Pathogenic MTOR somatic variant causing focal cortical dysplasia drives hyperexcitability via overactivation of neuronal GluN2C N-methyl-D-aspartate receptors.

OBJECTIVE: Genetic variations in proteins of the mechanistic target of rapamycin (mTOR) pathway cause a spectrum of neurodevelopmental disorders often associated with brain malformations and with intractable epilepsy. The mTORopathies are characterized by hyperactive mTOR pathway and comprise tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) type II. How hyperactive mTOR translates into abnormal neuronal activity and hypersynchronous network remains to be better understood. Previously, the role of upregulated GluN2C-containing glutamate-gated N-methyl-D-aspartate receptors (NMDARs) has been demonstrated for germline defects in the TSC genes. Here, we questioned whether this mechanism would expand to other mTORopathies in the different context of a somatic genetic variation of the MTOR protein recurrently found in FCD type II. METHODS: We used a rat model of FCD created by in utero electroporation of neural progenitors of dorsal telencephalon with expression vectors encoding either the wild-type or the pathogenic MTOR variant (p.S2215F). In this mosaic configuration, patch-clamp whole-cell recordings of the electroporated, spiny stellate neurons and extracellular recordings of the electroporated areas were performed in neocortical slices. Selective inhibitors were used to target mTOR activity and GluN2C-mediated currents. RESULTS: Neurons expressing the mutant protein displayed an excessive activation of GluN2C NMDAR-mediated spontaneous excitatory postsynaptic currents. GluN2C-dependent increase in spontaneous spiking activity was detected in the area of electroporated neurons in the mutant condition and was restricted to a critical time window between postnatal days P9 and P20. SIGNIFICANCE: Somatic MTOR pathogenic variant recurrently found in FCD type II resulted in overactivation of GluN2C-mediated neuronal NMDARs in neocortices of rat pups. The related and time-restricted local hyperexcitability was sensitive to subunit GluN2C-specific blockade. Our study suggests that GluN2C-related pathomechanisms might be shared in common by mTOR-related brain disorders.

Transient microstructural brain anomalies and epileptiform discharges in mice defective for epilepsy and language-related NMDA receptor subunit gene Grin2a.

OBJECTIVE: The epilepsy-aphasia spectrum (EAS) is a heterogeneous group of age-dependent childhood disorders characterized by sleep-activated discharges associated with infrequent seizures and language, cognitive, and behavioral deficits. Defects in the GRIN2A gene, encoding a subunit of glutamate-gated N-methyl-d-aspartate (NMDA) receptors, represent the most important cause of EAS identified so far. Neocortical or thalamic lesions were detected in a subset of severe EAS disorders, and more subtle anomalies were reported in patients with so-called "benign" phenotypes. However, whether brain structural alterations exist in the context of GRIN2A defects is unknown. METHODS: Magnetic resonance diffusion tensor imaging (MR-DTI) was used to perform longitudinal analysis of the brain at 3 developmental timepoints in living mice genetically knocked out (KO) for Grin2a. In addition, electroencephalography (EEG) was recorded using multisite extracellular electrodes to characterize the neocortical activity in vivo. RESULTS: Microstructural alterations were detected in the neocortex, the corpus callosum, the hippocampus, and the thalamus of Grin2a KO mice. Most MR-DTI alterations were detected at a specific developmental stage when mice were aged 30 days, but not at earlier (15 days) or later (2 months) ages. EEG analysis detected epileptiform discharges in Grin2a KO mice in the third postnatal week. SIGNIFICANCE: Grin2a KO mice replicated several anomalies found in patients with EAS disorders. Transient structural alterations detected by MR-DTI recalled the age-dependent course of EAS disorders, which in humans start during childhood and show variable outcome at the onset of adolescence. Together with the epileptiform discharges detected in young Grin2a KO mice, our data suggested the existence of early anomalies in the maturation of the neocortical and thalamocortical systems. Whereas the possible relationship of those anomalies with sleep warrants further investigations, our data suggest that Grin2a KO mice may serve as an animal model to study the neuronal mechanisms of EAS disorders and to design new therapeutic strategies.

Inactivation of Socs3 in the hypothalamus enhances the hindbrain response to endogenous satiety signals via oxytocin signaling.

Leptin is an adipocyte-derived hormone that controls energy balance by acting primarily in the CNS, but its action is lost in common forms of obesity due to central leptin resistance. One potential mechanism for such leptin resistance is an increased hypothalamic expression of Suppressor of cytokine signaling 3 (Socs3), a feedback inhibitor of the Jak-Stat pathway that prevents Stat3 activation. Ample studies have confirmed the important role of Socs3 in leptin resistance and obesity. However, the degree to which Socs3 participates in the regulation of energy homeostasis in nonobese conditions remains largely undetermined. In this study, using adult mice maintained under standard diet, we demonstrate that Socs3 deficiency in the mediobasal hypothalamus (MBH) reduces food intake, protects against body weight gain, and limits adiposity, suggesting that Socs3 is necessary for normal body weight maintenance. Mechanistically, MBH Socs3-deficient mice display increased hindbrain sensitivity to endogenous, meal-related satiety signals, mediated by oxytocin signaling. Thus, oxytocin signaling likely mediates the effect of hypothalamic leptin on satiety circuits of the caudal brainstem. This provides an anatomical substrate for the effect of leptin on meal size, and more generally, a mechanism for how the brain controls short-term food intake as a function of the energetic stores available in the organism to maintain energy homeostasis. Any dysfunction in this pathway could potentially lead to overeating and obesity.

The cell cycle-apoptosis connection revisited in the adult brain.

Adult neurogenesis is studied in vivo using thymidine analogues such as bromodeoxyuridine (BrdU) to label DNA synthesis during the S phase of the cell cycle. However, BrdU may also label DNA synthesis events not directly related to cell proliferation, such as DNA repair and/or abortive reentry into the cell cycle, which can occur as part of an apoptotic process in postmitotic neurons. In this study, we used three well-characterized models of injury-induced neuronal apoptosis and the combined visualization of cell birth (BrdU labeling) and death (Tdt-mediated dUTP-biotin nick end labeling) to investigate the specificity of BrdU incorporation in the adult mouse brain in vivo. We present evidence that BrdU is not significantly incorporated during DNA repair and that labeling is not detected in vulnerable or dying postmitotic neurons, even when a high dose of BrdU is directly infused into the brain. These findings have important implications for a controversy surrounding adult neurogenesis: the connection between cell cycle reactivation and apoptosis of terminally differentiated neurons.

In Utero Administration of Drugs Targeting Microglia Improves the Neurodevelopmental Outcome Following Cytomegalovirus Infection of the Rat Fetal Brain.

Congenital cytomegalovirus (CMV) infections represent one leading cause of neurodevelopmental disorders. Recently, we reported on a rat model of CMV infection of the developing brain in utero, characterized by early and prominent infection and alteration of microglia-the brain-resident mononuclear phagocytes. Besides their canonical function against pathogens, microglia are also pivotal to brain development. Here we show that CMV infection of the rat fetal brain recapitulated key postnatal phenotypes of human congenital CMV including increased mortality, sensorimotor impairment reminiscent of cerebral palsy, hearing defects, and epileptic seizures. The possible influence of early microglia alteration on those phenotypes was then questioned by pharmacological targeting of microglia during pregnancy. One single administration of clodronate liposomes in the embryonic brains at the time of CMV injection to deplete microglia, and maternal feeding with doxycyxline throughout pregnancy to modify microglia in the litters' brains, were both associated with dramatic improvements of survival, body weight gain, sensorimotor development and with decreased risk of epileptic seizures. Improvement of microglia activation status did not persist postnatally after doxycycline discontinuation; also, active brain infection remained unchanged by doxycycline. Altogether our data indicate that early microglia alteration, rather than brain CMV load per se, is instrumental in influencing survival and the neurological outcomes of CMV-infected rats, and suggest that microglia might participate in the neurological outcome of congenital CMV in humans. Furthermore this study represents a first proof-of-principle for the design of microglia-targeted preventive strategies in the context of congenital CMV infection of the brain.

Leukemia inhibitory factor promotes neural stem cell self-renewal in the adult brain.

Although neural stem cells (NSCs) persist in various areas of the adult brain, their contribution to brain repair after injury is very limited. Treatment with exogenous growth factors can mitigate this limitation, suggesting that the brain environment is normally deficient in permissive cues and that it may be possible to stimulate the latent regenerative potential of endogenous progenitors with appropriate signals. We analyzed the effects of overexpressing the cytokine leukemia inhibitory factor (LIF) on adult neurogenesis in the normal brain. We found that LIF reduces neurogenesis in the olfactory bulb and subventricular zone by acting directly on NSCs. LIF appears to promote NSC self-renewal, preventing the emergence of more differentiated cell types. This ultimately leads to an expansion of the NSC pool. Our results have implications for the development of therapeutic strategies for brain repair and suggest that LIF may be useful, in combination with other factors, in promoting regeneration in the adult brain.

Neurogenesis and neural stem cells in the dorsal vagal complex of adult rat brain: new vistas about autonomic regulations--a review.

The dorsal vagal complex (DVC) of the brainstem is the major reflex center of autonomic nervous system. Several neuroplasticity effectors have been identified in the DVC of adult rat, such as PSA-NCAM, GAP-43, BDNF and its receptor TrkB; moreover, acute vagal stimulation was found to induce c-fos and to down-regulate western-blot-assayed tissular concentration of PSA-NCAM. Adult neurogenesis was first shown in rat DVC by BrdU incorporation combined with phenotypic labelling in situ; new neurons are generated in equal proportions with new astrocytes and at a lower rate than in olfactory bulb or hippocampus. Intrinsic proliferative cells were then detected within the DVC of adult rat by means of Ki-67 immunohistochemistry and western-blot of D-cyclins. The presence of neural stem cells within DVC was directly demonstrated by applying the in vitro neurosphere assay on microdissected adult DVC explants; DVC-derived neurospheres display lower proliferation rate and neurogenic potential than forebrain ones. Vagotomy in adult promotes massive and transient increase of neurogenic and microglial proliferations within DVC, the kinetics and location of which were analyzed by Ki-67 immunohistochemistry and cyclin D western blot. These mechanisms shed light on so far unknown plasticity potential in DVC, which brings novel cues about physiological adaptations of autonomic reflexes in adult mammals.

Cytomegalovirus Infection of the Rat Developing Brain In Utero Prominently Targets Immune Cells and Promotes Early Microglial Activation.

BACKGROUND: Congenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells. OBJECTIVES AND METHODS: In order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15) and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments. RESULTS: Rat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b- lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1. CONCLUSION: In line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which role, whether favorable or detrimental, those putative double-edge swords events actually play.

An Early Postnatal Oxytocin Treatment Prevents Social and Learning Deficits in Adult Mice Deficient for Magel2, a Gene Involved in Prader-Willi Syndrome and Autism.

BACKGROUND: Mutations of MAGEL2 have been reported in patients presenting with autism, and loss of MAGEL2 is also associated with Prader-Willi syndrome, a neurodevelopmental genetic disorder. This study aimed to determine the behavioral phenotype of Magel2-deficient adult mice, to characterize the central oxytocin (OT) system of these mutant mice, and to test the curative effect of a peripheral OT treatment just after birth. METHODS: We assessed the social and cognitive behavior of Magel2-deficient mice, analyzed the OT system of mutant mice treated or not by a postnatal administration of OT, and determined the effect of this treatment on the brain. RESULTS: Magel2 inactivation induces a deficit in social recognition and social interaction and a reduced learning ability in adult male mice. In these mice, we reveal anatomical and functional modifications of the OT system and show that these defects change from birth to adulthood. Daily administration of OT in the first postnatal week was sufficient to prevent deficits in social behavior and learning abilities in adult mutant male mice. We show that this OT treatment partly restores a normal OT system. Thus, we report that an alteration of the OT system around birth has long-term consequences on behavior and on cognition. Importantly, an acute OT treatment of Magel2-deficient pups has a curative effect. CONCLUSIONS: Our study reveals that OT plays a crucial role in setting social behaviors during a period just after birth. An early OT treatment in this critical period could be a novel therapeutic approach for the treatment of neurodevelopmental disorders such as Prader-Willi syndrome and autism.

Cerebral cell renewal in adult mice controls the onset of obesity.

The hypothalamus plays a crucial role in the control of the energy balance and also retains neurogenic potential into adulthood. Recent studies have reported the severe alteration of the cell turn-over in the hypothalamus of obese animals and it has been proposed that a neurogenic deficiency in the hypothalamus could be involved in the development of obesity. To explore this possibility, we examined hypothalamic cell renewal during the homeostatic response to dietary fat in mice, i.e., at the onset of diet-induced obesity. We found that switching to high-fat diet (HFD) accelerated cell renewal in the hypothalamus through a local, rapid and transient increase in cell proliferation, peaking three days after introducing the HFD. Blocking HFD-induced cell proliferation by central delivery of an antimitotic drug prevented the food intake normalization observed after HFD introduction and accelerated the onset of obesity. This result showed that HFD-induced dividing brain cells supported an adaptive anorectic function. In addition, we found that the percentage of newly generated neurons adopting a POMC-phenotype in the arcuate nucleus was increased by HFD. This observation suggested that the maturation of neurons in feeding circuits was nutritionally regulated to adjust future energy intake. Taken together, these results showed that adult cerebral cell renewal was remarkably responsive to nutritional conditions. This constituted a physiological trait required to prevent severe weight gain under HFD. Hence this report highlighted the amazing plasticity of feeding circuits and brought new insights into our understanding of the nutritional regulation of the energy balance.

Alteration of forebrain neurogenesis after cervical spinal cord injury in the adult rat.

Spinal cord injury (SCI) triggers a complex cellular response at the injury site, leading to the formation of a dense scar tissue. Despite this local tissue remodeling, the consequences of SCI at the cellular level in distant rostral sites (i.e., brain), remain unknown. In this study, we asked whether cervical SCI could alter cell dynamics in neurogenic areas of the adult rat forebrain. To this aim, we quantified BrdU incorporation and determined the phenotypes of newly generated cells (neurons, astrocytes, or microglia) during the subchronic and chronic phases of injury. We find that subchronic SCI leads to a reduction of BrdU incorporation and neurogenesis in the olfactory bulb and in the hippocampal dentate gyrus. By contrast, subchronic SCI triggers an increased BrdU incorporation in the dorsal vagal complex of the hindbrain, where most of the newly generated cells are identified as microglia. In chronic condition 90 days after SCI, BrdU incorporation returns to control levels in all regions examined, except in the hippocampus, where SCI produces a long-term reduction of neurogenesis, indicating that this structure is particularly sensitive to SCI. Finally, we observe that SCI triggers an acute inflammatory response in all brain regions examined, as well as a hippocampal-specific decline in BDNF levels. This study provides the first demonstration that forebrain neurogenesis is vulnerable to a distal SCI.

Cytokine control of adult neural stem cells.

The neuropoietic cytokine family includes interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF), among others. These cytokines have been shown to alter neural stem cell (NSC) self-renewal and progenitor cell division and differentiation, which could be mediated by the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway. Using neurospheres from the adult mouse subventricular zone (SVZ), we found that acute or chronic exposure to LIF or CNTF differentially affects sphere development and sphere growth. Both cytokines also favor the amplification of NSCs. Contrasting results were obtained with IL-6 or leptin, although both cytokines also activate the JAK/STAT pathway. Stimulating NSC self-renewal in vivo could be of therapeutic interest for treating neurodegeneration. When applied to the adult mouse brain, chronic LIF stimulates NSC self-renewal but prevents the emergence of more differentiated progeny. On the other hand, acute LIF treatment stimulates SVZ regeneration, most likely through an increase in NSCs. These results reveal that cytokine effects could vary as a function of exposure duration and suggest that, in the search for strategies to promote brain repair, in vivo acute LIF treatment could promote cell replacement.

The neuropoietic cytokine family in development, plasticity, disease and injury.

Neuropoietic cytokines are well known for their role in the control of neuronal, glial and immune responses to injury or disease. Since this discovery, it has emerged that several of these proteins are also involved in nervous system development, in particular in the regulation of neurogenesis and stem cell fate. Recent data indicate that these proteins have yet more functions, as key modulators of synaptic plasticity and of various behaviours. In addition, neuropoietic cytokines might be a factor in the aetiology of psychiatric disorders.