Jean-Marie Mangin

A functional role for EGFR signaling in myelination and remyelination.

Cellular strategies for oligodendrocyte regeneration and remyelination involve characterizing endogenous neural progenitors that are capable of generating oligodendrocytes during normal development and after demyelination, and identifying the molecular signals that enhance oligodendrogenesis from these progenitors. Using both gain- and loss-of-function approaches, we explored the role of epidermal growth factor receptor (EGFR) signaling in adult myelin repair and in oligodendrogenesis. We show that 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) promoter–driven overexpression of human EGFR (hEGFR) accelerated remyelination and functional recovery following focal demyelination of mouse corpus callosum. Lesion repopulation by Cspg4+ (also known as NG2) Ascl1+ (also known as Mash1) Olig2+ progenitors and functional remyelination were accelerated in CNP-hEGFR mice compared with wild-type mice. EGFR overexpression in subventricular zone (SVZ) and corpus callosum during early postnatal development also expanded this NG2+Mash1+Olig2+ progenitor population and promoted SVZ-to-lesion migration, enhancing oligodendrocyte generation and axonal myelination. Analysis of hypomorphic EGFR-mutant mice confirmed that EGFR signaling regulates oligodendrogenesis and remyelination by NG2+Mash1+Olig2+ progenitors. EGFR targeting holds promise for enhancing oligodendrocyte regeneration and myelin repair.

Adult-born SVZ progenitors receive transient synapses during remyelination in corpus callosum.

We found that demyelinated axons formed functional glutamatergic synapses onto adult-born NG2+ oligodendrocyte progenitor cells (OPCs) migrating from the subventricular zone after focal demyelination of adult mice corpus callosum. This glutamatergic input was substantially reduced compared with endogenous callosal OPCs 1 week after lesion and was lost on differentiation into oligodendrocytes. Therefore, axon–oligodendrocyte progenitor synapse formation is a transient and regulated step that occurs during remyelination of callosal axons.

Experience-dependent regulation of NG2 progenitors in the developing barrel cortex

We found that, during the formation of the mouse barrel cortex, NG2 cells received glutamatergic synapses from thalamocortical fibers and preferentially accumulated along septa separating the barrels. Sensory deprivation reduced thalamocortical inputs on NG2 cells and increased their proliferation, leading to a more uniform distribution in the deprived barrels. Thus, early sensory experience regulates thalamocortical innervation on NG2 cells, as well as their proliferation and distribution during development.

Synapses on NG2-expressing progenitors in the brain: multiple functions?

Progenitor cells expressing the proteoglycan NG2 represent approximately 5% of the total cells in the adult brain, and are found both in grey and white matter regions where they give rise to oligodendrocytes. The finding that these cells receive synaptic contacts from excitatory and inhibitory neurons has not only raised major interest in the possible roles of these synapses, but also stimulated further research on the developmental and cellular functions of NG2‐expressing (NG2+) progenitors themselves in the context of neural circuit physiology. Here we review recent findings on the functional properties of the synapses on NG2+ cells in grey and white matter regions of the brain. In this review article we make an attempt to integrate current knowledge on the cellular and developmental properties of NG2+ progenitors with the functional attributes of their synapses, in order to understand the physiological relevance of neuron–NG2+ progenitor signal transmission. We propose that, although NG2+ progenitors receive synaptic contact in all brain regions where they are found, their synapses might have different developmental and functional roles, probably reflecting the distinct functions of NG2+ progenitors in the brain.

Gene Expression Profiling of Astrocytes from Hyperammonemic Mice Reveals Altered Pathways for Water and Potassium Homeostasis In Vivo

Acute hyperammonemia (HA) causes cerebral edema and brain damage in children with urea cycle disorders (UCDs) and in patients in acute liver failure. Chronic HA is associated with developmental delay and mental retardation in children with UCDs, and with neuropsychiatric symptoms in patients with chronic liver failure. Astrocytes are a major cellular target of hyperammonemic encephalopathy, and changes occurring in these cells are thought to be causally related to the brain edema of acute HA. To study the effect of HA on astrocytes in vivo, we crossed the Otcspf mouse, a mouse with the X‐linked UCD ornithine transcarbamylase (OTC) deficiency, with the hGFAP‐EGFP mouse, a mouse selectively expressing green fluorescent protein in astrocytes. We used FACS to purify astrocytes from the brains of hyperammonemic and healthy Otcspf/GFAP‐EGFP mice. RNA isolated from these astrocytes was used in microarray expression analyses and qRT‐PCR. When compared with healthy littermates, we observed a significant downregulation of the gap‐junction channel connexin 43 (Cx43) the water channel aquaporin 4 (Aqp4) genes, and the astrocytic inward‐rectifying potassium channel (Kir) genes Kir4.1 and Kir5.1 in hyperammonemic mice. Aqp4, Cx43, and Kir4.1/Kir5.1 are co‐localized to astrocytic end‐feet at the brain vasculature, where they regulate potassium and water transport. Since, NH4+ ions can permeate water and K+‐channels, downregulation of these three channels may be a direct effect of elevated blood ammonia levels. Our results suggest that alterations in astrocyte‐mediated water and potassium homeostasis in brain may be key to the development of the brain edema.

Kinetic properties of the α2 homo-oligomeric glycine receptor impairs a proper synaptic functioning

Ionotropic glycine receptors (GlyRs) are present in the central nervous system well before the establishment of synaptic contacts. Immature nerve cells are known, at least in the spinal cord, to express α2 homomeric GlyRs, the properties of which are relatively unknown compared to those of the adult synaptic form of the GlyR (mainly α1/β heteromeres). Here, the kinetics properties of GlyRs at the single‐channel level have been recorded in real‐time by means of the patch‐clamp technique in the outside‐out configuration coupled with an ultra‐fast flow application system (< 100 µs). Recordings were performed on chinese hamster ovary (CHO) cells stably transfected with the α2 GlyR subunit. We show that the onset, the relaxation and the desensitisation of α2 homomeric GlyR‐mediated currents are slower by one or two orders of magnitude compared to synaptic mature GlyRs and to other ligand‐gated ionotropic channels involved in fast synaptic transmission. First latency analysis performed on single GlyR channels revealed that their slow activation time course was due to delayed openings. When synaptic release of glycine was mimicked (1 mm glycine; 1 ms pulse duration), the opening probability of α2 homomeric GlyRs was low (Po≈ 0.1) when compared to mature synaptic GlyRs (Po= 0.9). This low Po is likely to be a direct consequence of the relatively slow activation kinetics of α2 homomeric GlyRs when compared to the activation kinetics of mature α1/β GlyRs. Such slow kinetics suggest that embryonic α2 homomeric GlyRs cannot be activated by fast neurotransmitter release at mature synapses but rather could be suited for a non‐synaptic paracrine‐like release of agonist, which is known to occur in the embryo.

Satellite NG2 Progenitor Cells Share Common Glutamatergic Inputs with Associated Interneurons in the Mouse Dentate Gyrus

Several studies have provided evidence that NG2-expressing (NG2+) progenitor cells are anatomically associated to neurons in gray matter areas. By analyzing the spatial distribution of NG2+ cells in the hilus of the mouse dentate gyrus, we demonstrate that NG2+ cells are indeed closely associated to interneurons. To define whether this anatomical proximity reflected a specific physiological interaction, we performed patch-clamp recordings on hilar NG2+ cells and interneurons between 3 and 21 postnatal days. We first observed that hilar NG2+ cells exhibit spontaneous glutamatergic EPSCs (sEPSCs) whose frequency and amplitude increase during the first 3 postnatal weeks. At the same time, the rise time and decay time of sEPSCs significantly decreased, suggesting that glutamatergic synapses in NG2+ cells undergo a maturation process that is reminiscent of what has been reported in neurons during the same time period. We also observed that hilar interneurons and associated NG2+ cells are similarly integrated into the local network, receiving excitatory inputs from both granule cells and CA3 pyramidal neurons. By performing pair recordings, we found that bursts of activity induced by GABAergic antagonists were strongly synchronized between both cell types and that the amplitude of these bursts was positively correlated. Finally, by applying carbachol to increase EPSC activity, we observed that closely apposed cells were more likely to exhibit synchronized EPSCs than cells separated by >200 μm. The finding that NG2+ cells are sensing patterns of activity arising in closely associated neurons suggests that NG2+ cell function is finely regulated by the local network.

Functional glycine receptor maturation in the absence of glycinergic input in dopaminergic neurones of the rat substantia nigra.

The postnatal maturation pattern of glycine receptor channels (GlyRs) expressed by dopaminergic (DA) neurones of the rat substantia nigra pars compacta (SNc) was investigated using single‐channel and whole‐cell patch‐clamp recordings in brain slices from rats aged 7–21 postnatal days (P). In neonatal rats (P7‐P10), GlyRs exhibited a main conductance state of 100–110 pS with a mean open time of 16 ms. In juvenile rats (P19‐P22), both the GlyR main conductance state (46‐55 pS) and the mean open time (6.8 ms) were decreased. In neonatal rats, application of 30 μm picrotoxin, which is known to block homomeric GlyRs, strongly reduced glycine‐evoked responses, while it was much less effective in juvenile rats. These results suggest that these GlyRs correspond functionally to α2 homomeric GlyRs in neonatal rats and α1/β heteromeric GlyRs in juvenile rats. A drastic but transient decrease in the glycine responsiveness of DA neurones occurred around P17 concomitant to the functional switch from the homomeric state to the heteromeric state. This age corresponds to a maturation phase for DA neurones. The application of 1 μm gabazine blocked spontaneous or evoked inhibitory synaptic current, while the addition of 1 μm strychnine had no effect, suggesting a lack of functional glycinergic synapses on DA neurones. Although it has been proposed that taurine is co‐released with GABA at GABAergic synapses on DA neurones, in the present study the stimulation of GABAergic fibres failed to activate GlyRs. Blockade of taurine transporters and applications of high K+ and hyposmotic solutions were also unable to induce any strychnine‐sensitive current. We conclude that functional maturation of GlyRs can occur in the absence of any detectable GlyR activation in DA neurones of the SNc.

The Curious Case of NG2 Cells: Transient Trend or Game Changer?

It has been 10 years since the seminal work of Dwight Bergles and collaborators demonstrated that NG2 (nerve/glial antigen 2)-expressing oligodendrocyte progenitor cells (NG2 cells) receive functional glutamatergic synapses from neurons (Bergles et al., 2000), contradicting the old dogma that only neurons possess the complex and specialized molecular machinery necessary to receive synapses. While this surprising discovery may have been initially shunned as a novelty item of undefined functional significance, the study of neuron-to-NG2 cell neurotransmission has since become a very active and exciting field of research. Many laboratories have now confirmed and extended the initial discovery, showing for example that NG2 cells can also receive inhibitory GABAergic synapses (Lin and Bergles, 2004) or that neuron-to-NG2 cell synaptic transmission is a rather ubiquitous phenomenon that has been observed in all brain areas explored so far, including white matter tracts (Kukley et al., 2007; Ziskin et al., 2007; Etxeberria et al., 2010). Thus, while still being in its infancy, this field of research has already brought many surprising and interesting discoveries, and has become part of a continuously growing effort in neuroscience to re-evaluate the long underestimated role of glial cells in brain function (Barres, 2008). However, this area of research is now reaching an important milestone and its long-term significance will be defined by its ability to uncover the still elusive function of NG2 cells and their synapses in the brain, rather than by its sensational but transient successes at upsetting the old order established by neuronal physiology. To participate in the effort to facilitate such a transition, here we propose a critical review of the latest findings in the field of NG2 cell physiology – discussing how they inform us on the possible function(s) of NG2 cells in the brain – and we present some personal views on new directions the field could benefit from in order to achieve lasting significance.

Chronic Perinatal Hypoxia Reduces Glutamate–Aspartate Transporter Function in Astrocytes through the Janus Kinase/Signal Transducer and Activator of Transcription Pathway

The cellular and molecular mechanisms that govern the response of the perinatal brain to injury remain largely unexplored. We investigated the role of white matter astrocytes in a rodent model of diffuse white matter injury produced by exposing neonatal mice to chronic hypoxia—a paradigm that mimics brain injury in premature infants. We demonstrate the absence of reactive gliosis in the immature white matter following chronic hypoxia, as determined by astrocyte proliferation index and glial fibrillary acidic protein levels. Instead, Nestin expression in astrocytes is transiently increased, and the glial-specific glutamate transporters glutamate–aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) are reduced. Finally, we demonstrate that Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling—which is important in both astrocyte development and response to injury—is reduced in the white matter following hypoxia, as well as in primary astrocytes exposed to hypoxia in vitro. Hypoxia and JAK/STAT inhibition reduce glutamate transporter expression in astrocytes, but unlike hypoxia JAK/STAT inhibition downregulates GLAST expression without affecting GLT-1, as demonstrated in vitro by treatment with JAK inhibitor I and in vivo by treatment with the JAK/STAT inhibitor AG490 [(E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide]. Our findings (1) demonstrate specific changes in astrocyte function after perinatal hypoxia, which might contribute to the particular pathogenesis of perinatal white matter injury, (2) provide evidence that at least part of these changes result from a disturbance of the JAK/STAT pathway by hypoxia, and (3) identify JAK/STAT signaling as a potential therapeutic target to restore normal GLAST expression and uptake of glutamate after perinatal brain injury.