Search

Brain development is a dynamic and lengthy process that includes cell proliferation, migration, neurogenesis, gliogenesis, synaptogenesis, and pruning. Disruption of any of these developmental events can result in long-term outcomes ranging from brain structural changes, to cognitive and behavioral abnormality, with the mechanisms largely unknown. Emerging evidence suggests non-coding RNAs (ncRNAs) as pivotal molecules that participate in normal brain development and neurodevelopmental disorders. NcRNAs such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are transcribed from the genome but not translated into proteins. Many ncRNAs have been implicated as tuners of cell fate. In this review, we started with an introduction of the current knowledge of lncRNAs and miRNAs, and their potential roles in brain development in health and disorders. We then reviewed and discussed the evidence of ncRNA involvement in abnormal brain development resulted from alcohol, anesthetic drugs, nicotine, and viral infections. The complex connections among these ncRNAs were also discussed, along with potential overlapping ncRNA mechanisms, possible pharmacological targets for therapeutic/neuroprotective interventions, and potential biomarkers for brain developmental disorders.

Neural stem cells (NSC) restrict their differentiation potential as the central nervous system develops. Experimental evidence suggests that the mechanisms governing the transition from the neurogenic to the gliogenic phase irreversibly affect the ability of NSC to generate neurons. Cell cycle regulation has been associated with cell fate in different models. In this work, we assessed the temporal correlation between the loss of the neurogenic potential and cell cycle lengthening of NSC obtained from embryonic mouse spinal cords, during the transition of the neurogenic to the gliogenic phase, using neurospheres. We also used the cell cycle inhibitor Olomoucine to increase cell cycle length by decreasing the proliferation rate. Our results show that neurospheres obtained from a neurogenic stage give rise mostly to neurons, whereas those obtained from later stages produce preferentially glial cells. During the transition from neurogenesis to gliogenesis, the proliferation rate dropped, and the cell cycle length increased 1.5 folds, as monitored by DNA BrdU incorporation. Interestingly, Olomoucine-treated neurogenic-neurospheres display a reduced proliferation rate and preserve their neurogenic potential. Our results suggest that the mechanisms that restrict the differentiation potential of NSC are independent of the proliferation control., Highlights • Neurosphere cultured, spinal cord NSC preserve their differentiation potential. • Neurogenic NSC divide faster than those giving rise to glial cells. • Cell cycle inhibitors increase in NSC transitioning from the neurogenic to the gliogenic phase. • Artificial cell cycle lengthening does not affect the differentiation potential of neurogenic NSC.

Graphical abstract, Highlights • NESTIN, SOX1, and SOX4 decreased progressively from prenatal to adult age. • KI67 and TBR2 reached zero expression level at adolescence. • NEUROD1, DCX, PSA NCAM remained unchanged post-childhood. • VEGF and FGF2 did not change significantly from prenatal to adult age. • BAX and TP53 decreased progressively from prenatal to adult age., Purpose Immunohistological investigations have given rise to divergent perspectives about adult hippocampal neurogenesis in humans. Therefore, this study aimed to examine whether a comprehensive transcriptomic analysis of signature markers of neurogenesis, supplemented with markers of gliogenesis, vasculogenesis, cell proliferation, and apoptosis, may help discern essential aspects of adult hippocampal neurogenesis in humans. Materials and Methods RNA expression data for salient marker genes of neurogenesis, gliogenesis, vasculogenesis, and apoptosis in post-mortem human hippocampal tissue [from prenatal (n = 15), child (n = 5), adolescent (n = 4), and adult (n = 6) brains] were downloaded from the Allen Human Brain Atlas database (http://www.brainspan.org/rnaseq/search/index.html). Gene expression data was categorized, median values were computed, and age group-specific differential expression was subjected to statistical analysis (significance level, α = 0.01). Results With the exception of the genes encoding GFAP, BLBP, SOX2, and PSA-NCAM (unchanged), and the post-mitotic late maturation markers CALB1, CALB2, MAP2, and NEUN as well as the pan-neuronal marker PROX1 which were persistently expressed throughout, expression of all other genes associated with neurogenesis was steeply and progressively downregulated between perinatal life and adulthood. Interestingly, expression of the classical proliferation marker KI67 and a progenitor cell marker TBR2 were found to have reached baseline expression levels (zero expression score) at adolescence while the expression of immature neuronal, post-mitotic early and late maturation markers remained at a constant level after childhood. In contrast, markers of gliogenesis (other than PDGFRA and Vimentin) were significantly upregulated between prenatal life and childhood. Expression of the vasculogenesis markers VEGFA and FGF2 did not differ across any of the age groups studied, whereas the expression of apoptotic markers was progressively decreased after prenatal life. Conclusions Our findings indicate that the progression of neurogenesis from progenitor cells is highly restricted in the human brain from childhood onwards. An alternative possibility that limited neurogenesis may be continued in adolescents and adults from a developmentally arrested pool of immature neurons needs to be examined further through experimental studies.

Astrocytes perform multiple essential functions in the developing and mature brain, including regulation of synapse formation, control of neurotransmitter release and uptake, and maintenance of extracellular ion balance. As a result, astrocytes have been implicated in the progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite these critical functions, the study of human astrocytes can be difficult because standard differentiation protocols are time-consuming and technically challenging, but a differentiation protocol recently developed in our laboratory enables the efficient derivation of astrocytes from human embryonic stem cells. We used this protocol along with microarrays, luciferase assays, electrophoretic mobility shift assays, and ChIP assays to explore the genes involved in astrocyte differentiation. We demonstrate that paired-like homeodomain transcription factor 1 (PITX1) is critical for astrocyte differentiation. PITX1 overexpression induced early differentiation of astrocytes, and its knockdown blocked astrocyte differentiation. PITX1 overexpression also increased and PITX1 knockdown decreased expression of sex-determining region Y box 9 (SOX9), known initiator of gliogenesis, during early astrocyte differentiation. Moreover, we determined that PITX1 activates the SOX9 promoter through a unique binding motif. Taken together, these findings indicate that PITX1 drives astrocyte differentiation by sustaining activation of the SOX9 promoter.

The RAS/RAF/MEK/ERK pathway promotes gliogenesis but the kinetic role of RAF1, a key RAF kinase, in the induction of astrocytogenesis remains to be elucidated. To address this challenge, we determine the temporal functional outcome of RAF1 during mouse neural progenitor cell differentiation using an optogenetic RAF1 system (OptoRAF1). OptoRAF1 allows for reversible activation of the RAF/MEK/ERK pathway via plasma membrane recruitment of RAF1 based on blue light-sensitive protein dimerizer CRY2/CIB1. We found that early light-induced OptoRAF1 activation in neural progenitor cells promotes cell proliferation and increased expression of glial markers and glia-enriched genes. However, delayed OptoRAF1 activation in differentiated neural progenitor had little effect on glia marker expression, suggesting that RAF1 is required to promote astrocytogenesis only within a short time window. In addition, activation of OptoRAF1 did not have a significant effect on neurogenesis, but was able to promote neuronal neurite growth.

Exposure to air pollution - and particularly to particulate matter (PM) - is strongly associated with higher risk of neurodevelopmental disorders, poor mental health and cognitive defects. In animal models, disruption of CNS development and disturbances of adult neurogenesis contribute to PM neurotoxicity. Recent studies show that gestational PM exposure not only affects embryonic neurodevelopment, but also disturbs postnatal brain growth and maturation, by interfering with neurogenic/gliogenic events, myelination and synaptogenesis. Similarly, adult neurogenesis is affected at many levels, from neural stem cell amplification up to the maturation and integration of novel neurons in the adult brain parenchyma. The underlying mechanisms are still by and large unknown. Beyond microglia activation and neuroinflammation, recent studies propose a role for novel epigenetic mechanisms, including DNA methylation and extracellular vesicles-associated microRNAs.

In the developing mammalian neocortex, neural stem cells (NSCs) gradually alter their characteristics as development proceeds. NSCs initially expand the progenitor pool by symmetric proliferative division and then shift to asymmetric neurogenic division to commence neurogenesis. NSCs sequentially give rise to deep layer neurons first and superficial layer neurons later through mid- to late-embryonic stages, followed by shifting to a gliogenic phase at perinatal stages. The precise mechanisms regulating developmental timing of the transition from symmetric to asymmetric division have not been fully elucidated; however, gradual elongation in cell cycle length and concomitant accumulation of determinants that promote neuronal differentiation may function as a biological clock that regulates the onset of asymmetric neurogenic division. On the other hand, epigenetic regulatory systems have been implicated in the regulation of transition timing of neurogenesis and gliogenesis; the polycomb group (PcG) complex and Hmga genes have been found to govern the developmental timing by modulating chromatin structure during neocortical development. Furthermore, we uncovered several factors and mechanisms underlying the regulation of timing of neocortical neurogenesis and gliogenesis. In this review, we discuss recent findings regarding the mechanisms that govern the temporal properties of NSCs and the precise transition timing during neocortical development.

Chlorpyrifos (CPF) is an organophosphate pesticide widely used in agriculture, whose traditional and well-known mechanism of action is the inhibition of the enzyme Acetylcholinesterase (AChE). Subacute exposures to CPF have been associated with alterations different from the inhibition of AChE. Because of the vulnerability of the developing nervous system, prenatal and early postnatal exposures are of special concern. Human neural stem cells (hNSC) provide the opportunity to study early stages of neural development and may be a valuable tool for developmental neurotoxicology (DNT). In the current work, the cell line hNS1 was used as a model system with the aim of validating this cell line as a reliable testing method. To evaluate the effects of CPF on early developmental stages, hNS1 cells were exposed to different concentrations of the pesticide and cell death, proliferation and cell fate specification were analyzed under differentiation conditions. Since hNS1 cells responded to CPF in a similar way to other human cell lines, we consider it may be a valid model for DNT chemical assessment. CPF induced apoptotic cell death only at the highest doses tested, suggesting that it is not toxic for the specific developmental stage here addressed under short term exposure. In addition, the higher doses of CPF promoted the generation of astroglial cells, without affecting neurogenesis.

The selective vulnerability of distinct regions of the brain is a critical factor in neurodegenerative disorders. In Alzheimer's disease (AD), neurons in hippocampus situated in medial temporal lobe are immensely damaged. Identifying tissue-specific variants is essential in order to perceive the selective vulnerability in AD. In current work, we aligned mRNA-seq data with HG19/HG38 genomic assembly and identified specific variations present in temporal, frontal and other lobes of the AD using sequence alignment map tools. We compared the results with the genome-wide association and gene expression quantitative trait loci studies of the various neurological disorders. We also distinguished variants and epitranscriptomic modifications through the RNA-modification database and evaluated the variant effect in the coding/UTR regions. In addition, we developed genetic and functional interaction networks to understand the relationship between predicted vulnerable variations and differentially expressed genes. We found that genes involved in gliogenesis, intermediate filament organization are altered in the temporal lobe. Oxidative phosphorylation, and calcium ion homeostasis are modified in the frontal lobe, and protein degradation, apoptotic signaling are altered in other lobes. From this study, we propose that disruption of glial cell structural integrity, defective gliogenesis, and failure in glia-neuron communication are the primary factors for selective vulnerability.

In the developing central nervous system, including its simple and accessible model retina, neurogenesis is followed by gliogenesis. However, the mechanism underlying the neurogliogenic switch remains poorly understood despite the identification of several regulatory genes, associated with the lineage identity and transition. The mechanism may involve cross talks between regulatory genes, facilitated through microRNAs. Here, we posit miR-29c as one of the regulatory miRNAs that may influence neuronal versus glial differentiation. We observed that the temporal patterns of miR-29c expression corresponded with late retinal histogenesis, the stage in the developing retina when neurogliogenic decision predominantly occurs. Examination of the effects of miR-29c on neurogliogenesis by the perturbation of function approach revealed that miR-29c preferentially facilitated differentiation of late RPCs into rod photoreceptors and bipolar cells, the late-born neurons, at the expense of Müller glia, the sole glia generated by retinal progenitor cells. We further observed that miR-29c facilitated neurogenesis and inhibited gliogenesis by regulating the expression of RE-1 silencing transcription factor (REST), which encodes a transcriptional repressor of cell cycle regulators and neuronal genes. Thus, miR-29c may influence neurogliogenic decision in the developing retina by regulating the instructive out put of a molecular axis helmed by REST.

Radial glial progenitors (RGPs) give rise to the vast majority of neurons and glia in the neocortex. Although RGP behavior and progressive generation of neocortical neurons have been delineated, the exact process of neocortical gliogenesis remains elusive. Here, we report the precise progenitor behavior and gliogenesis program at single-cell resolution in the mouse neocortex. Fractions of dorsal RGPs transition from neurogenesis to gliogenesis progressively, producing astrocytes, oligodendrocytes, or both in well-defined propensities of ∼60%, 15%, and 25%, respectively, by fate-restricted "intermediate" precursor cells (IPCs). Although the total number of IPCs generated by individual RGPs appears stochastic, the output of individual IPCs exhibit clear patterns in number and subtype and form discrete local subclusters. Clonal loss of tumor suppressor Neurofibromatosis type 1 leads to excessive production of glia selectively, especially oligodendrocyte precursor cells. These results quantitatively delineate the cellular program of neocortical gliogenesis and suggest the cellular and lineage origin of primary brain tumor.

Huntington’s disease (HD) is characterized by defective oligodendroglial differentiation and white matter disease. Here, we investigated the role of glial progenitor cell (GPC) dysfunction in adult myelin maintenance in HD. We first noted a progressive, age-related loss of myelin in both R6/2 and zQ175 HD mice, compared to wild-type controls. As adults, R6/2 mice then manifested a significant delay in remyelination following cuprizone demyelination. RNA-Sequencing of GPCs from both R6/2 and zQ175 mice revealed a systematic down-regulation of genes associated with oligodendrocyte differentiation and myelinogenesis relative to controls. Gene co-expression and network analysis predicted repressed TCF7L2 signaling as a major driver of this expression pattern. In vivo TCF7L2 overexpression proved sufficient to restore both myelin gene expression and normal remyelination in demyelinated R6/2 mice. These data causally link impaired TCF7L2-dependent transcription to the poor development and homeostatic retention of myelin in HD, and provide a mechanism for its therapeutic restoration.

The homeobox gene Gsx2 has previously been shown to inhibit oligodendroglial specification in dorsal lateral ganglionic eminence (dLGE) progenitors of the ventral telencephalon. The precocious specification of oligodendrocyte progenitor cells (OPCs) observed in Gsx2 mutants, however, is transient and begins to normalize by late stages of embryogenesis. Interestingly, this normalization correlates with the expansion of Gsx1, a close homolog of Gsx2, in a subset of progenitors in the Gsx2 mutant LGE. Here, we interrogated the mechanisms underlying oligodendroglial specification in Gsx2 mutants in relation to Gsx1. We found that Gsx1/2 double mutant embryos exhibit a more robust expansion of Olig2(+) cells (i.e. OPCs) in the subventricular zone (SVZ) of the dLGE than Gsx2 mutants. Moreover, misexpression of Gsx1 throughout telencephalic VZ progenitors from E15 and onward resulted in a significant reduction of cortical OPCs. These results demonstrate redundant roles of Gsx1 and Gsx2 in suppressing early OPC specification in LGE VZ progenitors. However, Gsx1/2 mutants did not show a significant increase in adjacent cortical OPCs at later stages compared to Gsx2 mutants. This is likely due to reduced proliferation of OPCs within the SVZ of the Gsx1/2 double mutant LGE, suggesting a novel role for Gsx1 in expansion of migrating OPCs in the ventral telencephalon. We further investigated the glial specification mechanisms downstream of Gsx2 by generating Olig2/Gsx2 double mutants. Consistent with the known essential role for Olig2 in OPC specification, ectopic production of cortical OPCs observed in Gsx2 mutants disappeared in Olig2/Gsx2 double mutants. These mutants, however, maintained the expanded expression of gliogenic markers Zbtb20 and Bcan in the VZ of the LGE similarly to Gsx2 single mutants, suggesting that Gsx2 suppresses gliogenesis via Olig2-dependent and -independent mechanisms.

The hypothalamic anteroventral periventricular nucleus (AVPV) is the major regulator of reproductive function within the hypothalamic-pituitary-gonadal (HPG) axis. Despite an understanding of the function of neuronal subtypes within the AVPV, little is known about the molecular mechanisms regulating their development. Previous work from our laboratory has demonstrated that Notch signaling is required in progenitor cell maintenance and formation of kisspeptin neurons of the arcuate nucleus (ARC) while simultaneously restraining POMC neuron number. Based on these findings, we hypothesized that the Notch signaling pathway may act similarly in the AVPV by promoting development of kisspeptin neurons at the expense of other neuronal subtypes. To address this hypothesis, we utilized a genetic mouse model with a conditional loss of Rbpj in Nkx2.1 expressing cells (Rbpj cKO). We noted an increase in cellular proliferation, as marked by Ki-67, in the hypothalamic ventricular zone (HVZ) in Rbpj cKO mice at E13.5. This corresponded to an increase in general neurogenesis and more TH-positive neurons. Additionally, an increase in OLIG2-positive early oligodendrocytic precursor cells was observed at postnatal day 0 in Rbpj cKO mice. By 5 weeks of age in Rbpj cKO mice, TH-positive cells were readily detected in the AVPV but few kisspeptin neurons were present. To elucidate the direct effects of Notch signaling on neuron and glia differentiation, an in vitro primary hypothalamic neurosphere assay was employed. We demonstrated that treatment with the chemical Notch inhibitor DAPT increased mKi67 and Olig2 mRNA expression while decreasing astroglial Gfap expression, suggesting Notch signaling regulates both proliferation and early glial fate decisions. A modest increase in expression of TH in both the cell soma and neurite extensions was observed after extended culture, suggesting that inhibition of Notch signaling alone is enough to bias progenitors towards a dopaminergic fate. Together, these data suggest that Notch signaling restricts early cellular proliferation and differentiation of neurons and oligodendrocytes both in vivo and in vitro and acts as a fate selector of kisspeptin neurons.

Rasmussen's encephalitis (RE) is a rare and devastating unilateral inflammatory brain disease that causes severe and intractable partial epilepsy. It has been shown that epilepsy and subsequent inflammation have deleterious influence on hippocampal cell survival and neurogenesis, but this still has not been systematically explored in human tissue. In this study, we investigated the correlation between inflammation and epilepsy as well as the rates of hippocampal gliogenesis and neurogenesis in a pediatric group of six patients with RE and six control cases. The dentate gyrus (DG) samples were obtained from patients who underwent surgery for intractable RE. Sections were processed for immunohistochemistry using antibodies against sex determining region Y-box 2 (Sox2), nestin, human protein encoded by MKI67 gen (Ki67), and brain-derived neurotrophic factor (BDNF). There was an increase in the number of Ki67-positive granule cells in the DG of patients with RE in comparison with the autopsy control group, but no statistical difference for Sox2-positive cells was observed between these groups. Nestin immunolabeling was less intense in the RE group while BDNF expression was increased. Neurons that were BDNF-positive were found in DG from patients with RE but not in the control group. In patients with RE, few nestin-positive cells in DG were also positive for BDNF, unlike in controls which showed no colocalization for these two markers. These results suggest a proliferation activity in the DG subfield of patients with RE, and also future studies are necessary to address the role of new cells in the hippocampus of patients with RE.

Fasudil is a clinically approved Rho-associated protein kinase (ROCK) inhibitor that has been used widely to treat cerebral consequences of subarachnoid hemorrhage. It is known to have a positive effect on animal models of neurological disorders including Parkinson's disease and stroke. However, its cellular effect on progenitor populations and differentiation is not clearly understood. While recent studies suggest that fasudil promotes the mobilization of neural stem cells (NSCs) from the subventricular zone in vivo and promotes the differentiation of the C17.2 cerebellar neuroprogenitor line in vitro, it is unclear whether fasudil is involved in the differentiation of primary NSCs.Here, we tested the effect of fasudil on mouse NSCs in vitro, and observed increased gliogenesis in NSCs derived from lateral ventricles. Upon treatment, fasudil promoted characteristics of neurogenesis including phenotypic changes in neural outgrowth and interkinetic nuclear-like movements as an immediate response, while Sox2 expression was maintained and GFAP expression increased. Moreover, the gliogenic response to fasudil medium was observed in both early postnatal and adult NSC cultures.Taken together, our results show that fasudil promotes the differentiation of NSCs into astroglial lineage, suggesting that it could be used to develop novel vitro gliogenesis models and regulate differentiation for neural repair. Keywords: Fasudil, ROCK inhibitor, Neural stem cells, Differentiation, Gliogenesis, Primary cultures

Therapeutic ultrasound is a widely used application in clinics. The response to ultrasound is tissue specific and the parameters of ultrasonic treatments are decisive. Here, the responses of primary enteric (ENS) and central nervous system (CNS) neural stem cells were investigated to different frequency – energy density – combination treatments between 510 kHz–3 W s/cm2 and 4.36 MHz–25 W s/cm2. Responses to ultrasonic treatments were both frequency and energy density dependent. Ultrasound enhanced the expansion and showed enlarged neurospheres for both ENS and CNS neural stem cell cultures. Differentiation was not impaired in regard to neuronal and glial cell number as well as neurite and glial fiber outgrowth. Ultrasound is a promising tool to expand neural stem cells in vitro neither influencing neurogenesis nor gliogenesis by applying specific frequency – energy density – combinations. Keywords: Enteric nervous system, Ultrasound, Neural stem cells

Summary Radial glia-like (RGL) stem cells persist in the adult mammalian hippocampus, where they generate new neurons and astrocytes throughout life. The process of adult neurogenesis is well documented, but cell-autonomous factors regulating neuronal and astroglial differentiation are incompletely understood. Here, we evaluate the functions of the transcription factor zinc-finger E-box binding homeobox 1 (ZEB1) in adult hippocampal RGL cells using a conditional-inducible mouse model. We find that ZEB1 is necessary for self-renewal of active RGL cells. Genetic deletion of Zeb1 causes a shift toward symmetric cell division that consumes the RGL cell and generates pro-neuronal progenies, resulting in an increase of newborn neurons and a decrease of newly generated astrocytes. We identify ZEB1 as positive regulator of the ets-domain transcription factor ETV5 that is critical for asymmetric division., Graphical abstract, Highlights • The stem cell transcription factor, ZEB1, demarcates active from quiescent RGL cells • ZEB1 drives the self-renewal of RGL cells by promoting asymmetric cell division • Loss of Zeb1 causes increased neurogenesis and decreased astrogliogenesis • ZEB1 induces Etv5 expression to regulate asymmetric cell division, Gupta et al. show that the stem cell transcription factor, ZEB1, labels activated hippocampal stem cells and regulates their self-renewal and astroglial fate specification. Mechanistically, ZEB1 promotes asymmetric cell divisions and induces the expression of ETV5, a transcriptional regulator of asymmetric divisions and astroglial lineage commitment.

Irradiation of the brain in early human life may set abnormal developmental events into motion that last a lifetime, leading to a poor quality of life for affected individuals. While the effect of irradiation at different early developmental stages on the late human life has not been investigated systematically, animal experimental studies suggest that acute postnatal irradiation with ⩾0.1Gy may significantly reduce neurogenesis in the dentate gyrus and endotheliogenesis in cerebral vessels and induce cognitive impairment and aging. Fractionated irradiation also reduces neurogenesis. Furthermore, irradiation induces hippocampal neuronal loss in CA1 and CA3 areas, neuroinflammation and reduces gliogenesis. The hippocampal neurovascular niche and the total number of microvessels are also changed after radiation exposures. Each or combination of these pathological changes may cause cognitive impairment and aging. Interestingly, acute irradiation of aged brain with a certain amount of radiation has also been reported to induce brain hormesis or neurogenesis. At molecular levels, inflammatory cytokines, chemokines, neural growth factors, neurotransmitters, their receptors and signal transduction systems, reactive oxygen species are involved in radiation-induced adverse effect on brain development and functions. Further study at different omics levels after low dose/dose rate irradiation may not only unravel the mechanisms of radiation-induced adverse brain effect or hormesis, but also provide clues for detection or diagnosis of radiation exposure and for therapeutic approaches to effectively prevent radiation-induced cognitive impairment and aging. Investigation focusing on radiation-induced changes of critical brain development events may reveal many previously unknown adverse effects.

Summary Hippocalcin (HPCA) is a calcium-binding protein that is restricted to nervous tissue and contributes to neuronal activity. Here we report that, in addition to inducing neurogenesis, HPCA inhibits astrocytic differentiation of neural stem cells. It promotes neurogenesis by regulating protein kinase Cα (PKCα) activation by translocating to the membrane and binding to phosphoinositide-dependent protein kinase 1 (PDK1), which induces PKCα phosphorylation. We also found that phospholipase D1 (PLD1) is implicated in the HPCA-mediated neurogenesis pathway; this enzyme promotes dephosphorylation of signal transducer and activator of transcription 3 (STAT3[Y705]), which is necessary for astrocytic differentiation. Moreover, we found that the SH2-domain-containing tyrosine phosphatase 1 (SHP-1) acts upstream of STAT3. Importantly, this SHP-1-dependent STAT3-inhibitory mechanism is closely involved in neurogenesis and suppression of gliogenesis by HPCA. Taken together, these observations suggest that HPCA promotes neuronal differentiation through activation of the PKCα/PLD1 cascade followed by activation of SHP-1, which dephosphorylates STAT3(Y705), leading to inhibition of astrocytic differentiation., Highlights • Hippocalcin is required for neuronal differentiation in neural stem cells • PKCα/PLD1 activation is required for hippocalcin-mediated neuronal differentiation • Blocking of STAT3(Y705) activity by hippocalcin decreases astrocytic differentiation • Hippocalcin promotes neurogenesis by inhibiting gliogenesis in neural stem cells, In this article, Han and colleagues investigate the role of hippocalcin in neuronal differentiation of neural stem cells. They show that hippocalcin promotes neuronal differentiation through activation of the PKCα/PLD1 cascade followed by activation of SHP-1, which dephosphorylates STAT3(Y705), leading to inhibition of astrocytic differentiation. These findings suggest that hippocalcin functions as an on-off switch and a main regulator for neuronal differentiation of neural stem cells.

There is high risk of fetal neurodevelopmental defects in pregestational diabetes mellitus (PGDM). However, the effective mechanism of hyperglycemia-induced neurodevelopmental negative effects, including neural stem cell self-renewal and differentiation, still remains obscure. Neuropoietic cytokines have been shown to play a vital part during nervous system development and in the coordination of neurons, gliocytes. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) dysfunction might be related to a reduction of self-protective response in brain malformation induced by hyperglycemia. We therefore evaluated the role of Nrf2 and neuropoietic cytokines in fetal neurodevelopmental defects induced by PGDM and determined the mechanisms involved. Our data reveal that PGDM dramatically impairs the developmental switch of neural stem cells from neurogenesis to gliogenesis, principally under the cooperative mediation of neuropoietic cytokine CNTF and Nrf2 antioxidative signaling. This indicates that CNTF and Nrf2 could be potentially used in the prevention or therapy of neurodevelopmental defects of PGDM offspring.

Notch-Delta signaling regulates many developmental processes, including tissue homeostasis, and maintenance of stem cells. This signaling pathway is thought to be unidirectional and is triggered by binding of Delta/Serrate/Lag2 (DSL) ligands to the Notch receptor, which leads to cleavage and activation of Notch intracellular domain (NICD). Although the cleavage of DSL ligands has also been reported, the functional significance remains poorly understood. Here, we show that reverse signaling is functional, by modulating the production of Delta like 1 intracellular domain (D1ICD). We find sustained D1ICD production abrogates cell proliferation and enhances neurogenesis, whereas inhibiting D1ICD production leads to promotion of cell proliferation and gliogenesis. D1ICD suppresses Notch signaling through up regulation of Numb, a mediator of lateral inhibition. In addition, D1ICD promotes neurogenesis through a Notch signaling independent manner by inhibiting Erk1/2 phosphorylation. Our results demonstrate that D1ICD has biological functions, and we propose Notch-Delta signaling is, in fact, bi-directional.

The heterogeneity and molecular characteristics of progenitor cells, especially glial progenitors, in the developing human cerebral cortex remain elusive. Here, we find that EGFR expression begins to sharply increase after gestational week (GW) 20, which corresponds to the beginning stages of human gliogenesis. In addition, EGFR+ cells are mainly distributed in the germinal zone and frequently colocalize with the stemness marker SOX2 during this period. Then, by performing single-cell RNA sequencing on these EGFR+ cells, we successfully enriched and characterized various glial- and neuronal-lineage progenitor cells and validated their phenotypes in fixed slices. Notably, we identified two subgroups with molecular characteristics similar to those of astrocytes, and the immunostaining results show that these cells are mainly distributed in the outer subventricular zone and might originate from the outer radial glial cells. In short, the EGFR-sorting strategy and molecular signatures in the diverse lineages provide insights into human glial development.

Adult neural stem cells (NSCs) are defined by their inherent capacity to self-renew and give rise to neurons, astrocytes, and oligodendrocytes. In vivo, however, hippocampal NSCs do not generate oligodendrocytes for reasons that have remained enigmatic. Here, we report that deletion of Drosha in adult dentate gyrus NSCs activates oligodendrogenesis and reduces neurogenesis at the expense of gliogenesis. We further find that Drosha directly targets NFIB to repress its expression independently of Dicer and microRNAs. Knockdown of NFIB in Drosha-deficient hippocampal NSCs restores neurogenesis, suggesting that the Drosha/NFIB mechanism robustly prevents oligodendrocyte fate acquisition in vivo. Taken together, our findings establish that adult hippocampal NSCs inherently possess multilineage potential but that Drosha functions as a molecular barrier preventing oligodendrogenesis.

Advances in the study of brain development over the last decades, especially recent findings regarding the evolutionary expansion of the human neocortex, and large-scale analyses of the proteome/transcriptome in the human brain, have offered novel insights into the molecular mechanisms guiding neural maturation, and the pathophysiology of multiple forms of neurological disorders. As a preamble to reviews of this issue, we provide an overview of the cellular, molecular and genetic bases of brain development with an emphasis on the major mechanisms associated with landmarks of normal neural development in the embryonic stage and early postnatal life, including neural stem/progenitor cell proliferation, cortical neuronal migration, evolution and folding of the cerebral cortex, synaptogenesis and neural circuit development, gliogenesis and myelination. We will only briefly depict developmental disorders that result from perturbations of these cellular or molecular mechanisms, and the most common perinatal brain injuries that could disturb normal brain development.

One of the well established aspects of the development of the central nervous system (CNS) is that neurogenesis precedes gliogenesis. However, the mechanism underlying this temporal switch between the two distinct lineages is poorly understood. It is thought that let-7, a heterochronic miRNA, may help neural stem cells (NSCs) choose between the neuronal and glial lineages. Here, we have tested the premise in the retina, a simple and accessible CNS model, where neurogliogenic decision takes place postnatally during late histogenesis. A positive correlation was observed between the temporal induction of let-7 expression and differentiation of late born neurons and Müller glia (MG), the sole glia generated by retinal progenitor cells (RPCs). Examination of let-7's involvement in late histogenesis by the perturbation of function approaches revealed that let-7 facilitated differentiation of both neurons and MG, without preference to a particular lineage. We demonstrate that let-7's positive influence on neuronal and MG differentiation is likely achieved by inhibiting the expression of Hmga2, the DNA architecture protein involved in the self-renewal of neural progenitors. Thus, our observations suggest that let-7 promotes differentiation, regardless of the neuronal or glial lineage, shifting the balance from RPCs' maintenance to their differentiation in the developing retina.

The brain is one of the most complex organs in the body, which emerges from a relatively simple set of basic ‘building blocks’ during early development according to complex cellular and molecular events orchestrated through a set of inherited instructions. Innovations in stem cell technologies have enabled modelling of neural cells using two- and three- dimensional cultures. In particular, cerebral (‘brain’) organoids have taken the center stage of brain development models that have the potential for providing meaningful insight into human neurodevelopmental and neurological disorders. We review the current understanding of cellular events during human brain organogenesis, and the events occurring during cerebral organoid differentiation. We highlight the strengths and weaknesses of this experimental model system. In particular, we explain evidence that organoids can mimic many aspects of early neural development, including neural induction, patterning, and broad neurogenesis and gliogenesis programs, offering the opportunity to study genetic regulation of these processes in a human context. Several shortcomings of the current culture methods limit the utility of cerebral organoids to spontaneously give rise to many important cell types, and to model higher order features of tissue organization. We suggest that future studies aim to improve these features in order to make them better models for the study of laminar organization, circuit formation and how disruptions of these processes relate to disease.

Zinc finger E-box binding homeobox (Zeb) 2 is a transcription factor, identified due its ability to bind Smad proteins, and consists of multiple functional domains which interact with a variety of transcriptional co-effectors. The complex nature of the Zeb2, both at its genetic and protein levels, underlie its multifunctional properties, with Zeb2 capable of acting individually or as part of a transcriptional complex to repress, and occasionally activate, target gene expression. This review introduces Zeb2 as an essential regulator of nervous system development. Zeb2 is expressed in the nervous system throughout its development, indicating its importance in neurogenic and gliogenic processes. Indeed, mutation of Zeb2 has dramatic neurological consequences both in animal models, and in humans with Mowat-Wilson syndrome, which results from heterozygous ZEB2 mutations. The mechanisms by which Zeb2 regulates the induction of the neuroectoderm (CNS primordium) and the neural crest (PNS primordium) are reviewed herein. We then describe how Zeb2 acts to direct the formation, delamination, migration and specification of neural crest cells. Zeb2 regulation of the development of a number of cerebral regions, including the neocortex and hippocampus, are then described. The diverse molecular mechanisms mediating Zeb2-directed development of various neuronal and glial populations are reviewed. The role of Zeb2 in spinal cord and enteric nervous system development is outlined, while its essential function in CNS myelination is also described. Finally, this review discusses how the neurodevelopmental defects of Zeb2 mutant mice delineate the developmental dysfunctions underpinning the multiple neurological defects observed in Mowat-Wilson syndrome patients.

Summary Mammalian neural stem/progenitor cells (NSPCs) sequentially generate neurons and glia during CNS development. Here we identified miRNA-153 (miR-153) as a modulator of the temporal regulation of NSPC differentiation. Overexpression (OE) of miR-153 delayed the onset of astrogliogenesis and maintained NSPCs in an undifferentiated state in vitro and in the developing cortex. The transcription factors nuclear factor I (NFI) A and B, essential regulators of the initiation of gliogenesis, were found to be targets of miR-153. Inhibition of miR-153 in early neurogenic NSPCs induced precocious gliogenesis, whereas NFIA/B overexpression rescued the anti-gliogenic phenotypes induced by miR-153 OE. Our results indicate that miR-mediated fine control of NFIA/B expression is important in the molecular networks that regulate the acquisition of gliogenic competence by NSPCs in the developing CNS., Graphical Abstract, Highlights • We identify miR-153 as a regulator for the acquisition of gliogenic competence • NFIA and NFIB are physiological targets of miR-153 • Inhibition of miR-153 confers gliogenic competence on early neurogenic NSPCs • Fine-tuning of NFIA/B expressions by miR-153 is involved in the timing of gliogenesis, Gliogenesis is tightly inhibited during the early neurogenic phase of central nervous system development. Okano, Shimazaki, and colleagues identified a microRNA, miR-153, as an inhibitor of gliogenesis in early neurogenic neural stem/progenitor cells (NSPCs). This study demonstrates that miR-153 controls the timing of the acquisition of gliogenic competence via direct repression of the gliogenic transcription factors NFIA and NFIB.

Spinal cord injury (SCI) evokes rapid deleterious and reparative glial reactions. Understanding the triggers for these responses is necessary for designing strategies to maximize repair. This study examined lesion formation and glial responses to vascular disruption and hemorrhage, a prominent feature of acute SCI. The specific role of hemorrhage is difficult to evaluate in trauma-induced lesions, because mechanical injury initiates many downstream responses. To isolate vascular disruption from trauma-induced effects, we created a novel and reproducible model of collagenase-induced intraspinal hemorrhage (ISH) and compared glial reactions between unilateral ISH and a hemi-contusion injury. Similar to contusion injuries, ISH lesions caused loss of myelin and axons and became filled with iron-laden macrophages. We hypothesized that intraspinal hemorrhage would also initiate reparative cellular responses including NG2+ oligodendrocyte progenitor cell (OPC) proliferation and oligodendrocyte genesis. Indeed, ISH induced OPC proliferation within 1d post-injury (dpi), which continued throughout the first week and resulted in a sustained elevation of NG2+ OPCs. ISH also caused oligodendrocyte loss within 4h that was sustained through 3d post-ISH. However, oligodendrogenesis, as determined by bromo-deoxyuridine (BrdU) positive oligodendrocytes, restored oligodendrocyte numbers by 7dpi, revealing that proliferating OPCs differentiated into new oligodendrocytes after ISH. The signaling molecules pERK1/2 and pSTAT3 were robustly increased acutely after ISH, with pSTAT3 being expressed in a portion of OPCs, suggesting that activators of this signaling cascade may initiate OPC responses. Aside from subtle differences in timing of OPC responses, changes in ISH tissue closely mimicked those in hemi-contusion tissue. These results are important for elucidating the contribution of hemorrhage to lesion formation and endogenous cell-mediated repair, and will provide the foundation for future studies geared toward identifying the role of specific blood components on injury and repair mechanisms. This understanding may provide new clinical targets for SCI and other devastating conditions such as intracerebral hemorrhage.

SummaryWe present a method to label and trace the lineage of multiple neural progenitors simultaneously in vertebrate animals via multiaddressable genome-integrative color (MAGIC) markers. We achieve permanent expression of combinatorial labels from new Brainbow transgenes introduced in embryonic neural progenitors with electroporation of transposon vectors. In the mouse forebrain and chicken spinal cord, this approach allows us to track neural progenitor’s descent during pre- and postnatal neurogenesis or perinatal gliogenesis in long-term experiments. Color labels delineate cytoarchitecture, resolve spatially intermixed clones, and specify the lineage of astroglial subtypes and adult neural stem cells. Combining colors and subcellular locations provides an expanded marker palette to individualize clones. We show that this approach is also applicable to modulate specific signaling pathways in a mosaic manner while color-coding the status of individual cells regarding induced molecular perturbations. This method opens new avenues for clonal and functional analysis in varied experimental models and contexts.

Glial cells which are indispensable for the central nervous system development and functioning, are proven to be vulnerable to a harmful influence of pathological cues and tissue misbalance. However, they are also highly sensitive to both in vitro and in vivo modulation of their commitment, differentiation, activity and even the fate-switch by different types of bioactive molecules. Since glial cells (comprising macroglia and microglia) are an abundant and heterogeneous population of neural cells, which are almost uniformly distributed in the brain and the spinal cord parenchyma, they all create a natural endogenous reservoir of cells for potential neurogenerative processes required to be initiated in response to pathophysiological cues present in the local tissue microenvironment. The past decade of intensive investigation on a spontaneous and enforced conversion of glial fate into either alternative glial (for instance from oligodendrocytes to astrocytes) or neuronal phenotypes, has considerably extended our appreciation of glial involvement in restoring the nervous tissue cytoarchitecture and its proper functions. The most effective modulators of reprogramming processes have been identified and tested in a series of pre-clinical experiments. A list of bioactive compounds which are potent in guiding in vivo cell fate conversion and driving cell differentiation includes a selection of transcription factors, microRNAs, small molecules, exosomes, morphogens and trophic factors, which are helpful in boosting the enforced neuro-or gliogenesis and promoting the subsequent cell maturation into desired phenotypes. Herein, an issue of their utility for a directed glial differentiation and transdifferentiation is discussed in the context of elaborating future therapeutic options aimed at restoring the diseased nervous tissue.

Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. To delineate the molecular mechanisms behind nociceptor hyperexcitability and small-fiber degeneration, it becomes important to uniquely identify changes in the nociceptor population from within the heterogeneous population of neurons in the DRG. To identify genes that are differentially expressed in PDN, we compared the RNA profile of mice fed a regular-diet (RD) or a high-fat diet (HFD), a commonly used model of PDN. To capture the translational state of these nociceptive neurons, we used transgenic mice expressing tagged ribosomal subunits (RiboTag) in the nociceptor population expressing the sodium channel-Nav1.8. Analysis of the RiboTagged mRNA between HFD and RD mice at 10 weeks revealed 84 upregulated genes and 70 downregulated genes. We identified candidate genes like ApoD, known to increase excitatory signaling through CXCR4/CXCL12. Previously our work demonstrated that CXCR4/CXCR12 signaling is critical for the development of mechanical allodynia and small-fiber degeneration in PDN. Further analysis implicated changes in pathways relating to extracellular matrix organization, gliogenesis and, complex 1 biogenesis. In addition to the translational profiling, we plan to perform single-cell RNA sequencing of the Nav1.8-positive DRG neuron population using Nav1.8-Cre;Ai9 mice fed an RD or an HFD. Single-cell RNA sequencing will give us a better understanding of the transcriptional status of subpopulations within the Nav1.8-positive DRG neurons and generate candidate genes at a higher resolution. This study will provide insight into the genes that are involved in the pathogenesis of neuropathic pain and small-fiber degeneration and yield potential translational targets for disease-modifying treatments for PDN.

Glial cells are present in all organisms with a CNS and, with increasing brain complexity, glial cells have undergone substantive increases in cell number, diversity, and functions. Invertebrates, such as Drosophila, possess glial subtypes with similarity to mammalian astrocytes in their basic morphology and function, representing fertile ground for unraveling fundamental aspects of glial biology. Although glial subtypes in simple organisms may be relatively homogenous, emerging evidence suggests the possibility that mammalian astrocytes might be highly diversified to match the needs of local neuronal subtypes. In this Perspective, we review classic and new roles identified for astrocytes and oligodendrocytes by recent studies. We propose that delineating genetic and developmental programs across species will be essential to understand the core functions of glia that allow enhanced neuronal function and to achieve new insights into glial roles in higher-order brain function and neurological disease.

The mouse Dach1 gene, involved in the development of the neocortex and the hippocampus, is expressed by neural stem cells (NSCs) during early neurogenesis, and its expression also continues in a subpopulation of cells in the dorsal part of the lateral ventricles (LV) of the adult mouse brain. In this study we aimed to elucidate the role of Dach1-expressing cells in adult neurogenesis/gliogenesis under physiological as well as post-ischemic conditions, employing transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the D6 promotor of the mouse Dach1 gene. A neurosphere-forming assay of GFP⁺ cells isolated from the dorsal part of the LV was carried out with subsequent differentiation in vitro. To elucidate the neurogenic/gliogenic potential of GFP⁺ cells in the dorsal part of the LV, in situ immunohistochemical/electrophysiological analyses of GFP⁺ cells in adult sham-operated brains (controls) and those after middle cerebral artery occlusion (MCAo) were performed. The GFP⁺ cells isolated from the dorsal part of the LV of controls formed neurospheres and differentiated solely into a glial phenotype, while those isolated after MCAo also gave rise to cells with the properties of neuronal precursors. In situ analyses revealed that GFP⁺ cells express the phenotype of adult NSCs or neuroblasts in controls as well as following ischemia. Following MCAo we found a significantly increased number of GFP⁺ cells expressing doublecortin as well as a number of GFP⁺ cells migrating through the rostral migratory stream into the olfactory bulb, where they probably differentiated into calretinin⁺ interneurons. Collectively, our results suggest the involvement of the mouse Dach1 gene in adult neurogenesis; cells expressing this gene exhibit the properties of adult NSCs or neuroblasts and respond to MCAo by enhanced neurogenesis.

Activity-based anorexia (ABA) is an animal model of anorexia nervosa that mimics core features of the clinical psychiatric disorder, including severe food restriction, weight loss, and hyperactivity. The ABA model is currently being used to study starvation-induced changes in the brain. Here, we examined hippocampal cell proliferation in animals with ABA (or the appropriate control conditions). Adolescent female Sprague-Dawley rats were assigned to 4 groups: control (24h/day food access), food-restricted (1h/day food access), exercise (24h/day food and wheel access), and ABA (1h/day food access, 24h/day wheel access). After 3 days of ABA, 5-bromo-2'-deoxyuridine (BrdU; 200mg/kg, i.p.) was injected and the rats were perfused 2h later. Brains were removed and subsequently processed for BrdU and Ki67 immunohistochemistry. The acute induction of ABA reduced cell proliferation in the dentate gyrus. This effect was significant in the hilus region of the dentate gyrus, but not in the subgranular zone, where adult neurogenesis occurs. Marked decreases in cell proliferation were also observed in the surrounding dorsal hippocampus and in the corpus callosum. These results indicate a primary effect on gliogenesis rather than neurogenesis following 3 days of ABA. For each brain region studied (except SGZ), there was a strong positive correlation between the level of cell proliferation and body weight/food intake. Future studies should examine whether these changes are maintained following long-term weight restoration and whether alterations in neurogenesis occur following longer exposures to ABA.

The Notch signaling pathway is essential for neuronal and glial specification during CNS development. Mind bomb-1 (Mib1) is an E3 ubiquitin ligase that ubiquitinates and promotes the endocytosis of Notch ligands. Although Mib1 is essential for transmitting the Notch signal, it is still unclear whether it is a primary regulator of Notch ligand activity in the developing spinal cord. In Mib1 conditional knock-out mice, we observed depletion of spinal progenitors, premature differentiation of neurons, and unbalanced specification of V2 interneurons, all of which mimic the conventional Notch phenotype. In agreement with this, the reduction of progenitors in the absence of Mib1 led to a loss of both astrocytes and oligodendrocytes. Late removal of Mib1 using a drug-inducible system suppressed glial differentiation, suggesting that Mib1 continues to play a role in the formation of late progenitors mainly designated for gliogenesis. Finally, misexpression of Mib1 or Mib1 deletion mutants revealed that the ring domain of Mib1 is required for the specification of V2 interneurons in the chick neural tube. Together, these findings suggest that Mib1 is a major component of the signal-sending cells required to provide Notch ligand activity for specifying neurons and glia in the spinal cord.

National Institutes of Health Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Provost's Office for Research of the University of São Paulo Programa de Incentivo a Pesquisa NAPNA-USP, Brazil International Rett Syndrome Foundation Emerald Foundation California Institute for Regenerative Medicine Grant Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. in the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. in agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression. Univ São Paulo, Inst Quim, Dept Bioquim, BR-05508000 São Paulo, Brazil Univ Calif San Diego, Dept Pediat, San Diego, CA 92093 USA Univ Calif San Diego, Dept Cellular & Mol Med, San Diego, CA 92093 USA Universidade Federal de São Paulo, Dept Biofis, BR-04023062 São Paulo, Brazil Univ Fed Rio de Janeiro, Inst Ciencias Biomed, BR-21941902 Rio de Janeiro, Brazil Universidade Federal de São Paulo, Dept Biofis, BR-04023062 São Paulo, Brazil National Institutes of Health: 1-DP2-OD006495-01 FAPESP: 2006/61285-9 Provost's Office for Research of the University of São Paulo Programa de Incentivo a Pesquisa: 2011.1.9333.1.3 International Rett Syndrome Foundation: 2517 California Institute for Regenerative Medicine Grant: TR2-01814 Web of Science

After trauma brain injury, a large number of cells die, releasing neurotoxic chemicals into the extracellular medium, decreasing cellular glutathione levels and increasing reactive oxygen species that affect cell survival and provoke an enlargement of the initial lesion. Alpha-lipoic acid is a potent antioxidant commonly used as a treatment of many degenerative diseases such as multiple sclerosis or diabetic neuropathy. Herein, the antioxidant effects of lipoic acid treatment after brain cryo-injury in rat have been studied, as well as cell survival, proliferation in the injured area, gliogenesis and angiogenesis. Thus, it is shown that newborn cells, mostly corresponded with blood vessels and glial cells, colonized the damaged area 15 days after the lesion. However, lipoic acid was able to stimulate the synthesis of glutathione, decrease cell death, promote angiogenesis and decrease the glial scar formation. All those facts allow the formation of new neural tissue. In view of the results herein, lipoic acid might be a plausible pharmacological treatment after brain injury, acting as a neuroprotective agent of the neural tissue, promoting angiogenesis and reducing the glial scar formation. These findings open new possibilities for restorative strategies after brain injury, stroke or related disorders.

A fundamental issue during nervous system development is how individual cells are formed from the undefined precursors. Differentiated neurons and glia, two major cell types mediating neuronal function, are acquired from immature precursors via a series of explicit controls exerted by transcription factors such as proteins in the family of Glial cells missing (Gcm). In mammals, Gcm proteins are involved in placenta and parathyroid gland development, whereas in the invertebrate organism Drosophila, Gcm proteins act as fate determinants for glial cell fate, regulate neural stem cell (NSC) induction and conversion, and promote glial proliferation. In particular, Gcm protein levels are carefully tuned for Drosophila gliogenesis and their stability is under precise control via the ubiquitin-proteasome system (UPS). Here we summarize recent advances on Gcm proteins function. In addition to describe various features of Gcm protein family, the significance of their functions in the developing nervous system is also discussed.

During the development of locomotion circuits it is essential that motoneurons with distinct subtype identities select the correct trajectories and target muscles. In vertebrates, the generation of motoneurons and myelinating glia depends on Olig2, one of the five Olig family bHLH transcription factors. We investigated the so far unknown function of the single Drosophila homolog Oli. Combining behavioral and genetic approaches, we demonstrate that oli is not required for gliogenesis, but plays pivotal roles in regulating larval and adult locomotion, and axon pathfinding and targeting of embryonic motoneurons. In the embryonic nervous system, Oli is primarily expressed in postmitotic progeny, and in particular, in distinct ventral motoneuron subtypes. oli mediates axonal trajectory selection of these motoneurons within the ventral nerve cord and targeting to specific muscles. Genetic interaction assays suggest that oli acts as part of a conserved transcription factor ensemble including Lim3, Islet and Hb9. Moreover, oli is expressed in postembryonic leg-innervating motoneuron lineages and required in glutamatergic neurons for walking. Finally, over-expression of vertebrate Olig2 partially rescues the walking defects of oli-deficient flies. Thus, our findings reveal a remarkably conserved role of Drosophila Oli and vertebrate family members in regulating motoneuron development, while the steps that require their function differ in detail., Highlights ► Drosophila Oli is expressed in embryonic ventral motoneuron subtypes. ► oli controls axonal trajectory selection and muscle targeting during embryogenesis. ► Oli acts as part of a conserved transcription factor ensemble that includes Hb9. ► Oli is expressed in postembryonic leg-innervating motoneuron lineages. ► Oli is required in glutamatergic neurons for adult locomotion.

Addiction is a chronic relapsing disorder associated with compulsive drug taking and drug seeking and a loss of control in limiting intake, reflected in three stages of a recurrent cycle: binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation (“craving”). This review discusses the role of adult-born neural and glial progenitors in drug-seeking associated with the different stages of the addiction cycle. A review of the current literature suggests that the loss of newly born progenitors, particularly in hippocampal and cortical regions, may play a role in determining vulnerability to relapse in rodent models of drug addiction. The normalization of drug-impaired neurogenesis or gliogenesis may help reverse neuroplasticity during abstinence, and thus may help reduce the vulnerability to relapse and aid recovery.

The purpose of this study was to investigate the microglia-driven apoptosis and the Aβ deposits triggered generation of new microglial cells in the neocortex of TgCRND8 mice. Three- and seven-month-old TgCRND8 mice, displaying an early and widespread amyloid deposition, respectively, were used. In 7-month-old TgCRND8 mice the Aβ-associated glial reaction was accompanied by an intense immunoreactivity of both TNF-α and inducible nitric oxide synthase, increased immunoreactivity of the pro-apoptotic protein Bax and a decrease in levels of the anti-apoptotic protein Bcl-2.Cortical and hippocampal neurons of TgCRND8 mice displayed higher immunoreactivity and higher nuclear expression of the transcription factor NF-kB than controls. It is possible that such an increase could represent a defence/compensatory response to degeneration. These findings indicate that Aβ deposits activate brain-resident microglia population and astrocytes, and induce overproduction of inflammatory mediators that enhance pro- and anti-apoptotic cascades. In both 3- and 7-month-old TgCRND8 mice apparent gliogenesis was present in the vicinity of Aβ plaques in the neocortex, indicating that microglia have a high proliferative rate which might play a more complex role than previously acknowledge.

Pluripotency and their neural crest origin make dental pulp stem cells (DPSCs) an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the integration of transplanted DPSC derived neuronal pecursors into the central nervous system. To address this issue, neuronally predifferentiated DPSCs, labeled with a vital cell dye Vybrant DiD were introduced into postnatal rat brain. DPSCs were transplanted into the cerebrospinal fluid of 3-day-old male Wistar rats. Cortical lesion was induced by touching a cold (-60°C) metal stamp to the calvaria over the forelimb motor cortex. Four weeks later cell localization was detected by fluorescent microscopy and neuronal cell markers were studied by immunohistochemistry. To investigate electrophysiological properties of engrafted, fluorescently labeled DPSCs, 300 μm-thick horizontal brain slices were prepared and the presence of voltage-dependent sodium and potassium channels were recorded by patch clamping. Predifferentiated donor DPSCs injected into the cerebrospinal fluid of newborn rats migrated as single cells into a variety of brain regions. Most of the cells were localized in the normal neural progenitor zones of the brain, the subventricular zone (SVZ), subgranular zone (SGZ) and subcallosal zone (SCZ). Immunohistochemical analysis revealed that transplanted DPSCs expressed the early neuronal marker N-tubulin, the neuronal specific intermediate filament protein NF-M, the postmitotic neuronal marker NeuN, and glial GFAP. Moreover, the cells displayed TTX sensitive voltage dependent (VD) sodium currents (I(Na)) and TEA sensitive delayed rectifier potassium currents (K(DR)). Four weeks after injury, fluorescently labeled cells were detected in the lesioned cortex. Neurospecific marker expression was increased in DPSCs found in the area of the cortical lesions compared to that in fluorescent cells of uninjured brain. TTX sensitive VD sodium currents and TEA sensitive K(DR) significantly increased in labeled cells of the cortically injured area. In conclusion, our data demonstrate that engrafted DPSC-derived cells integrate into the host brain and show neuronal properties not only by expressing neuron-specific markers but also by exhibiting voltage dependent sodium and potassium channels. This proof of concept study reveals that predifferentiated hDPSCs may serve as useful sources of neuro- and gliogenesis in vivo, especially when the brain is injured.

The neural stem cells that give rise to the neural lineages of the brain can generate their progeny directly or through transit amplifying intermediate neural progenitor cells (INPs). The INP-producing neural stem cells in Drosophila are called type II neuroblasts, and their neural progeny innervate the central complex, a prominent integrative brain center. Here we use genetic lineage tracing and clonal analysis to show that the INPs of these type II neuroblast lineages give rise to glial cells as well as neurons during postembryonic brain development. Our data indicate that two main types of INP lineages are generated, namely mixed neuronal/glial lineages and neuronal lineages. Genetic loss-of-function and gain-of-function experiments show that the gcm gene is necessary and sufficient for gliogenesis in these lineages. The INP-derived glial cells, like the INP-derived neuronal cells, make major contributions to the central complex. In postembryonic development, these INP-derived glial cells surround the entire developing central complex neuropile, and once the major compartments of the central complex are formed, they also delimit each of these compartments. During this process, the number of these glial cells in the central complex is increased markedly through local proliferation based on glial cell mitosis. Taken together, these findings uncover a novel and complex form of neurogliogenesis in Drosophila involving transit amplifying intermediate progenitors. Moreover, they indicate that type II neuroblasts are remarkably multipotent neural stem cells that can generate both the neuronal and the glial progeny that make major contributions to one and the same complex brain structure.

Alcohol-induced Wernicke–Korsakoff syndrome (WKS) culminates in bilateral diencephalic lesion and severe amnesia. Using the pyrithiamine-induced thiamine deficiency (PTD) animal paradigm of WKS, our laboratory has demonstrated hippocampal dysfunction in the absence of gross anatomical pathology. Extensive literature has revealed reduced hippocampal neurogenesis following a neuropathological insult, which might contribute to hippocampus-based learning and memory impairments. Thus, the current investigation was conducted to determine whether PTD treatment altered hippocampal neurogenesis in a stage-dependent fashion. Male Sprague–Dawley rats were assigned to one of 4 stages of thiamine deficiency based on behavioral symptoms: pre-symptomatic stage, ataxic stage, early post-opisthotonus stage, or the late post-opisthotonus stage. The S-phase mitotic marker 5′-bromo-2′-deoxyuridine (BrdU) was administered at the conclusion of each stage following thiamine restoration and subjects were perfused 24 hours or 28 days after BrdU to assess cellular proliferation or neurogenesis and survival, respectively. Dorsal hippocampal sections were immunostained for BrdU (proliferating cell marker), NeuN (neurons), GFAP (astrocytes), Iba-1 (microglia), and O4 (oligodendrocytes). The PTD treatment increased progenitor cell proliferation and survival during the early post-opisthotonus stage. However, levels of neurogenesis were reduced during this stage as well as the late post-opisthotonus stage where there was also an increase in astrocytogenesis. The diminished numbers of newly generated neurons (BrdU/NeuN co-localization) was paralleled by increased BrdU cells that did not co-localize with any of the phenotypic markers during these later stages. These data demonstrate that long-term alterations in neurogenesis and gliogenesis might contribute to the observed hippocampal dysfunction in the PTD model and human WKS.

Penetrating traumatic insult during pregnancy is a leading cause of human fetal demise; in particular, trauma to the brain may lead to devastating long-term cognitive sequelae. Perinatal brain injury involves glial precursors, but the neural mechanisms controlling astrocyte ontogeny after injury remain incompletely understood, partly due to a lack of appropriate markers and animal models. We analyzed astrocyte precursor response to injury at the beginning (E11) and peak (E15) of gliogenesis in an avian tectal model of penetrating embryonic brain trauma, without confounding maternal and sibling effects. At both ages, lateral ventricular dilatation, necrotic foci, periventricular cysts and intraventricular hemorrhages were observed distal to stab wounds two days after a unilateral stab injury to optic tecta. Neuronal (TUBB3) and oligodendrocyte precursor (PLP) markers were down-regulated, even far-removed from the wound site. In contrast, the mature astrocyte marker, GFAP, was up-regulated at the wound site, around necrotic areas and cysts, plus in usual areas of GFAP expression. Increased inflammatory response and apoptotic cell death were also confirmed in the injured tecta. Increased expression of NFIA, SOX9 and GLAST at the wound site and in the ventricular zone (VZ) of the injured tecta indicated an astroglial precursor response. However, cell division increased in the VZ only in early (E11) injury, but not later (E15), indicating that in late injury the astrogliogenesis occurring after acute injury is predominantly due to precursor differentiation rather than precursor proliferation. The inability to replenish the glial precursor pool during the critical period of vulnerability to injury may be an important cause of subsequent developmental abnormalities.

The role of radial glia cells (RGCs) as neural progenitors and as guides for migrating neurons is well established, whereas their precise contribution to adult hippocampal neurogenesis remains less understood. To precisely study the properties of hippocampal RGCs under normal conditions in vitro, here we acquired the hippocampal RGCs of postnatal 1 d rats under adherent conditions in vitro, identified their astroglia and stem/progenitor properties. We found that the neonatal rat hippocampal RGCs had longer processes than the RGCs from fetal cerebral cortices, and these cells could be double-labeled by BLBP, GFAP, Vimentin with Nestin and expressed some stem/progenitor genes, these cells also presented multiple differentiation potentialities, albeit the ability of gliogenesis far exceeded the neurogenesis under normal culture conditions in vitro. Taken together, we acquired and identified some properties of the RGCs from neonatal rat hippocampi in vitro.