NSC Proliferation Research Articles

Injectable Hydrogel Loaded with CDs and FTY720 Combined with Neural Stem Cells for the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) is an incurable and disabling event that is accompanied by complex inflammation-related pathological processes, such as the production of excessive reactive oxygen species (ROS) by infiltrating inflammatory immune cells and their release into the extracellular microenvironment, resulting in extensive apoptosis of endogenous neural stem cells. In this study, we noticed the neuroregeneration-promoting effect as well as the ability of the innovative treatment method of FTY720-CDs@GelMA paired with NSCs to increase motor function recovery in a rat spinal cord injury model. Carbon dots (CDs) and fingolimod (FTY720) were added to a hydrogel created by chemical cross-linking GelMA (FTY720-CDs@GelMA). The basic properties of FTY720-CDs@GelMA hydrogels were investigated using TEM, SEM, XPS, and FTIR. The swelling and degradation rates of FTY720-CDs@GelMA hydrogels were measured, and each group's ability to scavenge reactive oxygen species was investigated. The in vitro biocompatibility of FTY720-CDs@GelMA hydrogels was assessed using neural stem cells. The regeneration of the spinal cord and recovery of motor function in rats were studied following co-treatment of spinal cord injury using FTY720-CDs@GelMA hydrogel in combination with NSCs, utilising rats with spinal cord injuries as a model. Histological and immunofluorescence labelling were used to determine the regeneration of axons and neurons. The recovery of motor function in rats was assessed using the BBB score. The hydrogel boosted neurogenesis and axonal regeneration by eliminating excess ROS and restoring the regenerative environment. The hydrogel efficiently contained brain stem cells and demonstrated strong neuroprotective effects in vivo by lowering endogenous ROS generation and mitigating ROS-mediated oxidative stress. In a follow-up investigation, we discovered that FTY720-CDs@GelMA hydrogel could dramatically boost NSC proliferation while also promoting neuronal regeneration and synaptic formation, hence lowering cavity area. Our findings suggest that the innovative treatment of FTY720-CDs@GelMA paired with NSCs can effectively improve functional recovery in SCI patients, making it a promising therapeutic alternative for SCI.

Disrupted de novo pyrimidine biosynthesis impairs adult hippocampal neurogenesis and cognition in pyridoxine-dependent epilepsy.

Despite seizure control by early high-dose pyridoxine (vitamin B6) treatment, at least 75% of pyridoxine-dependent epilepsy (PDE) patients with ALDH7A1 mutation still suffer from intellectual disability. It points to a need for additional therapeutic interventions for PDE beyond pyridoxine treatment, which provokes us to investigate the mechanisms underlying the impairment of brain hemostasis by ALDH7A1 deficiency. In this study, we show that ALDH7A1-deficient mice with seizure control exhibit altered adult hippocampal neurogenesis and impaired cognitive functions. Mechanistically, ALDH7A1 deficiency leads to the accumulation of toxic lysine catabolism intermediates, α-aminoadipic-δ-semialdehyde and its cyclic form, δ-1-piperideine-6-carboxylate, which in turn impair de novo pyrimidine biosynthesis and inhibit NSC proliferation and differentiation. Notably, supplementation of pyrimidines rescues abnormal neurogenesis and cognitive impairment in ALDH7A1-deficient adult mice. Therefore, our findings not only define the important role of ALDH7A1 in the regulation of adult hippocampal neurogenesis but also provide a potential therapeutic intervention to ameliorate the defective mental capacities in PDE patients with seizure control.

Microvascular endothelial cells derived from spinal cord promote spinal cord injury repair.

Neural regeneration after spinal cord injury (SCI) closely relates to the microvascular endothelial cell (MEC)-mediated neurovascular unit formation. However, the effects of central nerve system-derived MECs on neovascularization and neurogenesis, and potential signaling involved therein, are unclear. Here, we established a primary spinal cord-derived MECs (SCMECs) isolation with high cell yield and purity to describe the differences with brain-derived MECs (BMECs) and their therapeutic effects on SCI. Transcriptomics and proteomics revealed differentially expressed genes and proteins in SCMECs were involved in angiogenesis, immunity, metabolism, and cell adhesion molecular signaling was the only signaling pathway enriched of top 10 in differentially expressed genes and proteins KEGG analysis. SCMECs and BMECs could be induced angiogenesis by different stiffness stimulation of PEG hydrogels with elastic modulus 50-1650Pa for SCMECs and 50-300Pa for BMECs, respectively. Moreover, SCMECs and BMECs promoted spinal cord or brain-derived NSC (SNSC/BNSC) proliferation, migration, and differentiation at different levels. At certain dose, SCMECs in combination with the NeuroRegen scaffold, showed higher effectiveness in the promotion of vascular reconstruction. The potential underlying mechanism of this phenomenon may through VEGF/AKT/eNOS- signaling pathway, and consequently accelerated neuronal regeneration and functional recovery of SCI rats compared to BMECs. Our findings suggested a promising role of SCMECs in restoring vascularization and neural regeneration.

FoxO3 Modulates Circadian Rhythms in Neural Stem Cells.

Both FoxO transcription factors and the circadian clock act on the interface of metabolism and cell cycle regulation and are important regulators of cellular stress and stem cell homeostasis. Importantly, FoxO3 preserves the adult neural stem cell population by regulating cell cycle and cellular metabolism and has been shown to regulate circadian rhythms in the liver. However, whether FoxO3 is a regulator of circadian rhythms in neural stem cells remains unknown. Here, we show that loss of FoxO3 disrupts circadian rhythmicity in cultures of neural stem cells, an effect that is mediated via regulation of Clock transcriptional levels. Using Rev-Erbα-VNP as a reporter, we then demonstrate that loss of FoxO3 does not disrupt circadian rhythmicity at the single cell level. A meta-analysis of published data revealed dynamic co-occupancy of multiple circadian clock components within FoxO3 regulatory regions, indicating that FoxO3 is a Clock-controlled gene. Finally, we examined proliferation in the hippocampus of FoxO3-deficient mice and found that loss of FoxO3 delayed the circadian phase of hippocampal proliferation, indicating that FoxO3 regulates correct timing of NSC proliferation. Taken together, our data suggest that FoxO3 is an integral part of circadian regulation of neural stem cell homeostasis.

Pregnenolone enhances the proliferation of mouse neural stem cells and promotes oligodendrogenesis, together with Sox10, and neurogenesis, along with Notch1 and Pax6.

Pregnenolone is a precursor of various steroid hormones involved in osteoblast proliferation, microtubules polymerization and cell survival protection. Previous reports focused on the effects of pregnenolone metabolites on stem cell proliferation and differentiation; however, the effects of pregnenolone itself has not been well explored. The present study aimed to investigate the role of pregnenolone on NSC proliferation and to determine the doses required for NSC differentiation as well as the various genes involved in its mechanism of action. NSCs were isolated from the embryonic cortex of E14 mice, incubated for 5 days, and then treated with pregnenolone doses of 2, 5, 10, 15 and 20μM for another 5 days. The number of neurospheres and neurosphere derived cells were then counted. Flow cytometry was used to evaluate the differentiation of NSCs into oligodendrocytes, astrocytes, and neurons. The expression level of Notch1, Pax6 and Sox10 genes were also measured by Real Time PCR after 5 days of treatment. Our data suggest that treatment with 10μM pregnenolone is optimal for NSC proliferation. In fact, this concentration caused the highest increase in the number of neurospheres and neurosphere derived cells, compared to the control group. In addition, treatment with low doses of pregnenolone (5 and 10μM) caused a significant increase in NSC differentiation towards immature (Olig2+) and mature (MBP+) oligodendrocyte cell populations, compared to controls. However, NSC differentiation into neurons (beta III tubulin+cells) increased in all treatment groups, with the highest and most significant increase obtained at 15μM concentration. It is worth noting that pregnenolone at the highest concentration of 15μM decreased the number of astrocytes (GFAP+). Furthermore, there was an increase of Sox10 expression with low pregnenolone doses, leading to oligodendrogenesis, whereas Notch1 and Pax6 gene expression increased in pregnenolone groups with more neurogenesis. Pregnenolone regulates NSCs proliferation in vitro. Treatment with low doses of pregnenolone caused an increase in the differentiation of NSCs into mature oligodendrocytes while higher doses increased the differentiation of NSCs into neurons. Oligodendrogenesis was accompanied by Sox10 while neurogenesis occurred together with Notch1 and Pax6 expression.

The Effects of Cuprizone on Murine Subventricular Zone-Derived Neural Stem Cells and Progenitor Cells Grown as Neurospheres.

Despite the extensive use of the cuprizone (CPZ) demyelination animal model, there is little evidence regarding the effects of CPZ on a cellular level. Initial studies have suggested that oligodendrocytes (OL) are the main cell targets for CPZ toxicity. However, recent data have revealed additional effects on neural stem cells and progenitor cells (NSC/NPC), which constitute a reservoir for OL regeneration during brain remyelination. We cultured NSC/NPC as neurospheres to investigate CPZ effects on cell mechanisms which are thought to be involved in demyelination and remyelination processes in vivo. Proliferating NSC/NPC cultures exposed to CPZ showed overproduction of intracellular reactive oxygen species and increased progenitor migration at the expense of a significant inhibition of cell proliferation. Although NSC/NPC survival was not affected by CPZ in proliferative conditions, we found that CPZ-treated cultures undergoing cell differentiation were more prone to cell death than controls. The commitment and cell differentiation towards neural lineages did not seem to be affected by CPZ, as shown by the conserved proportions of OL, astrocytes, and neurons. Nevertheless, when CPZ treatment was performed after cell differentiation, we detected a significant reduction in the number and the morphological complexity of OL, astrogliosis, and neuronal damage. We conclude that, in addition to damaging mature OL, CPZ also reduces NSC/NPC proliferation and activates progenitor migration. These results shed light on CPZ direct effects on NSC proliferation and the progression of in vitro differentiation.

GABAergic regulation of cell proliferation within the adult mouse spinal cord

Manipulation of neural stem cell proliferation and differentiation in the postnatal CNS is receiving significant attention due to therapeutic potential. In the spinal cord, such manipulations may promote repair in conditions such as multiple sclerosis or spinal cord injury, but may also limit excessive cell proliferation contributing to tumours such as ependymomas. We show that when ambient γ-aminobutyric acid (GABA) is increased in vigabatrin-treated or decreased by GAD67 allele haplodeficiency in glutamic acid decarboxylase67-green fluorescent protein (GAD67-GFP) mice of either sex, the numbers of proliferating cells respectively decreased or increased. Thus, intrinsic spinal cord GABA levels are correlated with the extent of cell proliferation, providing important evidence for manipulating these levels. Diazepam binding inhibitor, an endogenous protein that interacts with GABA receptors and its breakdown product, octadecaneuropeptide, which preferentially activates central benzodiazepine (CBR) sites, were highly expressed in spinal cord, especially in ependymal cells surrounding the central canal. Furthermore, animals with reduced CBR activation via treatment with flumazenil or Ro15-4513, or with a G2F77I mutation in the CBR binding site had greater numbers of Ethynyl-2′-deoxyuridine positive cells compared to control, which maintained their stem cell status since the proportion of newly proliferated cells becoming oligodendrocytes or astrocytes was significantly lower. Altering endogenous GABA levels or modulating GABAergic signalling through specific sites on GABA receptors therefore influences NSC proliferation in the adult spinal cord. These findings provide a basis for further study into how GABAergic signalling could be manipulated to enable spinal cord self-regeneration and recovery or limit pathological proliferative activity.

Phosphatase and Tensin Homology Deleted on Chromosome 10 Inhibitors Promote Neural Stem Cell Proliferation and Differentiation.

Phosphatase and tensin homology deleted on chromosome 10 (PTEN) is a tumor suppressor gene. Its encoded protein has phosphatase and lipid phosphatase activities, which regulate the growth, differentiation, migration, and apoptosis of cells. The catalytic activity of PTEN is crucial for controlling cell growth under physiological and pathological conditions. It not only affects the survival and proliferation of tumor cells, but also inhibits a variety of cell regeneration processes. The use of PTEN inhibitors is being explored as a potentially beneficial therapeutic intervention for the repair of injuries to the central nervous system. PTEN influences the proliferation and differentiation of NSCs by regulating the expression and phosphorylation of downstream molecular protein kinase B (Akt) and the mammalian target of rapamycin (mTOR). However, the role of PTEN inhibitors in the Akt/mTOR signaling pathway in NSC proliferation and differentiation is unclear. Dipotassium bisperoxo (picolinoto) oxovanadate (V) [bpv(pic)] is a biologically active vanadium compound that blocks PTEN dephosphorylation and suppresses its activity, and has been used as a PTEN lipid phosphatase inhibitor. Here, bpv(pic) intervention was found to significantly increase the number of rat NSCs, as determined by bromodeoxyuridine staining and the cell counting kit-8, and to increase the percentage of neurons undergoing differentiation, as shown by immunofluorescence staining. Bpv(pic) intervention also significantly increased PTEN and mTOR expression, as shown by real-time PCR analysis and western blotting. In conclusion, PTEN inhibitor bpv(pic) promotes the proliferation and differentiation of NSCs into neurons.

Neurogenesis Is Increased in Human Neural Stem Cells by Aβ40 Peptide.

Amyloid-β 40 peptides [Aβ1-40 (Aβ40)] are present within amyloid plaques in the brains of patients with Alzheimer’s disease (AD). Even though Aβ peptides are considered neurotoxic, they can mediate many biological processes, both in adult brains and throughout brain development. However, the physiological function of these Aβ peptides remains poorly understood, and the existing data are sometimes controversial. Here, we analyze and compare the effects of monomeric Aβ40 on the biology of differentiating human neural stem cells (human NSCs). For that purpose, we used a model of human NSCs called hNS1. Our data demonstrated that Aβ40 at high concentrations provokes apoptotic cellular death and the damage of DNA in human NSCs while also increasing the proliferation and favors neurogenesis by raising the percentage of proliferating neuronal precursors. These effects can be mediated, at least in part, by β-catenin. These results provide evidence of how Aβ modulate/regulate human NSC proliferation and differentiation, suggesting Aβ40 may be a pro-neurogenic factor. Our data could contribute to a better understanding of the molecular mechanisms involved in AD pathology and to the development of human NSC-based therapies for AD treatment, since these results could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.

Actin Alpha 2 Downregulation Inhibits Neural Stem Cell Proliferation and Differentiation into Neurons through Canonical Wnt/β-Catenin Signaling Pathway.

Our previous study has shown that actin alpha 2 (ACTA2) is expressed in NSC and ACTA2 downregulation inhibits NSC migration by increasing RhoA expression and decreasing the expression of Rac1 to curb actin filament polymerization. Given that proliferation and differentiation are the two main characteristics of NSC, the role of ACTA2 downregulation in the proliferation and differentiation of NSC remains elusive. Here, the results demonstrated that ACTA2 downregulation using ACTA2 siRNA held the potential of inhibiting NSC proliferation using cell counting kit-8 (CCK8) and immunostaining. Then, our data illustrated that ACTA2 downregulation attenuated NSC differentiation into neurons, while directing NSC into astrocytes and oligodendrocytes using immunostaining and immunoblotting. Thereafter, the results revealed that the canonical Wnt/β-catenin pathway was involved in the effect of ACTA2 downregulation on the proliferation and differentiation of NSC through upregulating p-β-catenin and decreasing β-catenin due to inactivating GSK-3β, while this effect could be partially abolished with administration of CHIR99012, a GSK-3 inhibitor. Collectively, these results indicate that ACTA2 downregulation inhibits NSC proliferation and differentiation into neurons through inactivation of the canonical Wnt/β-catenin pathway. The aim of the present study is to elucidate the role of ACTA2 in proliferation and differentiation of NSC and to provide an intervention target for promoting NSC proliferation and properly directing NSC differentiation.

Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus.

The reprogramming of cells into induced neural stem cells (iNSCs), which are faster and safer to generate than induced pluripotent stem cells, holds tremendous promise for fundamental and frontier research, as well as personalized cell-based therapies for neurological diseases. However, reprogramming cells with viral vectors increases the risk of tumor development due to vector and transgene integration in the host cell genome. To circumvent this issue, the Sendai virus (SeV) provides an alternative integration-free reprogramming method that removes the danger of genetic alterations and enhances the prospects of iNSCs from bench to bedside. Since pigs are among the most successful large animal models in biomedical research, porcine iNSCs (piNSCs) may serve as a disease model for both veterinary and human medicine. Here, we report the successful generation of piNSC lines from pig fibroblasts by employing the SeV. These piNSCs can be expanded for up to 40 passages in a monolayer culture and produce neurospheres in a suspension culture. These piNSCs express high levels of NSC markers (PAX6, SOX2, NESTIN, and VIMENTIN) and proliferation markers (KI67) using quantitative immunostaining and western blot analysis. Furthermore, piNSCs are multipotent, as they are capable of producing neurons and glia, as demonstrated by their expressions of TUJ1, MAP2, TH, MBP, and GFAP proteins. During the reprogramming of piNSCs with the SeV, no induced pluripotent stem cells developed, and the established piNSCs did not express OCT4, NANOG, and SSEA1. Hence, the use of the SeV can reprogram porcine somatic cells without first going through an intermediate pluripotent state. Our research produced piNSCs using SeV methods in novel, easily accessible large animal cell culture models for evaluating the efficacy of iNSC-based clinical translation in human medicine. Additionally, our piNSCs are potentially applicable in disease modeling in pigs and regenerative therapies in veterinary medicine.

LncRNA MEG3 inhibits the proliferation of neural stem cells after ischemic stroke via the miR-493–5P/MIF axis

ObjectiveThe proliferation of neural stem cells (NSCs11NSCs: neural stem cells.), or lack thereof, can have profound effects on brain tissue remodeling for ischemic stroke (IS22IS: ischemic stroke.). In this study, we aimed to reveal the influence of the lncRNA MEG3/miR-493–5p/MIF axis on NSC proliferation after IS. MethodsWe established an oxygen glucose-deprivation/reoxygenation (OGD/R33OGD/R: oxygen and glucose confinement/reoxygenation.) in vitro model of IS in NSCs. We evaluated NSC isolation efficiency and proliferation by NESTIN, SOX2, and PCNA immunofluorescence staining. MEG3 and miR-493–5P levels were assessed by quantitative real-time polymerase chain reaction (qRT-PCR44qRT-PCR: quantitative real-time polymerase chain reaction.). Changes in MIF protein expression levels were analyzed using Western blotting. We then evaluated the role of MEG3 and miR-493–5p by transfection of si-MEG3, a miR-493–5p mimic, or miR-493–5p inhibitor. NSC proliferation was quantified using Cell Counting Kit-8 analysis. ResultsNESTIN and SOX2 were co-expressed in endogenous NSCs. Following OGD/R, MEG3 and miR-493–5P were significantly upregulated in NSCs, while MIF levels decreased and proliferation was inhibited. Knockdown of MEG3 inhibited miR-493–5p and rescued expression of MIF and PCNA, restoring cellular proliferation levels. In NSCs transfected with a miR-493–5p mimic or inhibitor, MIF levels were down- or upregulated, respectively. Consistently, transfection of a miR-493–5p mimic reduced NSC proliferation, while transfection with a miR-493–5p inhibitor or si-MEG3 rescued the inhibitory effect of OGD/R on NSC proliferation. After co-transfection of si-MEG3 and a miR-493–5p mimic of OGD/R-induced NSCs, levels of PCNA, an indicator of cellular proliferation, were significantly reduced. Conclusion MEG3 inhibits NSC proliferation of after IS via positive regulation of miR-493–5p and potential subsequent downregulation of MIF.

Chronic unpredictable stress negatively regulates hippocampal neurogenesis and promote anxious depression-like behavior via upregulating apoptosis and inflammatory signals in adult rats

Psychological and physical stress play a pivotal role in etiology of anxiety and depression. Chronic psychological and physical stress modify various physiological phenomena, as a consequence of which oxidative stress, decreased neurotransmitter level, elevated corticosterone level and altered NSC homeostasis is observed. However, the precise mechanism by which chronic stress induce anxious depression and modify internal milieu is still unknown. Herein, we show that exposure to CUS increase oxidative stress, microgliosis, astrogliosis while it reduces hippocampal NSC proliferation, neuronal differentiation and maturation in adult rats. CUS exposure in rats reduce dopamine and serotonin level in cortex and hippocampus, which result in increased anxiety and depression-like phenotypes. We also found elevated level of NF-κB and TNF-α while decreased anti-inflammatory cytokine IL-10 level, that led to increased expression of Bax and cleaved Caspase-3 whereas down regulation of antiapoptotic protein Bcl2. Additionally, CUS altered adult hippocampal neurogenesis, increased gliosis and neuronal apoptosis in cerebral cortex and hippocampus which might be associated with reduced AKT and increased ERK signaling, as seen in the rat brain tissue. Taken together, these results indicate that CUS induce oxidative stress and neuroinflammation which directly affects NSC dynamics, monoamines levels and behavioral functions in adult rats.

MiR-31 promotes neural stem cell proliferation and restores motor function after spinal cord injury.

This study aims to examine whether miR-31 promotes endogenous NSC proliferation and be used for spinal cord injury management. In the present study, the morpholino knockdown of miR-31 induced abnormal neuronal apoptosis in zebrafish, resulting in impaired development of the tail. miR-31 agomir transfection in NSCs increased Nestin expression and decreased ChAT and GFAP expression levels. miR-31 induced the proliferation of mouse NSCs by upregulating the Notch signaling pathway, and more NSCs entered G1; Notch was inhibited by miR-31 inactivation. Injection of a miR-31 agomir into mouse models of spinal cord injury could effectively restore motor functions after spinal cord injury, which was achieved by promoting the proliferation of endogenous NSCs. After the injection of a miR-31 agomir in spinal cord injury mice, the expression of Nestin and GFAP increased, while GFAP expression decreased. In conclusion, the zebrafish experiments prove that a lack of miR-31 will block nervous system development. In spinal cord injury mouse models, miR-31 overexpression might promote spinal cord injury repair.

Cuprizone-induced demyelination under physiological and post-stroke condition leads to decreased neurogenesis response in adult mouse brain

Due to the limitation in treatment window of the rtPA (recombinant tissue plasminogen activator), the development of delayed treatment for stroke is needed. We previously reported that there is a difference in neurogenesis and neuroblast migration patterns in different mouse stroke models (proximal and distal middle cerebral artery occlusion models, pMCAo or dMCAo). Specifically, compared to robust neurogenesis and substantial migration of newly born neuroblasts in pMCAo model, dMCAo only illicit limited neurogenesis and migration of neuroblasts towards ischemic area. One potential reason for this difference is the relative location of ischemic area to white matter and the neurogenic niche (subventricular zone, SVZ). Specifically, white matter could serve as a physical barrier or inhibitory factor to neurogenesis and migration in the dMCAo model. Given that a major difference in human and rodent brains is the content of white matter in the brain, in this study, we further characterize these two models and test the important hypothesis that white matter is an important contributing inhibitory factor for the limited neurogenesis in the dMCAo model. We utilized a genetically inducible NSC-specific reporter mouse line (nestin-CreERT2-R26R-YFP) to label and track NSC proliferation, survival and differentiation in ischemic brain. To test whether myelin is inhibitory to neurogenesis in dMCAo model, we demyelinated mouse brains using cuprizone treatment after stroke and examined whether there is enhanced neurogenesis or migration of neuroblasts cells in stroke mice treated with cuprizone. Our data suggests that demyelination of the brain does not result in enhanced neurogenesis or migration of neuroblasts, supporting that myelin is not a major inhibitory factor for stroke-induced neurogenesis. In addition, our results suggest that in non-stroke mice, demyelination causes decreased neurogenesis in adult brain, indicating a potential positive role of myelin in maintenance of adult neural stem cell niche.

Graphene oxide-PLGA hybrid nanofibres for the local delivery of IGF-1 and BDNF in spinal cord repair

Spinal cord injury (SCI) can lead to permanent and severe functional impairment below the lesion level and is still one of the most challenging clinical problems. The treatment of SCI has progressed with the development of tissue engineering techniques. Insulin-like growth factor 1 (IGF-1) and brain-derived neurotrophic factor (BDNF) are growth factors closely related to nerve regeneration. In this study, IGF-1 and BDNF were successfully immobilized on biodegradable graphene oxide (GO)-incorporated PLGA (PLGA/GO) electrospun nanofibres. The effect of PLGA/GO nanofibres with immobilized IGF-1 and BDNF on neurogenesis was investigated in vitro and in vivo utilizing MTT assays, immunofluorescence, motor function detection and histology observations. We demonstrated that PLGA/GO nanofibres loaded with IGF-1 and BDNF not only protected NSCs from oxidative stress induced by H2O2 but also enhanced NSC proliferation and neuronal differentiation in vitro. The in vivo study of an SCI animal model demonstrated that local delivery of IGF-1 and BDNF immobilized to PLGA/GO nanofibres significantly improved functional locomotor recovery, reduced cavity formation and increased the number of neurons at the injury site. Our study indicated that PLGA/GO is an effective carrier for IGF-1 and BDNF delivery and that immobilization of IGF-1 and BDNF onto PLGA/GO nanofibres has a great potential as a nerve implant for spinal cord injury applications.

Co-administration of Anti microRNA-124 and -137 Oligonucleotides Prevents Hippocampal Neural Stem Cell Loss Upon Non-convulsive Seizures.

Convulsive seizures promote adult hippocampal neurogenesis (AHN) through a transient activation of neural stem/progenitor cells (NSPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG). However, in a significant population of epilepsy patients, non-convulsive seizures (ncSZ) are observed. The response of NSPCs to non-convulsive seizure induction has not been characterized before. We here studied first the short-term effects of controlled seizure induction on NSPCs fate and identity. We induced seizures of controlled intensity by intrahippocampally injecting increasing doses of the chemoconvulsant kainic acid (KA) and analyzed their effect on subdural EEG recordings, hippocampal structure, NSPC proliferation and the number and location of immature neurons shortly after seizure onset. After establishing a KA dose that elicits ncSZ, we then analyzed the effects of ncSZ on NSPC proliferation and NSC identity in the hippocampus. ncSZ specifically triggered neuroblast proliferation, but did not induce proliferation of NSPCs in the SGZ, 3 days post seizure onset. However, ncSZ induced significant changes in NSPC composition in the hippocampus, including the generation of reactive NSCs. Interestingly, intrahippocampal injection of a combination of two anti microRNA oligonucleotides targeting microRNA-124 and -137 normalized neuroblast proliferation and prevented NSC loss in the DG upon ncSZ. Our results show for the first time that ncSZ induce significant changes in neuroblast proliferation and NSC composition. Simultaneous antagonism of both microRNA-124 and -137 rescued seizure-induced alterations in NSPC, supporting their coordinated action in the regulation of NSC fate and proliferation and their potential for future seizure therapies.

Melatonin protected against the detrimental effects of microRNA‐363 in a rat model of vitamin A‐associated congenital spinal deformities: Involvement of Notch signaling

Congenital spinal deformities are a result of defective somitogenesis and are associated with vitamin A deficiency (VAD). However, the molecular mechanisms of VAD-associated congenital spinal deformities remain largely unknown. Increasing number of studies suggested that microRNAs and melatonin played important roles in the development of congenital spinal deformities. In this study, we showed that the whole-embryo expression of miR-363 was upregulated in VAD rats. Furthermore, we demonstrated that miR-363 inhibited the proliferation and neuronal differentiation of primary cultured NSCs, accompanied by downregulation of Notch1. To this end, melatonin suppressed miR-363 expression and rescued the effects of miR-363 on NSC proliferation and neuronal differentiation together with restoration of Notch signaling. The present study provided new insights into the mechanism of VAD-associated spinal deformities and the therapeutic effect of melatonin that may lead to novel understanding of the molecular mechanisms of congenital spinal deformities.

Neural stem cell-derived factors inhibit the growth and invasion of U87 stem-like cells in vitro.

Glioma is one of the most common and aggressive tumors in the brain. Significant attention has been paid to the potential use of neural stem/progenitor cells (NSCs/NPCs) as delivery vehicles to cure gliomas. However, whether the NSCs/NPCs or the factors they produced could make a contribution still remains to be seen. In this study, we focused on the inhibitory effects of the factors produced by NSCs/NPCs on the biological behavior of the glioma stem-like cell in vitro. The human glioma cell line U87 was selected and the U87 stem-like cells were addressed. After being cultured in the NSCconditionmedium (NSC-CM), the viability and proliferation of U87 stem-like cells were significantly reduced. The invasion of U87 stem-like cells and the migration of U87 cells were also significantly decreased. However, no significant change was observed in regard to the astrocytic differentiation of U87 stem-like cells. These indicated that NSCs/NPCs produced some factors and had aninhibitory effect on the growth and invasion but not the terminal differentiation of U87 stem-like cells. It is worth paying attention to NSCs/NPCs as ahigh-potential candidatefor glioma treatment.

Autophagy controls neonatal myogenesis by regulating the GH-IGF1 system through a NFE2L2- and DDIT3-mediated mechanism

ABSTRACTMacroautophagy/autophagy is emerging as an important process in adult muscle stem cells functions: it regulates metabolic reprogramming during activation from a quiescent state, maintains stemness and prevents senescence. We now show that autophagy is specifically required for neonatal myogenesis and muscle development. Specific deletion of Atg7 in PAX7+ (paired box 7) precursors led in mice to a dwarf phenotype, with an effect restricted to the neonatal phase of muscle development. Atg7 knockdown suppressed neonatal satellite cell (nSC) proliferation and differentiation, downregulating the GH-IGF1 functions. When we disrupted autophagy, NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) accumulated in muscle and nSCs and negatively modulated DDIT3/CHOP (DNA-damage inducible transcript 3) expression. Lower levels of DDIT3 were responsible for reduced GHR expression leading to impaired local production of IGF1. Our results conclusively identify a novel autophagy-dependent pathway that regulates nSC behavior and indicate that autophagy is required for skeletal muscle development in the neonatal phase.Abbreviations: AKT/protein kinase B: Thymoma viral proto-oncogene; ASCs: adult stem cells; ATF4: activating transcription factor 4; ATG7: autophagy related 7; BAT: brown adipose tissue; BMP: bone morphogenetic protein; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CSA: cross sectional area; CTNNB1: catenin (cadherin associated protein), beta 1; DDIT3: DNA-damage inducible transcript 3; DM: differentiation medium; E: embryonic stage; EIF2AK3/PERK; EIF4EBP1: eukaryotic translation initiation factor 2 alpha kinase 3; eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; FGF21: fibroblast growth factor 21; GH: growth hormone; GHR: growth hormone receptor; HSCs: hematopoietic stem cells; IGF1: insulin-like growth factor 1; ITGAM: integrin alpha M; KEAP1: kelch-like ECH-associated protein 1; LY6A/Sca-1; MAP1LC3: lymphocyte antigen 6 complex, locus A; microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; miRNAs: microRNAs; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; mtUPR: mitochondrial unfolded protein response; MYF5: myogenic factor 5; MYH: myosin, heavy polypeptide; MYOD1: myogenic differentiation 1; MYOG: myogenin; NFE2L2: nuclear factor, erythroid derived 2, like 2; nSC: neonatal satellite cells; NSCs: neuronal stem cells; P: postnatal day; PAX7: paired box 7; PECAM1: platelet/endothelial cell adhesion molecule 1; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SCs: adult satellite cells; SQSTM1: sequestosome 1; STAT5: signal transducer and activator of transcription 5; TGFB1: transforming growth factor beta 1; WAT: white adipose tissue; WT: wild type.