Apoptosis Of Neural Stem Cells Research Articles

DVL/GSK3/ISL1 pathway signaling: unraveling the mechanism of SIRT3 in neurogenesis and AD therapy

BackgroundThe established association between Alzheimer's disease (AD) and compromised neural regeneration is well-documented. In addition to the mitigation of apoptosis in neural stem cells (NSCs), the induction of neurogenesis has been proposed as a promising therapeutic strategy for AD. Our previous research has demonstrated the effective inhibition of NSC injury induced by microglial activation through the repression of oxidative stress and mitochondrial dysfunction by Sirtuin 3 (SIRT3). Nonetheless, the precise role of SIRT3 in neurogenesis remains incompletely understood.MethodsIn vivo, SIRT3 overexpression adenovirus was firstly injected by brain stereotaxic localization to affect the hippocampal SIRT3 expression in APP/PS1 mice, and then behavioral experiments were performed to investigate the cognitive improvement of SIRT3 in APP/PS1 mice, as well as neurogenic changes in hippocampal region by immunohistochemistry and immunofluorescence. In vitro, under the transwell co-culture condition of microglia and neural stem cells, the mechanism of SIRT3 improving neurogenesis of neural stem cells through DVL/GSK3/ISL1 axis was investigated by immunoblotting, immunofluorescence and other experimental methods.ResultsOur findings indicate that the overexpression of SIRT3 in APP/PS1 mice led to enhanced cognitive function and increased neurogenesis. Additionally, SIRT3 was observed to promote the differentiation of NSCs into neurons during retinoic acid (RA)-induced NSC differentiation in vitro, suggesting a potential role in neurogenesis. Furthermore, we observed the activation of the Wnt/ß-catenin signaling pathway during this process, with Glycogen Synthase Kinase-3a (GSK3a) primarily governing NSC proliferation and GSK3ß predominantly regulating NSC differentiation. Moreover, the outcomes of our study demonstrate that SIRT3 exerts a protective effect against microglia-induced apoptosis in neural stem cells through its interaction with DVLs.ConclusionsOur results show that SIRT3 overexpressing APP/PS1 mice have improved cognition and neurogenesis, as well as improved neurogenesis of NSC in microglia and NSC transwell co-culture conditions through the DVL/GSK3/ISL1 axis.

Cocaine amphetamine-regulated transcription peptide inhibits apoptosis in oxygen-glucose deprived neural stem cells.

Apoptosis has been recognized as a critical pathophysiological process during cerebral ischemia. The neuroprotective effect of CART on ischemic brain injury is determined. However, there is little research on the protective effect of CART on neural stem cells (NSCs). Primary cultured rat NSCs were utilized as the research subject. In vitro oxygen glucose deprivation (OGD) treatment was employed, and NSCs were extracted from SD pregnant rats following previous experimental protocols and identified through cell immunofluorescence staining. The appropriate concentration of CART affecting OGD NSCs was initially screened using Cell Counting Kit-8 (CCK-8) and Lactate Dehydrogenase (LDH) assays. EdU staining and Western blotting (WB) techniques were employed to assess the impact of the suitable CART concentration on the proliferation and apoptosis of OGD NSCs. Finally, Western blot analysis was conducted to investigate the cAMP-response element binding protein (CREB) pathway and expression levels of related proteins after KG-501 treatment in order to elucidate the mechanism underlying apoptosis and proliferation regulation in OGD NSCs. CCK-8 and LDH assays indicated that a concentration of 0.8 nM CART may be the optimal concentration for modulating the proliferation of OGD NSCs. Subsequently, cellular immunofluorescence and EdU detection experiments further confirmed the findings obtained from CCK-8 analysis. Western blot analysis of apoptosis-related protein expression also demonstrated that an appropriate concentration of CART could suppress the apoptosis of OGD NSCs. Finally, Western blotting was conducted to examine the CREB pathway and related protein expression after treatment with KG-501, revealing that an appropriate concentration of CART regulated both apoptosis and proliferation in OGD NSCs through CREB signaling. The concentration of CART at 0.8 nM may be deemed appropriate for inhibiting apoptosis and promoting proliferation in OGD NSCs in vitro. The mechanism maybe through activating the CREB pathway.

Bisphenol-F and Bisphenol-S (BPF and BPS) Impair the Stemness of Neural Stem Cells and Neuronal Fate Decision in the Hippocampus Leading to Cognitive Dysfunctions.

Neurogenesis occurs throughout life in the hippocampus of the brain, and many environmental toxicants inhibit neural stem cell (NSC) function and neuronal generation. Bisphenol-A (BPA), an endocrine disrupter used for surface coating of plastic products causes injury in the developing and adult brain; thus, many countries have banned its usage in plastic consumer products. BPA analogs/alternatives such as bisphenol-F (BPF) and bisphenol-S (BPS) may also cause neurotoxicity; however, their effects on neurogenesis are still not known. We studied the effects of BPF and BPS exposure from gestational day 6 to postnatal day 21 on neurogenesis. We found that exposure to non-cytotoxic concentrations of BPF and BPS significantly decreased the number/size of neurospheres, BrdU+ (proliferating NSC marker)and MAP-2+ (neuronal marker) cells and GFAP+ astrocytes in the hippocampus NSC culture, suggesting reduced NSC stemness and self-renewal and neuronal differentiation and increased gliogenesis. These analogs also reduced the number of BrdU/Sox-2+, BrdU/Dcx+, and BrdU/NeuN+ co-labeled cells in the hippocampus of the rat brain, suggesting decreased NSC proliferation and impaired maturation of newborn neurons. BPF and BPS treatment increases BrdU/cleaved caspase-3+ cells and Bax-2 and cleaved caspase protein levels, leading to increased apoptosis in hippocampal NSCs. Transmission electron microscopy studies suggest that BPF and BPS also caused degeneration of neuronal myelin sheath, altered mitochondrial morphology, and reduced number of synapses in the hippocampus leading to altered cognitive functions. These results suggest that BPF and BPS exposure decreased the NSC pool, inhibited neurogenesis, induced apoptosis of NSCs, caused myelin degeneration/synapse degeneration, and impaired learning and memory in rats.

Linc-NSC affects cell differentiation, apoptosis and proliferation in mouse neural stem cells and embryonic stem cells in vitro and in vivo

BackgroundStem cell therapy is a promising therapeutic strategy. In a previous study, we evaluated tumorigenicity by the stereotactic transplantation of neural stem cells (NSCs) and embryonic stem cells (ESCs) from experimental mice. Twenty-eight days later, there was no evidence of tumor formation or long-term engraftment in the NSCs transplantation group. In contrast, the transplantation of ESCs caused tumor formation; this was due to their high proliferative capacity. Based on transcriptome sequencing, we found that a long intergenic non-coding RNA (named linc-NSC) with unknown structure and function was expressed at 1100-fold higher levels in NSCs than in ESCs. This finding suggested that linc-NSC is negatively correlated with stem cell pluripotency and tumor development, but positively correlated with neurogenesis. In the present study, we investigated the specific role of linc-NSC in NSCs/ESCs in tumor formation and neurogenesis.MethodsWhole transcriptome profiling by RNA sequencing and bioinformatics was used to predict lncRNAs that are widely associated with enhanced tumorigenicity. The expression of linc-NSC was assessed by quantitative real-time PCR. We also performed a number of in vitro methods, including cell proliferation assays, differentiation assays, immunofluorescence assays, flow cytometry, along with in vivo survival and immunofluorescence assays to investigate the impacts of linc-NSC on tumor formation and neurogenesis in NSCs and ESCs.ResultsFollowing the knockdown of linc-NSC in NSCs, NSCs cultured in vitro and those transplanted into the cortex of mice showed stronger survival ability (P < 0.0001), enhanced proliferation(P < 0.001), and reduced apoptosis (P < 0.05); the opposite results were observed when linc-NSC was overexpressed in ESCs. Furthermore, the overexpression of linc-NSC in ECSs induced enhanced apoptosis (P < 0.001) and differentiation (P < 0.01), inhibited tumorigenesis (P < 0.05) in vivo, and led to a reduction in tumor weight (P < 0.0001).ConclusionsOur analyses demonstrated that linc-NSC, a promising gene-edited target, may promote the differentiation of mouse NSCs and inhibit tumorigenesis in mouse ESCs. The knockdown of linc-NSC inhibited the apoptosis in NSCs both in vitro and in vivo, and prevented tumor formation, revealing a new dimension into the effect of lncRNA on low survival NSCs and providing a prospective gene manipulation target prior to transplantation. In parallel, the overexpression of linc-NSC induced apoptosis in ESCs both in vitro and in vivo and attenuated the tumorigenicity of ESCs in vivo, but did not completely prevent tumor formation.

Transplantation of Neural Stem Cells Loaded in an IGF-1 Bioactive Supramolecular Nanofiber Hydrogel for the Effective Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) leads to massive cell death, disruption, and demyelination of axons, resulting in permanent motor and sensory dysfunctions. Stem cell transplantation is a promising therapy for SCI. However, owing to the poor microenvironment that develops following SCI, the bioactivities of these grafted stem cells are limited. Cell implantation combined with biomaterial therapies is widely studied for the development of tissue engineering technology. Herein, an insulin-like growth factor-1 (IGF-1)-bioactive supramolecular nanofiber hydrogel (IGF-1 gel) is synthesized that can activate IGF-1 downstream signaling, prevent the apoptosis of neural stem cells (NSCs), improve their proliferation, and induce their differentiation into neurons and oligodendrocytes. Moreover, implantation of NSCs carried out with IGF-1 gels promotes neurite outgrowth and myelin sheath regeneration at lesion sites following SCI. In addition, IGF-1 gels can enrich extracellular vesicles (EVs) derived from NSCs or from nerve cells differentiated from these NSCs via miRNAs related to axonal regeneration and remyelination, even in an inflammatory environment. These EVs are taken up by autologous endogenous NSCs and regulate their differentiation. This study provides adequate evidence that combined treatment with NSCs and IGF-1 gels is a potential therapeutic strategy for treating SCI.

Retraction Note: Effect of apoptosis in neural stem cells treated with sevoflurane

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s12871-024-02448-2.

Deubiquitinase Mysm1 regulates neural stem cell proliferation and differentiation by controlling Id4 expression

Neural stem cells (NSCs) are critical for brain development and maintenance of neurogenesis. However, the molecular mechanisms that regulate NSC proliferation and differentiation remain unclear. Mysm1 is a deubiquitinase and is essential for the self-renewal and differentiation of several stem cells. It is unknown whether Mysm1 plays an important role in NSCs. Here, we found that Mysm1 was expressed in NSCs and its expression was increased with age in mice. Mice with Mysm1 knockdown by crossing Mysm1 floxed mice with Nestin-Cre mice exhibited abnormal brain development with microcephaly. Mysm1 deletion promoted NSC proliferation and apoptosis, resulting in depletion of the stem cell pool. In addition, Mysm1-deficient NSCs skewed toward neurogenesis instead of astrogliogenesis. Mechanistic investigations with RNA sequencing and genome-wide CUT&Tag analysis revealed that Mysm1 epigenetically regulated Id4 transcription by regulating histone modification at the promoter region. After rescuing the expression of Id4, the hyperproliferation and imbalance differentiation of Mysm1-deficient NSCs was reversed. Additionally, knockdown Mysm1 in aged mice could promote NSC proliferation. Collectively, the present study identified a new factor Mysm1 which is essential for NSC homeostasis and Mysm1-Id4 axis may be an ideal target for proper NSC proliferation and differentiation.

The role of microRNA-325-3p as a critical player in cell death in NSCs and astrocytes.

Neural stem cells (NSCs) are defined by their ability to self-renew and generate various cell types within the nervous system. Understanding the underlying mechanism by which NSCs proliferate and differentiate is crucial for the efficient modulation of in vivo neurogenesis. MicroRNAs are small non-coding RNAs controlling gene expression concerned in post-transcriptional control by blocking messenger RNA (mRNA) translation or degrading mRNA. MicroRNAs play a role as modulators by matching target mRNAs. Recent studies have discussed the biological mechanism of microRNA regulation in neurogenesis. To investigate the role of microRNAs in NSCs and NSC-derived glial cells, we screened out NSC-specific microRNAs by using miRNome-wide screening. Then, we induced downregulation by the sponge against the specific microRNA to evaluate the functional role of the microRNA in proliferation, differentiation, and apoptosis in NSCs and NSC-derived astrocytes. We found that microRNA-325-3p is highly expressed in NSCs and astrocytes. Furthermore, we showed that microRNA-325-3p is a regulator of apoptosis by targeting brain-specific angiogenesis inhibitor (BAI1), which is a receptor for apoptotic cells and expressed in the brain and cultured astrocytes. Downregulation of microRNA-325-3p using an inducible sponge system induced cell death by regulating BAI1 in NSCs and NSC-derived astrocytes. Overall, our findings can provide an insight into the potential roles of NSC-specific microRNAs in brain neurogenesis and suggest the possible usage of the microRNAs as biomarkers of neurodegenerative disease.

Retracted: Resetting Proteostasis of CIRBP with ISRIB Suppresses Neural Stem Cell Apoptosis under Hypoxic Exposure.

[This retracts the article DOI: 10.1155/2022/3627026.].

Deletion of the adaptor protein p66ShcA prevents the apoptosis of neural stem cells in response to mitogen deprivation

AbstractBackgroundAlzheimer’s disease (AD) has been associated with altered hippocampal neurogenesis and the depletion of the neural stem cell (NSC) niche. NSCs rely on cues from their extracellular environment to mediate their survival and maturation. The Shc adaptor family mediates this mitogenic signaling in response to a wide range of growth factors and is necessary for embryonic neurogenesis. A member of this family, p66ShcA has been found to antagonize mitogenic signalling, however its role in neurogenesis has not been established. Moreover, the genetic ablation of p66ShcA, has a been reported to reverse cognitive decline in mouse models of AD, despite its minimal expression in mature neurons.MethodTo evaluate p66ShcA’s role in the fate of NSCs, we generated and characterized a p66ShcA‐null murine NSC line (p66KO‐NSC). The NSCs were characterized by assessing their differentiation competency and viability in response to alterations in epidermal growth factor (EGF) signalling.ResultThe p66KO‐NSCs exhibited enhanced neurogenesis in response to EGF withdrawal, relative to WT NSCs. The pharmacologic inhibition of either EGFR or ERK induced the apoptosis of the WT NSCs via the release of cytochrome C and caspase activation. Surprisingly, the inhibition of EGFR or ERK had a negligible effect on the survival of the p66KO‐NPCs and instead induced their neuronal differentiation.Conclusionp66ShcA mediates NSC apoptosis in response to the loss of mitogenic cues. The deletion of p66ShcA increases neurogenesis by facilitating the survival of NSCs during their neuronal differentiation.

Kidins220 sets the threshold for survival of neural stem cells and progenitors to sustain adult neurogenesis

In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.

Parental Folate Deficiency Inhibits Proliferation and Increases Apoptosis of Neural Stem Cells in Rat Offspring: Aggravating Telomere Attrition as a Potential Mechanism

The effect of maternal folate status on the fetal central nervous system (CNS) is well recognized, while evidence is emerging that such an association also exists between fathers and offspring. The biological functions of telomeres and telomerase are also related to neural cell proliferation and apoptosis. The study aimed to investigate the effect of parental folate deficiency on the proliferation and apoptosis of neural stem cells (NSCs) in neonatal offspring and the role of telomeres in this effect. In this study, rats were divided into four groups: maternal folate-deficient and paternal folate-deficient diet (D-D) group; maternal folate-deficient and paternal folate-normal diet (D-N) group; maternal folate-normal and paternal folate-deficient diet (N-D) group; and the maternal folate-normal and paternal folate-normal diet (N-N) group. The offspring were sacrificed at postnatal day 0 (PND0), and NSCs were cultured from the hippocampus and striatum tissues of offspring for future assay. The results revealed that parental folate deficiency decreased folate levels, increased homocysteine (Hcy) levels of the offspring’s brain tissue, inhibited proliferation, increased apoptosis, shortened telomere length, and aggravated telomere attrition of offspring NSCs in vivo and in vitro. In vitro experiments further showed that offspring NSCs telomerase activity was inhibited due to parental folate deficiency. In conclusion, parental folate deficiency inhibited the proliferation and increased apoptosis of offspring NSCs, maternal folate deficiency had more adverse effects than paternal, and the mechanisms may involve the telomere attrition of NSCs.

IGF1c mimetic peptide-based supramolecular hydrogel microspheres synergize with neural stem cells to promote functional recovery from spinal cord injury

Spinal cord injuries (SCI) often cause severe inflammatory response and neuronal loss, which consequently deteriorates motor function. Although exogenous stem cells are considered to be vital seed cells for cell replacement therapy after SCI, their low survival rate and low efficiency of neuronal differentiation after in vivo transplantation limit their widespread application in clinical practice. To this end, this study proposed a new strategy for dramatically improving the efficiency of insulin-like growth factor 1 (IGF1c) mimetic peptide to activate its receptor by fabricating IGF1c mimetic peptide supramolecular hydrogel microspheres (IGF1c-PHM) using an innovatively designed piezoelectric ceramic-driven thermal electrospray device. IGF1c-PHM displayed an average size of 10 µm and could activate the IGF1R of neural stem cells (NSCs) more effectively than IGF1c peptide block hydrogel (IGF1c-PH). In vitro, IGF1c-PHM exerted strong neuroprotective effects by maintaining the proliferation, inhibiting the apoptosis of NSCs, and promoting the efficiency of differentiation into neurons in presence of myelin extracts. In vivo, IGF1c-PHM improved the inflammatory environment by suppressing the recruitment of the inflammatory cells, altering the subtypes of the macrophages, and promoting the survival rate of endogenous cells and NSCs in the injury area. In particular, IGF1c-PHM enhanced the proliferation ability and neuronal differentiation of NSCs in vivo after SCI. In the presence of IGF1c-PHM, the transplanted NSCs could differentiate into several mature neurons at the injury site, and the axons regenerated in the injury site were remyelinated, all of which closely correlate with the improvement of the electrophysiological indices after SCI. Finally, at 8 weeks post-SCI, the gait analysis and behavioral scores confirmed the significant improvement in motor function in rats treated with IGF1c-PHM and NSCs. Therefore, IGF1c-PHM combined with NSCs is a promising therapy for treating SCI.

Inhibition of the NLRP3 Inflammasome Activation/Assembly through the Activation of the PI3K Pathway by Naloxone Protects Neural Stem Cells from Ischemic Condition.

Naloxone is a well-known opioid antagonist and has been suggested to have neuroprotective effects in cerebral ischemia. We investigated whether naloxone exhibits anti-inflammatory and neuroprotective effects in neural stem cells (NSCs) injured by oxygen-glucose deprivation (OGD), whether it affects the NOD-like receptor protein 3 (NLRP3) inflammasome activation/assembly, and whether the role of the phosphatidylinositol 3-kinase (PI3K) pathway is important in the control of NLRP3 inflammasome activation/assembly by naloxone. Primary cultured NSCs were subjected to OGD and treated with different concentrations of naloxone. Cell viability, proliferation, and the intracellular signaling proteins associated with the PI3K pathway and NLRP3 inflammasome activation/assembly were evaluated in OGD-injured NSCs. OGD significantly reduced survival, proliferation, and migration and increased apoptosis of NSCs. However, treatment with naloxone significantly restored survival, proliferation, and migration and decreased apoptosis of NSCs. Moreover, OGD markedly increased NLRP3 inflammasome activation/assembly and cleaved caspase-1 and interleukin-1β levels in NSCs, but naloxone significantly attenuated these effects. These neuroprotective and anti-inflammatory effects of naloxone were eliminated when cells were treated with PI3K inhibitors. Our results suggest that NLRP3 inflammasome is a potential therapeutic target and that naloxone reduces ischemic injury in NSCs by inhibiting NLRP3 inflammasome activation/assembly mediated by the activation of the PI3K signaling pathway.

Metformin attenuates sevoflurane-induced neurogenesis damage and cognitive impairment: involvement of the Nrf2/G6PD pathway.

Anesthetics such as sevoflurane are commonly administered to infants and children. However, the possible neurotoxicity caused by prolonged or repetitive exposure to it should be a concern. The neuroprotective effects of metformin are observed in many models of neurological disorders. In this study, we investigated whether metformin could reduce the developmental neurotoxicity induced by sevoflurane exposure in neonatal rats and the potential mechanism. Postnatal day 7 (PND 7) Sprague-Dawley rats and neural stem cells (NSCs) were treated with normal saline or metformin before sevoflurane exposure. The Morris water maze (MWM) was used to observe spatial memory and learning at PND 35-42. Immunofluorescence staining was used to detect neurogenesis in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus at PND 14. MTT assays, immunofluorescence staining, and TUNEL staining were used to assess the viability, proliferation, differentiation, and apoptosis of NSCs. Western blotting and ELISA were used to assess the protein expression of cleaved caspase-3, nuclear factor erythroid 2-related factor 2 (Nrf2), and glucose-6-phosphate dehydrogenase (G6PD) pathway-related molecules. Exposure to sevoflurane resulted in late cognitive defects, impaired neurogenesis in both the SVZ and SGZ, reduced NSC viability and proliferation, increased NSC apoptosis, and decreased protein expression of G6PD in vitro. Metformin pretreatment attenuated sevoflurane-induced cognitive functional decline and neurogenesis inhibition. Metformin pretreatment also increased the protein expression of Nrf2 and G6PD. However, treatment with the Nrf2 inhibitor, ML385 or the G6PD inhibitor, dehydroepiandrosterone (DHEA) reversed the protective effect of metformin on sevoflurane-induced NSC damage in vitro. Our findings suggested that metformin could reduce sevoflurane-induced neurogenesis damage and neurocognitive defects in the developing rat brain by influencing the Nrf2/G6PD signaling pathways.

A Novel Multicellular Placental Barrier Model to Investigate the Effect of Maternal Aflatoxin B1 Exposure on Fetal-Side Neural Stem Cells.

Ingestion of food toxins such as aflatoxin B1 (AFB1) during pregnancy may impair fetal neurodevelopment. However, animal model results may not be accurate due to the species' differences, and testing on humans is ethically impermissible. Here, we developed an in vitro human maternal-fetal multicellular model composed of a human hepatic compartment, a bilayer placental barrier, and a human fetal central nervous system compartment using neural stem cells (NSCs) to investigate the effect of AFB1 on fetal-side NSCs. AFB1 passed through the HepG2 hepatocellular carcinoma cells to mimic the maternal metabolic effects. Importantly, even at the limited concentration (0.0641 ± 0.0046 μM) of AFB1, close to the national safety level standard of China (GB-2761-2011), the mixture of AFB1 crossing the placental barrier induced NSC apoptosis. The level of reactive oxygen species in NSCs was significantly elevated and the cell membrane was damaged, causing the release of intracellular lactate dehydrogenase (p < 0.05). The comet experiment and γ-H2AX immunofluorescence assay showed that AFB1 caused significant DNA damage to NSCs (p < 0.05). This study provided a new model for the toxicological evaluation of the effect of food mycotoxin exposure during pregnancy on fetal neurodevelopment.

Assessment of neurotransmitter release in human iPSC-derived neuronal/glial cells: a missing in vitro assay for regulatory developmental neurotoxicity testing.

Human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) and their differentiated neuronal/glial derivatives have been recently considered suitable to assess in vitro developmental neurotoxicity (DNT) triggered by exposure to environmental chemicals. The use of human-relevant test systems combined with in vitro assays specific for different neurodevelopmental events, enables a mechanistic understanding of the possible impact of environmental chemicals on the developing brain, avoiding extrapolation uncertainties associated with in vivo studies. Currently proposed in vitro battery for regulatory DNT testing accounts for several assays suitable to study key neurodevelopmental processes, including NSC proliferation and apoptosis, differentiation into neurons and glia, neuronal migration, synaptogenesis, and neuronal network formation. However, assays suitable to measure interference of compounds with neurotransmitter release or clearance are at present not included, which represents a clear gap of the biological applicability domain of such a testing battery. Here we applied a HPLC-based methodology to measure the release of neurotransmitters in a previously characterized hiPSC-derived NSC model undergoing differentiation towards neurons and glia. Glutamate release was assessed in control cultures and upon depolarization, as well as in cultures repeatedly exposed to some known neurotoxicants (BDE47 and lead) and chemical mixtures. Obtained data indicate that these cells have the ability to release glutamate in a vesicular manner, and that both glutamate clearance and vesicular release concur in the maintenance of extracellular glutamate levels. In conclusion, analysis of neurotransmitter release is a sensitive readout that should be included in the envisioned battery of in vitro assays for DNT testing.

Folic acid protects against age-associated apoptosis and telomere attrition of neural stem cells in senescence-accelerated mouse prone 8.

Folic acid (FA) could improve cognitive performances and attenuate brain cell injury in the aging brain, FA supplementation is also associated with inhibiting neural stem cells (NSCs) apoptosis. However, its role in age-associated telomere attrition remains unclear. We hypothesized that FA supplementation attenuates age-associated apoptosis of NSCs via alleviating telomere attrition in senescence-accelerated mouse prone 8 (SAMP8). In this study, 4-month-old male SAMP8 mice were assigned equal numbers to four different diet groups (n=15). Fifteen age-matched senescence-accelerated mouse resistant 1 (SAMR1) (Con-R) were used as the standard aging control group, feeding the FA-normal diet. After folic acid treatment 6-month, all mice were sacrificed. NSCs apoptosis, proliferation, oxidative damage and telomere length have been evaluated by immunofluorescence and Q-fluorescent in situ hybridization. The results showed that FA supplementation inhibited age-associated NSCs apoptosis and prevented telomere attrition in the cerebral cortex of SAMP8 mice. Importantly, this effect might be interpreted by the decreased levels of oxidative damage. In conclusion, we demonstrate it may be one of the mechanisms that FA inhibiting age-associated NSCs apoptosis by alleviating telomere length shortening.

Supramolecular Hydrogel Microspheres of Platelet-Derived Growth Factor Mimetic Peptide Promote Recovery from Spinal Cord Injury.

Neural stem cells (NSCs) are considered to be prospective replacements for neuronal cell loss as a result of spinal cord injury (SCI). However, the survival and neuronal differentiation of NSCs are strongly affected by the unfavorable microenvironment induced by SCI, which critically impairs their therapeutic ability to treat SCI. Herein, a strategy to fabricate PDGF-MP hydrogel (PDGF-MPH) microspheres (PDGF-MPHM) instead of bulk hydrogels is proposed to dramatically enhance the efficiency of platelet-derived growth factor mimetic peptide (PDGF-MP) in activating its receptor. PDGF-MPHM were fabricated by a piezoelectric ceramic-driven thermal electrospray device, had an average size of 9 μm, and also had the ability to activate the PDGFRβ of NSCs more effectively than PDGF-MPH. In vitro, PDGF-MPHM exerted strong neuroprotective effects by maintaining the proliferation and inhibiting the apoptosis of NSCs in the presence of myelin extracts. In vivo, PDGF-MPHM inhibited M1 macrophage infiltration and extrinsic or intrinsic cells apoptosis on the seventh day after SCI. Eight weeks after SCI, the T10 SCI treatment results showed that PDGF-MPHM + NSCs significantly promoted the survival of NSCs and neuronal differentiation, reduced lesion size, and considerably improved motor function recovery in SCI rats by stimulating axonal regeneration, synapse formation, and angiogenesis in comparison with the NSCs graft group. Therefore, our findings provide insights into the ability of PDGF-MPHM to be a promising therapeutic agent for SCI repair.

Histone acetyltransferase KAT2A modulates neural stem cell differentiation and proliferation by inducing degradation of the transcription factor PAX6

Neural stem cells (NSCs) proliferation and differentiation rely on proper expression and post-translational modification of transcription factors involved in the determination of cell fate. Further characterization is needed to connect modifying enzymes with their transcription factor substrates in the regulation of these processes. Here, we demonstrated that the inhibition of KAT2A, a histone acetyltransferase, leads to a phenotype of small eyes in the developing embryo of zebrafish, which is associated with enhanced proliferation and apoptosis of NSCs in zebrafish eyes. We confirmed that this phenotype is mediated by the evaluated level of PAX6 protein. We further verified that KAT2A negatively regulates PAX6 at the protein level in cultured neural stem cells of rat cerebral cortex. We revealed that PAX6 is a novel acetylation substrate of KAT2A, and the acetylation of PAX6 promotes its ubiquitination mediated by the E3 ligase RNF8 that facilitated PAX6 degradation. Our study proposes that KAT2A inhibition results in accelerated proliferation, delayed differentiation, or apoptosis, depending on the context of PAX6 dosage. Thus, the KAT2A/PAX6 axis plays an essential role to keep a balance between the self-renewal and differentiation of NSCs.