogenous BDNF promotes motoneuron survival in explants. Our results demonstrate, for the first time, that NGF can induce embryonic motoneuron apoptosis through its receptor p75NTR.

Macrophage-Derived Tumor Necrosis Factor α, an Early Developmental Signal for Motoneuron Death

Mechanisms inducing neuronal death at defined times during embryogenesis remain enigmatic. We show in explants that a developmental switch occurs between embryonic day 12 (E12) and E13 in rats that is 72-48 hr before programmed cell death. Half the motoneurons isolated from peripheral tissues at E12 escape programmed cell death, whereas 90% of motoneurons isolated at E13 enter a death program. The surrounding somite commits E12 motoneurons to death. This effect requires macrophage cells, is mimicked by tumor necrosis factor α (TNFα), and is inhibited by anti-TNFα antibodies. In vivo, TNFα is detected within somite macrophages, and TNF receptor 1 (TNFR1) is detected within motoneurons precisely between E12 and E13. Although motoneuron cell death occurs normally in TNFα-/- mice, this process is significantly reduced in explants from TNFα-/- and TNFR1 -/- mice. Thus, embryonic motoneurons acquire the competence to die, before the onset of programmed cell death, from extrinsic signals such as macrophage-derived TNFα

Microglial control of neuronal activity.

Fine-tuning of neuronal activity was thought to be a neuron-autonomous mechanism until the discovery that astrocytes are active players of synaptic transmission. The involvement of astrocytes has changed our understanding of the roles of non-neuronal cells and shed new light on the regulation of neuronal activity. Microglial cells are the macrophages of the brain and they have been mostly investigated as immune cells. However, recent data discussed in this review support the notion that, similarly to astrocytes, microglia are involved in the regulation of neuronal activity. For instance, in most, if not all, brain pathologies a strong temporal correlation has long been known to exist between the pathological activation of microglia and dysfunction of neuronal activity. Recent studies have convincingly shown that alteration of microglial function is responsible for pathological neuronal activity. This causal relationship has also been demonstrated in mice bearing loss-of-function mutations in genes specifically expressed by microglia. In addition to these long-term regulations of neuronal activity, recent data show that microglia can also rapidly regulate neuronal activity, thereby acting as partners of neurotransmission.

Is astrocyte calcium signaling relevant for synaptic plasticity

Astrocytes constitute a major group of glial cells which were long regarded as passive elements, fulfilling nutritive and structural functions for neurons. Calcium rise in astrocytes propagating to neurons was the first demonstration of direct interaction between the two cell types. Since then, calcium has been widely used, not only as an indicator of astrocytic activity but also as a stimulator switch to control astrocyte physiology. As a result, astrocytes have been elevated from auxiliaries to neurons, to cells involved in processing synaptic information. Curiously, while there is evidence that astrocytes play an important role in synaptic plasticity, the data relating to calciums pivotal role are inconsistent. In this review, we will detail the various mechanisms of calcium flux in astrocytes, then briefly present the calcium-dependent mechanisms of gliotransmitter release. Finally, we will discuss the role of calcium in plasticity and present alternative explanations that could reconcile the conflicting results published recently.