Human neural stem cells.

'Human neural stem cells' jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human neural stem cells (hNSCs) in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hNSCs, which is applicable to the quality control for hNSCs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that publication of the guideline will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hNSCs for clinical development and therapeutic applications.

Abnormal chromatin remodeling caused by ARID1A deletion leads to malformation of the dentate gyrus.

ARID1A, an SWI/SNF chromatin-remodeling gene, is commonly mutated in cancer and hypothesized to be a tumor suppressor. Recently, loss-of-function of ARID1A gene has been shown to cause intellectual disability. Here we generate Arid1a conditional knockout mice and investigate Arid1a function in the hippocampus. Disruption of Arid1a in mouse forebrain significantly decreases neural stem/progenitor cells (NSPCs) proliferation and differentiation to neurons within the dentate gyrus (DG), increasing perinatal and postnatal apoptosis, leading to reduced hippocampus size. Moreover, we perform single-cell RNA sequencing (scRNA-seq) to investigate cellular heterogeneity and reveal that Arid1a is necessary for the maintenance of the DG progenitor pool and survival of post-mitotic neurons. Transcriptome and ChIP-seq analysis data demonstrate that ARID1A specifically regulates Prox1 by altering the levels of histone modifications. Overexpression of downstream target Prox1 can rescue proliferation and differentiation defects of NSPCs caused by Arid1a deletion. Overall, our results demonstrate a critical role for Arid1a in the development of the hippocampus and may also provide insight into the genetic basis of intellectual disabilities such as Coffin-Siris syndrome, which is caused by germ-line mutations or microduplication of Arid1a.

Discovery of toxoflavin, a potent IRE1α inhibitor acting through structure-dependent oxidative inhibition.

Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC50 value of 0.226 µM. Its inhibitory potencies on IRE1α kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α's role in ER stress.

KW-2449 and VPA exert therapeutic effects on human neurons and cerebral organoids derived from MECP2-null hESCs.

BACKGROUND: Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MECP2), is one of the most prevalent neurodevelopmental disorders in girls. However, the underlying mechanism of MECP2 remains largely unknown and currently there is no effective treatment available for RTT. METHODS: We generated MECP2-KO human embryonic stem cells (hESCs), and differentiated them into neurons and cerebral organoids to investigate phenotypes of MECP2 loss-of-function, potential therapeutic agents, and the underlying mechanism by transcriptome sequencing. RESULTS: We found that MECP2 deletion caused reduced number of hESCs-derived neurons and simplified dendritic morphology. Moreover, MECP2-KO cortical organoids exhibited fewer neural progenitor cells and neurons at day 60. Electrophysiological recordings showed that MECP2 deletion altered synaptic activity in organoids. Transcriptome analysis of organoids identified many genes in the PI3K-AKT pathway downregulated following MECP2 deletion. Treatment with either KW-2449 or VPA, small molecules for the activation of PI3K-AKT signaling pathway, alleviated neuronal deficits and transcriptome changes in MECP2-KO human neuronal models. CONCLUSIONS: These findings suggest that KW-2449 and VPA might be promising drugs for RTT treatment.

The epigenetic state of EED-Gli3-Gli1 regulatory axis controls embryonic cortical neurogenesis.

Mutations in the embryonic ectoderm development (EED) cause Weaver syndrome, but whether and how EED affects embryonic brain development remains elusive. Here, we generated a mouse model in which Eed was deleted in the forebrain to investigate the role of EED. We found that deletion of Eed decreased the number of upper-layer neurons but not deeper-layer neurons starting at E16.5. Transcriptomic and genomic occupancy analyses revealed that the epigenetic states of a group of cortical neurogenesis-related genes were altered in Eed knockout forebrains, followed by a decrease of H3K27me3 and an increase of H3K27ac marks within the promoter regions. The switching of H3K27me3 to H3K27ac modification promoted the recruitment of RNA-Pol2, thereby enhancing its expression level. The small molecule activator SAG or Ptch1 knockout for activating Hedgehog signaling can partially rescue aberrant cortical neurogenesis. Taken together, we proposed a novel EED-Gli3-Gli1 regulatory axis that is critical for embryonic brain development.

PRMT1 promotes neuroblastoma cell survival through ATF5.

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.

c-Myc controls the fate of neural progenitor cells during cerebral cortex development.

The anatomical structure of the mammalian cerebral cortex is the essential foundation for its complex neural activity. This structure is developed by proliferation, differentiation, and migration of neural progenitor cells (NPCs), the fate of which is spatially and temporally regulated by the proper gene. This study was used in utero electroporation and found that the well-known oncogene c-Myc mainly promoted NPCs' proliferation and their transformation into intermediate precursor cells. Furthermore, the obtained results also showed that c-Myc blocked the differentiation of NPCs to postmitotic neurons, and the expression of telomere reverse transcriptase was controlled by c-Myc in the neocortex. These findings indicated c-Myc as a key regulator of the fate of NPCs during the development of the cerebral cortex.

Telomerase Reverse Transcriptase and p53 Regulate Mammalian Peripheral Nervous System and CNS Axon Regeneration Downstream of c-Myc.

Although several genes have been identified to promote axon regeneration in the CNS, our understanding of the molecular mechanisms by which mammalian axon regeneration is regulated is still limited and fragmented. Here by using female mouse sensory axon and optic nerve regeneration as model systems, we reveal an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. We also provide evidence that TERT and p53 act downstream of c-Myc to control sensory axon regeneration. More importantly, overexpression of p53 in sensory neurons and retinal ganglion cells is sufficient to promote sensory axon and optic never regeneration, respectively. The study reveals a novel c-Myc-TERT-p53 signaling pathway, expanding horizons for novel approaches promoting CNS axon regeneration.SIGNIFICANCE STATEMENT Despite significant progress during the past decade, our understanding of the molecular mechanisms by which mammalian CNS axon regeneration is regulated is still fragmented. By using sensory axon and optic nerve regeneration as model systems, the study revealed an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. The results also delineated a c-Myc-TERT-p53 pathway in controlling axon growth. Last, our results demonstrated that p53 alone was sufficient to promote sensory axon and optic nerve regeneration in vivo Collectively, the study not only revealed a new mechanisms underlying mammalian axon regeneration, but also expanded the pool of potential targets that can be manipulated to enhance CNS axon regeneration.

GA-binding protein GABPß1 is required for the proliferation of neural stem/progenitor cells.

GA binding protein (GABP) is a ubiquitously expressed transcription factor that regulates the development of multiple cell types, including osteoblast, hematopoietic stem cells, B cells and T cells. However, so little is known about its biological function in the development of central nervous system. In this report, we show that GABP is highly expressed in neural stem/progenitor cells (NSPCs) and down-regulated in neurons, and that GABPß1 is required for the proper proliferation of NSPCs. Knockdown of GABPα resulted in an elevated expression level of GABPß1, and GABPß1 down-regulation significantly decreased the proliferation of NSPCs, whereas GABPß2 knockdown did not result in any changes in the proliferation of NSPCs. We observed that there was nearly a 21-fold increase of the GABPß1S mRNA level in GABPß1L KO NSPCs compared to WT cells, and knocking down of GABPß1S in GABPß1L KO NSPCs could further reduce their proliferation potential. We also found that knockdown of GABPß1 promoted neuronal and astrocytic differentiation of NSPCs. Finally, we identified dozens of downstream target genes of GABPß1, which are closely associated with the cell proliferation and differentiation. Collectively, our results suggest that both GABPß1L and GABPß1S play an essential role in regulating the proper proliferation and differentiation of NSPCs.

Regulation of adult mammalian intrinsic axonal regeneration by NF-κB/STAT3 signaling cascade.

The inflammatory response is a critical regulator for the regeneration of axon following nervous system injury. Nuclear factor-kappa B (NF-κB) is characteristically known for its ubiquitous role in the inflammatory response. However, its functional role in adult mammalian axon growth remains elusive. Here, we found that the NF-κB signaling pathway is activated in adult sensory neurons through peripheral axotomy. Furthermore, inhibition of NF-κB in peripheral sensory neurons attenuated their axon growth in vitro and in vivo. Our results also showed that NF-κB modulated axon growth by repressing the phosphorylation of STAT3. Furthermore, activation of STAT3 significantly promoted adult optic nerve regeneration. Taken together, the findings of our study indicated that NF-κB/STAT3 cascade is a critical regulator of intrinsic axon growth capability in the adult nervous system.

Lin28 Signaling Supports Mammalian PNS and CNS Axon Regeneration.

RNA-binding proteins Lin28a/b regulate cellular growth and tissue regeneration. Here, we investigated the role of Lin28 in the control of axon regeneration in postmitotic neurons. We find that Lin28a/b are both necessary and sufficient for supporting axon regeneration in mature sensory neurons through their regulatory partners, let-7 microRNAs (miRNAs). More importantly, overexpression of Lin28a in mature retinal ganglion cells (RGCs) produces robust and sustained optic nerve regeneration. Additionally, combined overexpression of Lin28a and downregulation of Pten in RGCs act additively to promote optic nerve regeneration, potentially by reducing the backward turning of regenerating RGC axons. Our findings not only reveal a vital role of Lin28 signaling in regulating mammalian axon regeneration but also identify a signaling pathway that can promote axon regeneration in the central nervous system (CNS).

Wnt3 and Gata4 regulate axon regeneration in adult mouse DRG neurons.

Neurons in the adult central nervous system (CNS) have a poor intrinsic axon growth potential after injury, but the underlying mechanisms are largely unknown. Wingless-related mouse mammary tumor virus integration site (WNT) family members regulate neural stem cell proliferation, axon tract and forebrain development in the nervous system. Here we report that Wnt3 is an important modulator of axon regeneration. Downregulation or overexpression of Wnt3 in adult dorsal root ganglion (DRG) neurons enhances or inhibits their axon regeneration ability respectively in vitro and in vivo. Especially, we show that Wnt3 modulates axon regeneration by repressing mRNA translation of the important transcription factor Gata4 via binding to the three prime untranslated region (3'UTR). Downregulation of Gata4 could restore the phenotype exhibited by Wnt3 downregulation in DRG neurons. Taken together, these data indicate that Wnt3 is a key intrinsic regulator of axon growth ability of the nervous system.

MiR-203 Interplays with Polycomb Repressive Complexes to Regulate the Proliferation of Neural Stem/Progenitor Cells.

The polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are two distinct polycomb group (PcG) proteins that maintain the stable silencing of specific sets of genes through chromatin modifications. Although the PRC2 component EZH2 has been known as an epigenetic regulator in promoting the proliferation of neural stem/progenitor cells (NSPCs), the regulatory network that controls this process remains largely unknown. Here we show that miR-203 is repressed by EZH2 in both embryonic and adult NSPCs. MiR-203 negatively regulates the proliferation of NSPCs. One of PRC1 components, Bmi1, is a downstream target of miR-203 in NSPCs. Conditional knockout of Ezh2 results in decreased proliferation ability of both embryonic and adult NSPCs. Meanwhile, ectopic overexpression of BMI1 rescues the proliferation defects exhibited by miR-203 overexpression or EZH2 deficiency in NSPCs. Therefore, this study provides evidence for coordinated function of the EZH2-miR-203-BMI1 regulatory axis that regulates the proliferation of NSPCs.

Involvement of transcription factor XBP1s in the resistance of HDAC6 inhibitor Tubastatin A to superoxidation via acetylation-mediated proteasomal degradation.

HDAC6 is a major cytoplasmic deacetylase. XBP1s is a basic-region leucine zipper (bZIP) transcriptional factor. Despite their mutual involvement in the anti-oxidative process, there are no reports about their inter-protein interactions so far. Here we identified a direct link between HDAC6 inhibition and XBP1s transcription activity in anti-oxidative damage. We showed that the specific HDAC6 inhibitor Tubastatin A could up-regulate XBP1s transcriptional activity, thereby increasing anti-oxidative genes expression. Moreover, knock down of XBP1s could significantly abolish the cell growth protection afforded by Tubastatin A. We hypothesize that Tubastatin A acts to increase XBP1s protein levels that are dependent on its HDAC6 deacetylase inhibition via a mechanism involving acetylation-mediated proteasomal degradation, providing novel mechanistic insight into the anti-oxidative effects of HDAC6 inhibition.

Akt-independent GSK3 inactivation downstream of PI3K signaling regulates mammalian axon regeneration.

Inactivation of glycogen synthase kinase 3 (GSK3) has been shown to mediate axon growth during development and regeneration. Phosphorylation of GSK3 by the kinase Akt is well known to be the major mechanism by which GSK3 is inactivated. However, whether such regulatory mechanism of GSK3 inactivation is used in neurons to control axon growth has not been directly studied. Here by using GSK3 mutant mice, in which GSK3 is insensitive to Akt-mediated inactivation, we show that sensory axons regenerate normally in vitro and in vivo after peripheral axotomy. We also find that GSK3 in sensory neurons of the mutant mice is still inactivated in response to peripheral axotomy and such inactivation is required for sensory axon regeneration. Lastly, we provide evidence that GSK3 activity is negatively regulated by PI3K signaling in the mutant mice upon peripheral axotomy, and the PI3K-GSK3 pathway is functionally required for sensory axon regeneration. Together, these results indicate that in response to peripheral nerve injury GSK3 inactivation, regulated by an alternative mechanism independent of Akt-mediated phosphorylation, controls sensory axon regeneration.

Berberine combined with 2-deoxy-d-glucose synergistically enhances cancer cell proliferation inhibition via energy depletion and unfolded protein response disruption.

BACKGROUND: Targeting multiple aspects of cellular metabolism, such as both aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), has the potential to improve cancer therapeutics. Berberine (BBR), a widely used traditional Chinese medicine, exerts its antitumor effects by inhibiting OXPHOS. 2-Deoxy-d-glucose (2-DG) targets aerobic glycolysis and demonstrates potential anticancer effects in the clinic. We hypothesized that BBR in combination with 2-DG would be more efficient than either agent alone against cancer cell growth. METHODS: The effects of BBR and 2-DG on cancer cell growth were evaluated using the Sulforhodamine B (SRB) method. Cell death was detected with the PI uptake assay, and Western blot, Q-PCR and luciferase reporter assays were used for signaling pathway detection. An adenovirus system was used for gene overexpression. RESULTS: BBR combined with 2-DG synergistically enhanced the growth inhibition of cancer cells in vitro. Further mechanistic studies showed that the combination drastically enhanced ATP depletion and strongly disrupted the unfolded protein response (UPR). Overexpressing GRP78 partially prevented the cancer cell inhibition induced by both compounds. CONCLUSIONS: Here, we report for the first time that BBR and 2-DG have a synergistic effect on cancer cell growth inhibition related to ATP energy depletion and disruption of UPR. GENERAL SIGNIFICANCE: Our results propose the potential use of BBR and 2-DG in combination as an anticancer treatment, reinforcing the hypothesis that targeting both aerobic glycolysis and OXPHOS provides more effective cancer therapy and highlighting the important role of UPR in the process.

The association between the Survivin A9194G exon polymorphisms and papillary thyroid carcinoma risk in the Han Chinese population.

The dysregulation of apoptosis plays a key role in carcinogenesis. This study was designed to investigate the association of apoptosis-related gene survivin A9194G single-nucleotide polymorphisms (SNPs) with papillary thyroid carcinoma (PTC) susceptibility. A case-control study of 126 patients and 198 controls was performed to investigate the association between Survivin A9194G polymorphisms and PTC susceptibility by polymerase chain reaction (PCR) following DNA sequencing methods. Moreover, the distribution of genotype frequency and the association of genotype with clinicopathologic characteristics were analyzed. We found that the survivin A9194G genotype was at a decreased risk of PTC (P=0.003; odds ratio (OR)=0.56). Furthermore, compared to the PTCs of the AA and AG phenotypes, the GG genotype thyroid cancers were significantly more common in younger patients and in cases of lower pathologic stages. In conclusion, the survivin (A9194G) polymorphism was found to play a protective role in the susceptibility to PTC. Nevertheless, further investigation with a larger sample size is needed to support our results.

Antiproliferative effects of selective cyclooxygenase-2 inhibitor modulated by nimotuzumab in estrogen-dependent breast cancer cells.

Breast cancer is the most common malignancy in women, and many breast cancer patients fail conventional treatment strategies of chemotherapy, radiation, and antiestrogen therapy. Research into the molecular pathways and biomarkers involved in the development of breast cancer should yield information that will guide therapeutic decisions. Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) are involved in the carcinogenesis of breast cancer and exist tight crosstalk with estrogen receptor (ER) pathway. Combination of EGFR and COX-2 inhibitors, therefore, could be an effective strategy for reducing cell growth in estrogen-dependent breast cancer. In order to verify the effects of EGFR and COX-2 inhibitors, breast cancer cells MCF-7 and SKBR-3 were characterized for receptors status and then treated with respective inhibitors (nimotuzumab and celecoxib) alone and in combination. Both cell lines were sensitive to celecoxib, but not to nimotuzumab. However, combination of two drugs demonstrated synergistic effects on cell killing. Moreover, association of two drugs resulted in SKBR-3 cells, a further G0/G1 phase arrest than one drug alone. Downregulation of p-EGFR, p-Akt, p-mTOR, and amplified in breast cancer 1 (AIB1) were observed in both cell lines, and upregulation of E-cadherin was only found in MCF-7, after treatment with single agent or in combination. These studies suggest that nimotuzumab and celecoxib exert synergistic antiproliferation effects in breast cancer, which partly correlates with ER status. Due to Akt/mTOR, EMT and AIB1 pathways participate in this process, therefore, E-cadherin and AIB1 may be considered as possible biomarkers to predict response in ER-positive breast cancer cells treated with EGFR and COX-2 inhibitors.

Role of Caspase 8, Caspase 9 and Bcl-2 polymorphisms in papillary thyroid carcinoma risk in Han Chinese population.

Dysregulation of apoptosis plays a key role in carcinogenesis. This study was designed to investigate the association of apoptosis-related gene Caspase 8, Caspase 9 and Bcl-2 polymorphisms with papillary thyroid carcinoma (PTC) susceptibility. We undertook a case-control study of 118 patients and 213 controls to investigate the association between Caspase 8 (-652 6 N ins/del), Caspase 9 (-1263 A>G) and Bcl-2 (-938 C>A) polymorphisms and PTC susceptibility by polymerase chain reaction restriction-fragment length polymorphism and DNA sequencing methods. We further analyzed the distribution of genotype frequency, as well as the association of genotype with clinicopathological characteristics. Overall, no statistically significant association was observed in Caspase 8 (-652 6 N ins/del). Nevertheless, Caspase 9 -1263 GG genotype was at increased risk of PTC (P=0.045; odds ratio (OR)=1.12). Furthermore, GG genotype thyroid cancers were significantly more common in older patients than AA or AG genotypes PTC and in cases of advanced pathological stages. However, Bcl-2 -938 AA genotype demonstrated a protective effect in PTCs (P=0.004; OR=0.35). Polymorphism in Caspase 9 (-1263 A>G) was observed to be associated with susceptibility of PTC. However, Bcl-2 (-938 C>A) polymorphism indicated to play a protective role in susceptibility to PTC. Nevertheless, further investigation with a larger sample size is needed to support our results.