Loss of microglial Arid1a exacerbates microglial scar formation via elevated CCL5 after traumatic brain injury.

Traumatic brain injury (TBI) is an acquired insult to the brain caused by an external mechanical force, potentially resulting in temporary or permanent impairment. Microglia, the resident immune cells of the central nervous system, are activated in response to TBI, participating in tissue repair process. However, the underlying epigenetic mechanisms in microglia during TBI remain poorly understood. ARID1A (AT-Rich Interaction Domain 1 A), a pivotal subunit of the multi-protein SWI/SNF chromatin remodeling complex, has received little attention in microglia, especially in the context of brain injury. In this study, we generated a Arid1a cKO mouse line to investigate the potential roles of ARID1A in microglia in response to TBI. We found that glial scar formation was exacerbated due to increased microglial migration and a heightened inflammatory response in Arid1a cKO mice following TBI. Mechanistically, loss of ARID1A led to an up-regulation of the chemokine CCL5 in microglia upon the injury, while the CCL5-neutralizing antibody reduced migration and inflammatory response of LPS-stimulated Arid1a cKO microglia. Importantly, administration of auraptene (AUR), an inhibitor of CCL5, repressed the microglial migration and inflammatory response, as well as the glial scar formation after TBI. These findings suggest that ARID1A is critical for microglial response to injury and that AUR has a therapeutic potential for the treatment of TBI.

Lhx2 promotes axon regeneration of adult retinal ganglion cells and rescues neurodegeneration in mouse models of glaucoma.

The axons of retinal ganglion cells (RGCs) form the optic nerve, transmitting visual information from the eye to the brain. Damage or loss of RGCs and their axons is the leading cause of visual functional defects in traumatic injury and degenerative diseases such as glaucoma. However, there are no effective clinical treatments for nerve damage in these neurodegenerative diseases. Here, we report that LIM homeodomain transcription factor Lhx2 promotes RGC survival and axon regeneration in multiple animal models mimicking glaucoma disease. Furthermore, following N-methyl-D-aspartate (NMDA)-induced excitotoxicity damage of RGCs, Lhx2 mitigates the loss of visual signal transduction. Mechanistic analysis revealed that overexpression of Lhx2 supports axon regeneration by systematically regulating the transcription of regeneration-related genes and inhibiting transcription of Semaphorin 3C (Sema3C). Collectively, our studies identify a critical role of Lhx2 in promoting RGC survival and axon regeneration, providing a promising neural repair strategy for glaucomatous neurodegeneration.

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.

Human midbrain dopaminergic progenitors.

Human midbrain dopaminergic progenitors (mDAPs) are one of the most representative cell types in both basic research and clinical applications. However, there are still many challenges for the preparation and quality control of mDAPs, such as the lack of standards. Therefore, the establishment of critical quality attributes and technical specifications for mDAPs is largely needed. "Human midbrain dopaminergic progenitor" jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human mDAPs in China. This standard specifies the technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for human mDAPs, which is applicable to the quality control for human mDAPs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will facilitate the institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human mDAPs for clinical development and therapeutic applications.

Histone methyltransferase Ezh2 coordinates mammalian axon regeneration via regulation of key regenerative pathways.

Current treatments for neurodegenerative diseases and neural injuries face major challenges, primarily due to the diminished regenerative capacity of neurons in the mammalian CNS as they mature. Here, we investigated the role of Ezh2, a histone methyltransferase, in regulating mammalian axon regeneration. We found that Ezh2 declined in the mouse nervous system during maturation but was upregulated in adult dorsal root ganglion neurons following peripheral nerve injury to facilitate spontaneous axon regeneration. In addition, overexpression of Ezh2 in retinal ganglion cells in the CNS promoted optic nerve regeneration via both histone methylation-dependent and -independent mechanisms. Further investigation revealed that Ezh2 fostered axon regeneration by orchestrating the transcriptional silencing of genes governing synaptic function and those inhibiting axon regeneration, while concurrently activating various factors that support axon regeneration. Notably, we demonstrated that GABA transporter 2, encoded by Slc6a13, acted downstream of Ezh2 to control axon regeneration. Overall, our study underscores the potential of modulating chromatin accessibility as a promising strategy for promoting CNS axon regeneration.

Agomir-331 Suppresses Reactive Gliosis and Neuroinflammation after Traumatic Brain Injury.

Traumatic brain injury usually triggers glial scar formation, neuroinflammation, and neurodegeneration. However, the molecular mechanisms underlying these pathological features are largely unknown. Using a mouse model of hippocampal stab injury (HSI), we observed that miR-331, a brain-enriched microRNA, was significantly downregulated in the early stage (0-7 days) of HSI. Intranasal administration of agomir-331, an upgraded product of miR-331 mimics, suppressed reactive gliosis and neuronal apoptosis and improved cognitive function in HSI mice. Finally, we identified IL-1ß as a direct downstream target of miR-331, and agomir-331 treatment significantly reduced IL-1ß levels in the hippocampus after acute injury. Our findings highlight, for the first time, agomir-331 as a pivotal neuroprotective agent for early rehabilitation of HSI.

CHIT1-positive microglia drive motor neuron ageing in the primate spinal cord.

Ageing is a critical factor in spinal-cord-associated disorders1, yet the ageing-specific mechanisms underlying this relationship remain poorly understood. Here, to address this knowledge gap, we combined single-nucleus RNA-sequencing analysis with behavioural and neurophysiological analysis in non-human primates (NHPs). We identified motor neuron senescence and neuroinflammation with microglial hyperactivation as intertwined hallmarks of spinal cord ageing. As an underlying mechanism, we identified a neurotoxic microglial state demarcated by elevated expression of CHIT1 (a secreted mammalian chitinase) specific to the aged spinal cords in NHP and human biopsies. In the aged spinal cord, CHIT1-positive microglia preferentially localize around motor neurons, and they have the ability to trigger senescence, partly by activating SMAD signalling. We further validated the driving role of secreted CHIT1 on MN senescence using multimodal experiments both in vivo, using the NHP spinal cord as a model, and in vitro, using a sophisticated system modelling the human motor-neuron-microenvironment interplay. Moreover, we demonstrated that ascorbic acid, a geroprotective compound, counteracted the pro-senescent effect of CHIT1 and mitigated motor neuron senescence in aged monkeys. Our findings provide the single-cell resolution cellular and molecular landscape of the aged primate spinal cord and identify a new biomarker and intervention target for spinal cord degeneration.

X chromosome encoded histone demethylase UTX regulates mammalian axon regeneration via microRNA-124.

Neurons in the mammalian central nervous system (CNS) gradually lose their intrinsic regeneration capacity during maturation mainly because of altered transcription profile. Recent studies have made great progress by identifying genes that can be manipulated to enhance CNS regeneration. However, as a complex process involving many genes and signaling networks, it is of great importance to deciphering the underlying neuronal chromatin and transcriptomic landscape coordinating CNS regeneration. Here we identify UTX, an X-chromosome associated gene encoding a histone demethylase, as a novel regulator of mammalian neural regeneration. We demonstrate that UTX acts as a repressor of spontaneous axon regeneration in the peripheral nerve system (PNS). In the CNS, either knocking out or pharmacological inhibiting UTX in retinal ganglion cells (RGCs) leads to significantly enhanced neuronal survival and optic nerve regeneration. RNA-seq profiling revealed that deleting UTX switches the RGC transcriptomics into a developmental-like state. Moreover, microRNA-124, one of the most abundant microRNAs in mature neurons, is identified as a downstream target of UTX and blocking endogenous microRNA124-5p results in robust optic nerve regeneration. These findings revealed a novel histone modification-microRNA epigenetic signaling network orchestrating transcriptomic landscape supporting CNS neural regeneration.

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.

ASH2L regulates postnatal neurogenesis through Onecut2-mediated inhibition of TGF-ß signaling pathway.

The ability of neural stem/progenitor cells (NSPCs) to proliferate and differentiate is required through different stages of neurogenesis. Disturbance in the regulation of neurogenesis causes many neurological diseases, such as intellectual disability, autism, and schizophrenia. However, the intrinsic mechanisms of this regulation in neurogenesis remain poorly understood. Here, we report that Ash2l (Absent, small or homeotic discs-like 2), one core component of a multimeric histone methyltransferase complex, is essential for NSPC fate determination during postnatal neurogenesis. Deletion of Ash2l in NSPCs impairs their capacity for proliferation and differentiation, leading to simplified dendritic arbors in adult-born hippocampal neurons and deficits in cognitive abilities. RNA sequencing data reveal that Ash2l primarily regulates cell fate specification and neuron commitment. Furthermore, we identified Onecut2, a major downstream target of ASH2L characterized by bivalent histone modifications, and demonstrated that constitutive expression of Onecut2 restores defective proliferation and differentiation of NSPCs in adult Ash2l-deficient mice. Importantly, we identified that Onecut2 modulates TGF-ß signaling in NSPCs and that treatment with a TGF-ß inhibitor rectifies the phenotype of Ash2l-deficient NSPCs. Collectively, our findings reveal the ASH2L-Onecut2-TGF-ß signaling axis that mediates postnatal neurogenesis to maintain proper forebrain function.

Neuronal Histone Methyltransferase EZH2 Regulates Neuronal Morphogenesis, Synaptic Plasticity, and Cognitive Behavior in Mice.

The histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)-mediated trimethylation of histone H3 lysine 27 (H3K27me3) regulates neural stem cell proliferation and fate specificity through silencing different gene sets in the central nervous system. Here, we explored the function of EZH2 in early post-mitotic neurons by generating a neuron-specific Ezh2 conditional knockout mouse line. The results showed that a lack of neuronal EZH2 led to delayed neuronal migration, more complex dendritic arborization, and increased dendritic spine density. Transcriptome analysis revealed that neuronal EZH2-regulated genes are related to neuronal morphogenesis. In particular, the gene encoding p21-activated kinase 3 (Pak3) was identified as a target gene suppressed by EZH2 and H3K27me3, and expression of the dominant negative Pak3 reversed Ezh2 knockout-induced higher dendritic spine density. Finally, the lack of neuronal EZH2 resulted in impaired memory behaviors in adult mice. Our results demonstrated that neuronal EZH2 acts to control multiple steps of neuronal morphogenesis during development, and has long-lasting effects on cognitive function in adult mice.

Microglial transglutaminase 2 deficiency causes impaired synaptic remodelling and cognitive deficits in mice.

Microglia are the primary source of transglutaminase 2 (TGM2) in the brain; however, the roles of microglial TGM2 in neural development and disease are still not well known. The aim of this study is to elucidate the role and mechanisms of microglial TGM2 in the brain. A mouse line with a specific knockout of Tgm2 in microglia was generated. Immunohistochemistry, Western blot and qRT-PCR assays were performed to evaluate the expression levels of TGM2, PSD-95 and CD68. Confocal imaging, immunofluorescence staining and behavioural analyses were conducted to identify phenotypes of microglial TGM2 deficiency. Finally, RNA sequencing, qRT-PCR and co-culture of neurons and microglia were used to explore the potential mechanisms. Deletion of microglial Tgm2 causes impaired synaptic pruning, reduced anxiety and increased cognitive deficits in mice. At the molecular level, the phagocytic genes, such as Cq1a, C1qb and Tim4, are significantly down-regulated in TGM2-deficient microglia. This study elucidates a novel role of microglial TGM2 in regulating synaptic remodelling and cognitive function, indicating that microglia Tgm2 is essential for proper neural development.

The Role and Mechanism of Transglutaminase 2 in Regulating Hippocampal Neurogenesis after Traumatic Brain Injury.

Traumatic brain injury usually results in neuronal loss and cognitive deficits. Promoting endogenous neurogenesis has been considered as a viable treatment option to improve functional recovery after TBI. However, neural stem/progenitor cells (NSPCs) in neurogenic regions are often unable to migrate and differentiate into mature neurons at the injury site. Transglutaminase 2 (TGM2) has been identified as a crucial component of neurogenic niche, and significantly dysregulated after TBI. Therefore, we speculate that TGM2 may play an important role in neurogenesis after TBI, and strategies targeting TGM2 to promote endogenous neural regeneration may be applied in TBI therapy. Using a tamoxifen-induced Tgm2 conditional knockout mouse line and a mouse model of stab wound injury, we investigated the role and mechanism of TGM2 in regulating hippocampal neurogenesis after TBI. We found that Tgm2 was highly expressed in adult NSPCs and up-regulated after TBI. Conditional deletion of Tgm2 resulted in the impaired proliferation and differentiation of NSPCs, while Tgm2 overexpression enhanced the abilities of self-renewal, proliferation, differentiation, and migration of NSPCs after TBI. Importantly, injection of lentivirus overexpressing TGM2 significantly promoted hippocampal neurogenesis after TBI. Therefore, TGM2 is a key regulator of hippocampal neurogenesis and a pivotal therapeutic target for intervention following TBI.

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.

Acetate supplementation restores cognitive deficits caused by ARID1A haploinsufficiency in excitatory neurons.

Mutations in AT-rich interactive domain-containing protein 1A (ARID1A) cause Coffin-Siris syndrome (CSS), a rare genetic disorder that results in mild to severe intellectual disabilities. However, the biological role of ARID1A in the brain remains unclear. In this study, we report that the haploinsufficiency of ARID1A in excitatory neurons causes cognitive impairment and defects in hippocampal synaptic transmission and dendritic morphology in mice. Similarly, human embryonic stem cell-derived excitatory neurons with deleted ARID1A exhibit fewer dendritic branches and spines, and abnormal electrophysiological activity. Importantly, supplementation of acetate, an epigenetic metabolite, can ameliorate the morphological and electrophysiological deficits observed in mice with Arid1a haploinsufficiency, as well as in ARID1A-null human excitatory neurons. Mechanistically, transcriptomic and ChIP-seq analyses demonstrate that acetate supplementation can increase the levels of H3K27 acetylation at the promoters of key regulatory genes associated with neural development and synaptic transmission. Collectively, these findings support the essential roles of ARID1A in the excitatory neurons and cognition and suggest that acetate supplementation could be a potential therapeutic intervention for CSS.

Correction: He et al. Intranasal Administration of Agomir-let-7i Improves Cognitive Function in Mice with Traumatic Brain Injury. Cells 2022, 11, 1348.

The authors wish to make the following changes to their paper [...].

A Mouse Model of Neurodegeneration Induced by Blade Penetrating Stab Wound to the Hippocampus.

Traumatic brain injury (TBI) is closely associated with the later development of neurodegenerative and psychiatric diseases which are still incurable. Although various animal TBI models have been generated, they usually have weaknesses in standardization, survivability and/or reproducibility. In the present study, we investigated whether applying a blade penetrating stab wound to the hippocampus would create an animal model of cognitive deficits. Open-field, Morris water maze and Barnes maze tests were used to evaluate the animal behaviors. The immunofluorescence staining of NeuN, GFAP, IBA1, and TUNEL was conducted to analyze the changes in neurons, astrocytes, and microglia, as well as cell death. Mice with a hippocampal blade stab injury (HBSI) displayed the activation of microglia and astrocytes, inflammation, neuronal apoptosis, and deficits in spatial learning and memory. These findings suggest that HBSI is an easy approach to generate a reliable in vivo model of TBI to capture hemorrhage, neuroinflammation, reactive gliosis, and neural death, as well as cognitive deficits observed in human patients.