Immature Astrocytes Research Articles

Breast carcinoma-amplified sequence 2 regulates adult neurogenesis via \u03b2-catenin

BackgroundBreast carcinoma-amplified sequence 2 (BCAS2) regulates β-catenin gene splicing. The conditional knockout of BCAS2 expression in the forebrain (BCAS2 cKO) of mice confers impaired learning and memory along with decreased β-catenin expression. Because β-catenin reportedly regulates adult neurogenesis, we wondered whether BCAS2 could regulate adult neurogenesis via β-catenin.MethodsBCAS2-regulating neurogenesis was investigated by characterizing BCAS2 cKO mice. Also, lentivirus-shBCAS2 was intracranially injected into the hippocampus of wild-type mice to knock down BCAS2 expression. We evaluated the rescue effects of BCAS2 cKO by intracranial injection of adeno-associated virus encoding BCAS2 (AAV-DJ8-BCAS2) and AAV-β-catenin gene therapy.ResultsTo show that BCAS2-regulating adult neurogenesis via β-catenin, first, BCAS2 cKO mice showed low SRY-box 2-positive (Sox2+) neural stem cell proliferation and doublecortin-positive (DCX+) immature neurons. Second, stereotaxic intracranial injection of lentivirus-shBCAS2 knocked down BCAS2 in the hippocampus of wild-type mice, and we confirmed the BCAS2 regulation of adult neurogenesis via β-catenin. Third, AAV-DJ8-BCAS2 gene therapy in BCAS2 cKO mice reversed the low proliferation of Sox2+ neural stem cells and the decreased number of DCX+ immature neurons with increased β-catenin expression. Moreover, AAV-β-catenin gene therapy restored neuron stem cell proliferation and immature neuron differentiation, which further supports BCAS2-regulating adult neurogenesis via β-catenin. In addition, cells targeted by AAV-DJ8 injection into the hippocampus included Sox2 and DCX immature neurons, interneurons, and astrocytes. BCAS2 may regulate adult neurogenesis by targeting Sox2+ and DCX+ immature neurons for autocrine effects and interneurons or astrocytes for paracrine effects.ConclusionsBCAS2 can regulate adult neurogenesis in mice via β-catenin.

Hemisynapse Formation Between Target Astrocytes and Cortical Neuron Axons in vitro.

One of the most fundamental organizing principles in the mammalian brain is that neurons do not establish synapses with the other major cell type, the astrocytes. However, induced synapse formation between neurons and astrocytes appears conceivable, because astrocytes are well known to express functional ionotropic glutamate receptors. Here, we attempted to trigger synapse formation between co-cultured neurons and astrocytes by overexpressing the strongly synaptogenic adhesion protein LRRTM2 in astrocytes physically contacted by cortical axons. Interestingly, control experiments with immature cortical astrocytes without any overexpression resulted in the induction of synaptic vesicle clustering in contacting axons (hemisynapse formation). This synaptogenic activity correlated with the endogenous expression of the synaptogenic protein Neuroligin1. Hemisynapse formation was further enhanced upon overexpression of LRRTM2 in cortical astrocytes. In contrast, cerebellar astrocytes required overexpression of LRRTM2 for induction of synaptic vesicle clustering in contacting axons. We further addressed, whether hemisynapse formation was accompanied by the appearance of fully functional glutamatergic synapses. We therefore attempted to record AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in innervated astrocytes using the whole-cell patch-clamp technique. Despite the endogenous expression of the AMPA receptor subunits GluA2 and to a lesser extent GluA1, we did not reliably observe spontaneous AMPA mEPSCs. In conclusion, overexpression of the synaptogenic protein LRRTM2 induced hemisynapse formation between co-cultured neurons and astrocytes. However, the formation of fully functional synapses appeared to require additional factors critical for nano-alignment of presynaptic vesicles and postsynaptic receptors.

Overexpressed ski efficiently promotes neurorestoration, increases neuronal regeneration, and reduces astrogliosis after traumatic brain injury.

Traumatic brain injury (TBI) survivors suffer from long-term disability and neuropsychiatric sequelae due to irreparable brain tissue destruction. However, there are still few efficient therapies to promote neurorestoration in damaged brain tissue. This study aimed to investigate whether the pro-oncogenic gene ski can promote neurorestoration after TBI. We established a ski-overexpressing experimental TBI mouse model using adenovirus-mediated overexpression through immediate injection after injury. Hematoxylin-eosin staining, MRI-based 3D lesion volume reconstruction, neurobehavioral tests, and analyses of neuronal regeneration and astrogliosis were used to assess neurorestorative efficiency. The effects of ski overexpression on the proliferation of cultured immature neurons and astrocytes were evaluated using imaging flow cytometry. The Ski protein level increased in the perilesional region at 3 days post injury. ski overexpression further elevated Ski protein levels up to 14 days post injury. Lesion volume was attenuated by approximately 36-55% after ski overexpression, with better neurobehavioral recovery, more newborn immature and mature neurons, and less astrogliosis in the perilesional region. Imaging flow cytometry results showed that ski overexpression elevated the proliferation rate of immature neurons and reduced the proliferation rate of astrocytes. These results show that ski can be considered a novel neurorestoration-related gene that effectively promotes neurorestoration, facilitates neuronal regeneration, and reduces astrogliosis after TBI.

Neurovascular Coupling in Development and Disease: Focus on Astrocytes.

Neurovascular coupling is a crucial mechanism that matches the high energy demand of the brain with a supply of energy substrates from the blood. Signaling within the neurovascular unit is responsible for activity-dependent changes in cerebral blood flow. The strength and reliability of neurovascular coupling form the basis of non-invasive human neuroimaging techniques, including blood oxygen level dependent (BOLD) functional magnetic resonance imaging. Interestingly, BOLD signals are negative in infants, indicating a mismatch between metabolism and blood flow upon neural activation; this response is the opposite of that observed in healthy adults where activity evokes a large oversupply of blood flow. Negative neurovascular coupling has also been observed in rodents at early postnatal stages, further implying that this is a process that matures during development. This rationale is consistent with the morphological maturation of the neurovascular unit, which occurs over a similar time frame. While neurons differentiate before birth, astrocytes differentiate postnatally in rodents and the maturation of their complex morphology during the first few weeks of life links them with synapses and the vasculature. The vascular network is also incomplete in neonates and matures in parallel with astrocytes. Here, we review the timeline of the structural maturation of the neurovascular unit with special emphasis on astrocytes and the vascular tree and what it implies for functional maturation of neurovascular coupling. We also discuss similarities between immature astrocytes during development and reactive astrocytes in disease, which are relevant to neurovascular coupling. Finally, we close by pointing out current gaps in knowledge that must be addressed to fully elucidate the mechanisms underlying neurovascular coupling maturation, with the expectation that this may also clarify astrocyte-dependent mechanisms of cerebrovascular impairment in neurodegenerative conditions in which reduced or negative neurovascular coupling is noted, such as stroke and Alzheimer’s disease.

The neuroendocrine effects of dehydroepiandrosterone and 17β-estradiol in the in vitro preterm hyperoxia infant model

Preterm birth causes neurological deficits. Previously, we demonstrated that fetal zone steroids reduce hyperoxia-mediated cell death in vitro. In immature oligodendrocytes (OLN-93 cells), dehydroepiandrosterone + 17β-estradiol co-treatment had synergistic beneficial effects while signals were transduced through different receptors. In immature astrocytes (C6 cells), both hormones compete for the same receptor and no synergistic effects were observed. 17β-estradiol and progesterone drastically decrease while fetal zone steroids, mainly dehydroepiandrosterone, remain persistently high within preterm infants until term. Substitution of 17β-estradiol and progesterone does not improve neurological outcomes. We investigated the influence of dehydroepiandrosterone, 17β-estradiol or dehydroepiandrosterone + 17β-estradiol treatment in C6 or OLN-93 cells on steroid receptor availability and activation of intracellular signaling molecules in hyperoxic cell culture. We sought explanations of the observed synergistic effect in preliminary study. In C6 cells, the generated signaling of dehydroepiandrosterone + 17β-estradiol treatment has no synergistic effects. The combined effect on this particular pathway does not potentiate cell survival. In OLN-93 cells, we observed significant differences in the early generated signaling of 17β-estradiol + dehydroepiandrosterone treatment to either 17β-estradiol dehydroepiandrosterone alone but never to both at the same time. The latter finding needs, therefore, further investigation to explain synergistic effects. Nevertheless, we add insight into the receptor and signaling cascade alterations induced by 17β-estradiol, dehydroepiandrosterone or 17β-estradiol + dehydroepiandrosterone treatment of C6 and OLN-93 cells in hyperoxia.

Влияние L-альфа-аминоадипиновой кислоты на нейроны и глиальные клетки стриатума мозга крыс

Introduction. Few published studies have examined the role of glia-neuron interactions in neurodegen-eration. In this regard, the search for new experimental models is important. L-alpha-aminoadipic acid (L-AA), being a structural analogue of glutamate with a selective toxic effect on astroglia, is of particular interest. However, morphological and neurochemical changes caused by L-AA still remain unclear. The aim of the study was to characterize immunomorphological changes of glia and neurons in the striata of rats after L-AA administration. Materials and methods. On days 3 and 12 after L-AA stereotaxic administration, we studied astrocytic pro-teins localization using immunofluorescence methods: GFAP, vimentin, glutamine synthetase, along with oligodendroglia (by cyclonucleotidphosphatase expression), microglia (IBA1 Ca-binding protein), neuronal nuclear protein NeuN, and the astroglia proliferative activity (based on Ki67 localization). Results. We detected astrocyte death and a decrease in glutamine synthetase immunoreactivity in the le-sioned area, but no changes in the microglia reaction and the L-AA effect on neurons and oligodendroglia. The astrocyte loss was replenished by proliferation and migration of newly formed immature astrocytes, and a glial scar formed on day 12 after the surgery. Conclusion. L-AA administration, which causes the death of striatum astrocytes in the injection area, can serve as a convenient model for studying reactive changes in astroglia and astrocytic dysfunction while revealing the pathogenetic patterns of neurodegenerative processes. Keywords: astrocytes, striatum, L-alpha-aminoadipic acid, gliosis, glial toxin

Microglia Susceptibility to Free Bilirubin Is Age-Dependent.

Increased concentrations of unconjugated bilirubin (UCB), namely its free fraction (Bf), in neonatal life may cause transient or definitive injury to neurons and glial cells. We demonstrated that UCB damages neurons and glial cells by compromising oligodendrocyte maturation and myelination, and by activating astrocytes and microglia. Immature neurons and astrocytes showed to be especially vulnerable. However, whether microglia susceptibility to UCB is also age-related was never investigated. We developed a microglia culture model in which cells at 2 days in vitro (2DIV) revealed to behave as the neonatal microglia (amoeboid/reactive cells), in contrast with those at 16DIV microglia that performed as aged cells (irresponsive/dormant cells). Here, we aimed to unveil whether UCB-induced toxicity diverged from the young to the long-cultured microglia. Cells were isolated from the cortical brain of 1- to 2-day-old CD1 mice and incubated for 24 h with 50/100 nM Bf levels, which were associated to moderate and severe neonatal hyperbilirubinemia, respectively. These concentrations of Bf induced early apoptosis and amoeboid shape in 2DIV microglia, while caused late apoptosis in 16DIV cells, without altering their morphology. CD11b staining increased in both, but more markedly in 2DIV cells. Likewise, the gene expression of HMGB1, a well-known alarmin, as well as HMGB1 and GLT-1–positive cells, were enhanced as compared to long-maturated microglia. The CX3CR1 reduction in 2DIV microglia was opposed to the 16DIV cells and suggests a preferential Bf-induced sickness response in younger cells. In conformity, increased mitochondrial mass and NO were enhanced in 2DIV cells, but unchanged or reduced, respectively, in the 16DIV microglia. However, 100 nM Bf caused iNOS gene overexpression in 2DIV and 16DIV cells. While only arginase 1/IL-1β gene expression levels increased upon 50/100 nM Bf treatment in long-maturated microglia, MHCII/arginase 1/TNF-α/IL-1β/IL-6 (>10-fold) were upregulated in the 2DIV microglia. Remarkably, enhanced inflammatory-associated microRNAs (miR-155/miR-125b/miR-21/miR-146a) and reduced anti-inflammatory miR-124 were found in young microglia by both Bf concentrations, while remained unchanged (miR/21/miR-125b) or decreased (miR-155/miR-146a/miR-124) in aged cells. Altogether, these findings support the neurodevelopmental susceptibilities to UCB-induced neurotoxicity, the most severe disabilities in premature babies, and the involvement of immune-inflammation neonatal microglia processes in poorer outcomes.

Differential Susceptibility and Vulnerability of Brain Cells in C57BL/6 Mouse to Mitochondrial Dysfunction Induced by Short-Term Cuprizone Exposure.

Cuprizone (CPZ) is a chemical chelator toxic to mitochondria of cells. While inducing oligodendrocyte (OL) loss and demyelination, CPZ caused no fatal damage to the other brain cells (neurons, astrocytes, and microglia) in previous studies, suggesting differential susceptibility and vulnerability of brain cells to the CPZ intoxication. To demonstrate this interpretation, C57BL/6 mice were fed rodent chow without or with CPZ (0.2%, w/w) for 7 days. One day later, mitochondrial function of brain cells was assessed by proton magnetic resonance spectroscopy (1H-MRS) and biochemical analysis. Another batch of mice were processed to localize the CPZ-induced damage to mitochondrial DNA, label brain cells, and identify apoptotic cells. Compared to controls, CPZ-exposed mice showed significantly lower levels of N-acetyl-L-aspartate, phosphocreatine, and ATP detected by 1H-MRS, indicating mitochondrial dysfunction in brain cells. Susceptibility analysis showed an order of OLs, microglia, and astrocytes from high to low, in terms of the proportion of 8-OHdG labeled cells in each type of these cells in corpus callosum. Vulnerability analysis showed the highest proportion of caspase-3 positive cells in labeled OLs in cerebral cortex and hippocampus, where neurons showed no caspase-3 labeling, but the highest proportion of 8-OHdG labeling, indicating a lowest vulnerability but highest susceptibility to CPZ-induced mitochondrial dysfunction. Immature OLs, microglia, and astrocytes showed adaptive changes in proliferation and activation in response to CPZ-exposure. These data for the first time demonstrated the CPZ-induced mitochondria dysfunction in brain cells of living mouse and specified the differential susceptibility and vulnerability of brain cells to the CPZ intoxication.

Laser Lesion in the Mouse Visual Cortex Induces a Stem Cell Niche-Like Extracellular Matrix, Produced by Immature Astrocytes.

The mammalian central nervous system (CNS) is characterized by a severely limited regeneration capacity. Comparison with lower species like amphibians, which are able to restore even complex tissues after damage, indicates the presence of an inhibitory environment that restricts the cellular response in mammals. In this context, signals provided by the extracellular matrix (ECM) are important regulators of events like cell survival, proliferation, migration, differentiation or neurite outgrowth. Therefore, knowledge of the post-lesional ECM and of cells that produce these factors might support development of new treatment strategies for patients suffering from traumatic brain injury and other types of CNS damage. In the present study, we analyzed the surround of focal infrared laser lesions of the adult mouse visual cortex. This lesion paradigm avoids direct contact with the brain, as the laser beam passes the intact bone. Cell type-specific markers revealed a distinct spatial distribution of different astroglial subtypes in the penumbra after injury. Glial fibrillary acidic protein (GFAP) as marker for reactive astrocytes was found broadly up-regulated, whereas the more immature markers vimentin and nestin were only expressed by a subset of cells. Dividing astrocytes could be identified via the proliferation marker Ki-67. Different ECM molecules, among others the neural stem cell-associated glycoprotein tenascin-C and the DSD-1 chondroitin sulfate epitope, were found on astrocytes in the penumbra. Wisteria floribunda agglutinin (WFA) and aggrecan as markers for perineuronal nets, a specialized ECM limiting synaptic plasticity, appeared normal in the vicinity of the necrotic lesion core. In sum, expression of progenitor markers by astrocyte subpopulations and the identification of proliferating astrocytes in combination with an ECM that contains components typically associated with neural stem/progenitor cells suggest that an immature cell fate is facilitated as response to the injury.

Foetal onset of EIF2B related disorder in two siblings: cerebellar hypoplasia with absent Bergmann glia and severe hypomyelination

Bi-allelic pathogenic variants in genes of the EIF2B family are responsible for Childhood Ataxia with Central nervous system Hypomyelination/Vanishing White Matter disease, a progressive neurodegenerative disorder of the central white matter. Only seven molecularly proven cases with antenatal onset have been reported so far. We report for the first time the neuropathological findings obtained from two foetuses harbouring deleterious variants in the EIF2B5 gene who presented in utero growth retardation and microcephaly with simplified gyral pattern that led to a medical termination of the pregnancy at 27 and 32 weeks of gestation. Neuropathological examination confirmed microcephaly with delayed gyration, periventricular pseudo-cysts and severe cerebellar hypoplasia. Histologically, the cerebellar cortex was immature, the dentate nuclei were fragmented and myelin stains revealed almost no myelination of the infratentorial structures. Bergmann glia was virtually absent associated to a drastic decreased number of mature astrocytes in the cerebellar white matter, multiple nestin-positive immature astrocytes as well as increased numbers of PDGRFα-positive oligodendrocyte precursors. Whole exome sequencing performed in the two foetuses and their parents allowed the identification of two EIF2B5 compound heterozygous variants in the two foetuses: c.468C > G p.Ile156Met and c.1165G > A p.Val389Met, the parents being heterozygous carriers. These variants are absent in the genome Aggregation Database (gnomAD r2.0.2). Contrary to the variant Ile156Met already described in a patient with CACH syndrome, the variant p.Val389Met is novel and predicted to be deleterious using several softwares. Neuropathological findings further expand the phenotypic spectrum of the disease that very likely occurs during early gestation and may manifest from the second half of pregnancy by a severe impairment of cerebral and cerebellar development.

Endothelial Yes-Associated Protein 1 Promotes Astrocyte Proliferation and Maturation via Cytoplasmic Leukemia Inhibitory Factor Secretion in Oxygen-Induced Retinopathy.

PurposePurpose The role of endothelial Yes-associated protein 1 (YAP) in the pathogenesis of retinal angiogenesis and the astrocyte network in the mouse oxygen-induced retinopathy (OIR) model is unknown.MethodsFor in vivo studies, OIR was induced in conditional endothelial YAP knockout mice and their wild-type littermates. Retinal vascularization and the astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro experiments were performed in astrocytes cultured with human microvascular endothelial cell-1–conditioned medium to analyze the mechanisms underlying the effect of endothelial YAP on astrocytes.ResultsEndothelial YAP deletion not only impaired retinal blood vessels, but also caused a sparse and disrupted astrocyte network in response to OIR. Levels of the immature astrocyte marker (platelet-derived growth factor A) in the retina were substantially increased owing to YAP deficiency, suggesting a possible failure in astrocyte maturation, whereas retinal expression of leukemia inhibitory factor (LIF) was decreased. In vitro studies suggested that loss or overexpression of YAP resulted in elevated or decreased LIF secretion by human microvascular endothelial cell-1, respectively. Increased LIF levels in the culture medium promoted astrocyte maturation and proliferation and rescued YAP inhibition-induced astrocyte loss. Finally, activating YAP could protect against the pathology of the astrocyte network and even suppress pathologic retinal vascularization in control OIR mice, but not in endothelial YAP-deficient OIR mice.ConclusionsEndothelial YAP regulation of LIF secretion is required for normalized astrocyte network formation in OIR, thereby providing a novel target for protecting the astrocyte network and thus benefiting retinal blood vessels.

Retinal blood vessel-origin yes-associated protein (YAP) governs astrocytic maturation via leukaemia inhibitory factor (LIF).

ObjectivesTo testify that endothelial cells (ECs) induce astrocyte maturation by leukaemia inhibitory factor (LIF) secretion.Materials and MethodsIn vivo experiments, mice bearing floxed alleles of YAP were crossed with mice expressing a Cre recombinase driven by the endothelial Tek promoter (Tek‐Cre) to finally obtain the following three genotypes: YAP f/f, Tek‐Cre; YAP f/w, Tek‐Cre; and YAP f/f. Retinal vascularization and astrocyte network were evaluated by whole‐mount fluorescence and Western blotting. In vitro, experiments were performed in an astrocyte and human microvascular endothelial cell (HMEC‐1) coculture model to analyse the mechanisms underlying the effect of endothelial YAP on astrocytes.ResultsIn vivo, YAPf/f;Tek‐Cre mice showed delayed angiogenesis, sparse vessels and decreased glial fibrillary acidic protein (GFAP)+ astrocytes but aberrant growth of endothelial networks and immature astrocytes (platelet‐derived growth factor A, PDGFRA+ astrocytes) overgrowth. In vitro, Yap deletion attenuated the LIF release that delayed the maturation of retinal astrocyte which was consistent with the results of HMEC‐1—astrocyte coculture. The effect of YAP overexpression on LIF‐LIFR axis in HMEC‐1 interferes the GFAP expression of astrocyte. In contrast, LIF protein rescues the astrocytic GFAP expression when EC YAP was inhibited by siRNAs.ConclusionsWe show that EC yes‐associated protein (YAP) is not only a critical coactivator of Hippo signalling in retinal vessel development but also plays an essential role in retinal astrocyte maturation by regulating LIF production.

Vimentin Phosphorylation Is Required for Normal Cell Division of Immature Astrocytes.

Vimentin (VIM) is an intermediate filament (nanofilament) protein expressed in multiple cell types, including astrocytes. Mice with VIM mutations of serine sites phosphorylated during mitosis (VIMSA/SA) show cytokinetic failure in fibroblasts and lens epithelial cells, chromosomal instability, facilitated cell senescence, and increased neuronal differentiation of neural progenitor cells. Here we report that in vitro immature VIMSA/SA astrocytes exhibit cytokinetic failure and contain vimentin accumulations that co-localize with mitochondria. This phenotype is transient and disappears with VIMSA/SA astrocyte maturation and expression of glial fibrillary acidic protein (GFAP); it is also alleviated by the inhibition of cell proliferation. To test the hypothesis that GFAP compensates for the effect of VIMSA/SA in astrocytes, we crossed the VIMSA/SA and GFAP−/− mice. Surprisingly, the fraction of VIMSA/SA immature astrocytes with abundant vimentin accumulations was reduced when on GFAP−/− background. This indicates that the disappearance of vimentin accumulations and cytokinetic failure in mature astrocyte cultures are independent of GFAP expression. Both VIMSA/SA and VIMSA/SAGFAP−/− astrocytes showed normal mitochondrial membrane potential and vulnerability to H2O2, oxygen/glucose deprivation, and chemical ischemia. Thus, mutation of mitotic phosphorylation sites in vimentin triggers formation of vimentin accumulations and cytokinetic failure in immature astrocytes without altering their vulnerability to oxidative stress.

Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1.

Brain-derived neurotrophic factor (BDNF) is a critical growth factor involved in the maturation of the CNS, including neuronal morphology and synapse refinement. Herein, we demonstrate astrocytes express high levels of BDNF's receptor, TrkB (in the top 20 of protein-coding transcripts), with nearly exclusive expression of the truncated isoform, TrkB.T1, which peaks in expression during astrocyte morphological maturation. Using a novel culture paradigm, we show that astrocyte morphological complexity is increased in the presence of BDNF and is dependent upon BDNF/TrkB.T1 signaling. Deletion of TrkB.T1, globally and astrocyte-specifically, in mice revealed morphologically immature astrocytes with significantly reduced volume, as well as dysregulated expression of perisynaptic genes associated with mature astrocyte function. Indicating a role for functional astrocyte maturation via BDNF/TrkB.T1 signaling, TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function. These data suggest a significant role for BDNF/TrkB.T1 signaling in astrocyte morphological maturation, a critical process for CNS development.

EIF2B Mutations Cause Mitochondrial Malfunction in Oligodendrocytes.

Vanishing white matter (VWM) disease (OMIM#306896) is an autosomal recessive neurodegenerative leukodystrophy caused by hypomorphic mutations in any of the five genes encoding the subunits of eukaryotic translation initiation factor 2B (eIF2B). The disease is manifested by loss of cerebral white matter and progressive deterioration upon exposure to environmental and physiological stressors. "Foamy" oligodendrocytes (OLG), increased numbers of oligodendrocytes precursor cells (OPC), and immature defective astrocytes are major neuropathological denominators. Our recent work using Eif2b5R132H/R132H mice uncovered a fundamental link between eIF2B and mitochondrial function. A decrease in oxidative phosphorylation capacity was observed in mutant astrocytes and fibroblasts. While an adaptive increase in mitochondria abundance corrects the phenotype of mutant fibroblasts, it is not sufficient to compensate for the high-energy demand of astrocytes, explaining their involvement in the disease. To date, astrocytes are marked as central for the disease while eIF2B-mutant OLG are currently assumed to lack a cellular phenotype on their own. Here we show a reduced capacity of eIF2B-mutant OPC isolated from Eif2b5R132H/R132H mice to conduct oxidative respiration despite the adaptive increase in their mitochondrial abundance. We also show their impaired ability to efficiently complete critical differentiation steps towards mature OLG. The concept that defective differentiation of eIF2B-mutant OPC could be a consequence of mitochondrial malfunction is in agreement with numerous studies indicating high dependency of differentiating OLG on accurate mitochondrial performance and ATP availability.

RGC-32 regulates expression of insulin-like growth factor binding proteins in astrocytes

Abstract Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects and activation of cell cycle. During experimental autoimmune encephalomyelitis (EAE), RGC-32 knock-out (KO) mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from wild-type (WT) mice had a reactive, hypertrophied phenotype, suggesting a role for RGC-32 in astrocyte differentiation. To gain more insight into the role played by RGC-32 in astrocytes, we investigated the transcriptional profile induced by TGF-β in astrocytes using qPCR arrays. The majority of genes differentially expressed were related to cell growth, extracellular matrix remodeling and signal transduction. Among them, a group of genes belonging to the family of insulin-like growth factor binding proteins (IGFBPs) and insulin-like growth factors (IGFs) were found to be most significantly regulated by RGC-32. The IGFBP category includes IGFBP2, IGFBP3, IGFBP6 and connective tissue growth factor (CTGF). Using Real-time PCR and Western Blotting, we confirmed the differential expression of these genes in RGC-32 KO astrocytes after TGF-β stimulation. When glial fibrillary acidic protein (GFAP) and CTGF levels were compared between WT and KO astrocytes after TGF-β stimulation, significantly lower levels of mRNA and protein expression were found in RGC-32 KO astrocytes. These data suggest that TGF-β stimulation of CTGF is RGC-32 dependent and that RGC-32 plays a role in the regulation of CTGF downstream effects. The differential regulation of IGFBPs and GFAP suggests that they might be responsible for the lack of astrocyte differentiation seen in RGC-32 KO mice during EAE.

Astrocyte-to-astrocyte contact and a positive feedback loop of growth factor signaling regulate astrocyte maturation.

Astrocytes are critical for the development and function of the central nervous system. In developing brains, immature astrocytes undergo morphological, molecular, cellular, and functional changes as they mature. Although the mechanisms that regulate the maturation of other major cell types in the central nervous system such as neurons and oligodendrocytes have been extensively studied, little is known about the cellular and molecular mechanisms that control astrocyte maturation. Here, we identified molecular markers of astrocyte maturation and established an in vitro assay for studying the mechanisms of astrocyte maturation. Maturing astrocytes in vitro exhibit similar molecular changes and represent multiple molecular subtypes of astrocytes found in vivo. Using this system, we found that astrocyte‐to‐astrocyte contact strongly promotes astrocyte maturation. In addition, secreted signals from microglia, oligodendrocyte precursor cells, or endothelial cells affect a small subset of astrocyte genes but do not consistently change astrocyte maturation. To identify molecular mechanisms underlying astrocyte maturation, we treated maturing astrocytes with molecules that affect the function of tumor‐associated genes. We found that a positive feedback loop of heparin‐binding epidermal growth factor‐like growth factor (HBEGF) and epidermal growth factor receptor (EGFR) signaling regulates astrocytes maturation. Furthermore, HBEGF, EGFR, and tumor protein 53 (TP53) affect the expression of genes important for cilium development, the circadian clock, and synapse function. These results revealed cellular and molecular mechanisms underlying astrocytes maturation with implications for the understanding of glioblastoma.

A Semi-stochastic Numerical Model of Adult Hippocampal Neurogenesis

Adult neurogenesis in dentate gyrus (DG) is a prominent contributor in the dynamics of hippocampal memory networks. This discrete model aims to estimate the temporal changes in the neural progenitor cell (NPC) populations in DG, together with the products of differentiation – immature neurons, astrocytes and oligodendrocytes. The dynamics are described in an ideal environment, where there is no limit for the total volume and all required chemical and physical cues that direct neurogenesis are continuously available. The system works independently on three levels. Each level is defined as the dynamics in a stage of neurogenesis with three types of NPCs: type I cell (radial glia), type II cell (transiently amplifying cells) and type III cell (neuroblasts). Cell fate was introduced as a semi-stochastic process (a choice) with a population limit for each cell type. Although it is based on discrete processes and has a rather simplistic approach, the simulations successfully provide a numerical template for adult neurogenesis, which can be further modified and implemented in a hippocampal trisynaptic loop network.

Astrocytes and radial glia-like cells, but not neurons, display a nonapoptotic increase in caspase-3 expression following exercise.

BackgroundExercise induces plasticity in the hippocampus, which includes increases in neurogenesis, the proliferation of new neurons, and angiogenesis, the sprouting of new capillaries from preexisting blood vessels. Following exercise, astrocytes also undergo morphological changes that parallel the events occurring in the neurovascular system. Interestingly, there have also been reports of apoptosis in the hippocampus following aerobic exercise. This experiment aimed to identify which population of hippocampal cells undergoes apoptosis after an acute bout of exercise.MethodsCleaved caspase‐3, a terminal protein in the apoptotic cascade, was initially used to identify apoptotic cells in the hippocampus after rats completed an acute bout of exercise. Next, the proportion of immature neurons, adult neurons, astrocytes, or radial glia‐like cells expressing cleaved caspase‐3 was quantified. TUNEL staining was completed as a second measure of apoptosis.ResultsFollowing exercise, cleaved caspase‐3 expression was increased in the CA1 and DG regions of the hippocampus. Cleaved caspase‐3 was not highly expressed in neuronal populations, and expression was not increased in these cells postexercise. Instead, cleaved caspase‐3 was predominantly expressed in astrocytes. Following exercise, there was an increased number of cleaved caspase‐3 positive astrocytes in DG and CA1, and cleaved caspase‐3 positive radial glia‐like cells located in the subgranular zone. To determine whether cleaved caspase‐3 expression in these glial cells was associated with apoptosis, a TUNEL assay was completed. TUNEL staining was negligible in all groups and did not mirror the pattern of caspase‐3 labeling.ConclusionsCleaved caspase‐3 expression was detected largely in non‐neuronal cell populations, and the pattern of cleaved caspase‐3 expression did not match that of TUNEL. This suggests that after exercise, cleaved caspase‐3 expression may serve a nonapoptotic role in these hippocampal astrocytes and radial glia‐like cells. It will be important to identify the function of exercise‐induced cleaved caspase‐3 expression in the future experiments.

RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis

Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-β-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-β stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-β-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-β-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.