MiR-181a-5p promotes neural stem cell proliferation and enhances the learning and memory of aged mice.

Hippocampal neural stem cell (NSC) proliferation is known to decline with age, which is closely linked to learning and memory impairments. In the current study, we found that the expression level of miR-181a-5p was decreased in the hippocampal NSCs of aged mice and that exogenous overexpression of miR-181a-5p promoted NSC proliferation without affecting NSC differentiation into neurons and astrocytes. The mechanistic study revealed that phosphatase and tensin homolog (PTEN), a negative regulator of the AKT signaling pathway, was the target of miR-181a-5p and knockdown of PTEN could rescue the impairment of NSC proliferation caused by low miR-181a-5p levels. Moreover, overexpression of miR-181a-5p in the dentate gyrus enhanced the proliferation of NSCs and ameliorated learning and memory impairments in aged mice. Taken together, our findings indicated that miR-181a-5p played a functional role in NSC proliferation and aging-related, hippocampus-dependent learning and memory impairments.

Cmarr/miR-540-3p axis promotes cardiomyocyte maturation transition by orchestrating Dtna expression.

The immature phenotype of embryonic stem cell-derived cardiomyocytes (ESC-CMs) limits their application. However, the molecular mechanisms of cardiomyocyte maturation remain largely unexplored. This study found that overexpression of long noncoding RNA (lncRNA)-Cmarr, which was highly expressed in cardiomyocytes, promoted the maturation change and physiological maturation of mouse ESC-CMs (mESC-CMs). Moreover, transplantation of cardiac patch overexpressing Cmarr exhibited better retention of mESC-CMs, reduced infarct area by enhancing vascular density in the host heart, and improved cardiac function in mice after myocardial infarction. Mechanism studies identified that Cmarr acted as a competitive endogenous RNA to impede the repression of miR-540-3p on Dtna expression and promoted the binding of the dystrophin-glycoprotein complex (DGC) and yes-associated protein (YAP), which in turn reduced the proportion of nuclear YAP and the expression of YAP target genes. Therefore, this study revealed the function and mechanism of Cmarr in promoting cardiomyocyte maturation and provided a lncRNA that can be used as a functional factor in the construction of cardiac patches for the treatment of myocardial infarction.

Linc1548 Promotes the Transition of Epiblast Stem Cells Into Neural Progenitors by Engaging OCT6 and SOX2.

The transition of embryonic stem cells from the epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs), called the neural induction process, is crucial for cell fate determination of neural differentiation. However, the mechanism of this transition is unclear. Here, we identified a long non-coding RNA (linc1548) as a critical regulator of neural differentiation of mouse embryonic stem cells (mESCs). Knockout of linc1548 did not affect the conversion of mESCs to EpiSCs, but delayed the transition from EpiSCs to NPCs. Moreover, linc1548 interacts with the transcription factors OCT6 and SOX2 forming an RNA-protein complex to regulate the transition from EpiSCs to NPCs. Finally, we showed that Zfp521 is an important target gene of this RNA-protein complex regulating neural differentiation. Our findings prove how the intrinsic transcription complex is mediated by a lncRNA linc1548 and can better understand the intrinsic mechanism of neural fate determination.

LncRNA SOX1-OT V1 acts as a decoy of HDAC10 to promote SOX1-dependent hESC neuronal differentiation.

Long noncoding RNAs (lncRNAs) are abundantly expressed in the nervous system, but their regulatory roles in neuronal differentiation are poorly understood. Using a human embryonic stem cell (hESC)-based 2D neural differentiation approach and a 3D cerebral organoid system, we show that SOX1-OT variant 1 (SOX1-OT V1), a SOX1 overlapping noncoding RNA, plays essential roles in both dorsal cortical neuron differentiation and ventral GABAergic neuron differentiation by facilitating SOX1 expression. SOX1-OT V1 physically interacts with HDAC10 through its 5' region, acts as a decoy to block HDAC10 binding to the SOX1 promoter, and thus maintains histone acetylation levels at the SOX1 promoter. SOX1 in turn activates ASCL1 expression and promotes neuronal differentiation. Taken together, we identify a SOX1-OT V1/HDAC10-SOX1-ASCL1 axis, which promotes neurogenesis, highlighting a role for lncRNAs in hESC neuronal differentiation.

Fetal growth restriction impairs hippocampal neurogenesis and cognition via Tet1 in offspring.

Fetal growth restriction (FGR) increases the risk for impaired cognitive function later in life. However, the precise mechanisms remain elusive. Using dexamethasone-induced FGR and protein restriction-influenced FGR mouse models, we observe learning and memory deficits in adult FGR offspring. FGR induces decreased hippocampal neurogenesis from the early post-natal period to adulthood by reducing the proliferation of neural stem cells (NSCs). We further find a persistent decrease of Tet1 expression in hippocampal NSCs of FGR mice. Mechanistically, Tet1 downregulation results in hypermethylation of the Dll3 and Notch1 promoters and inhibition of Notch signaling, leading to reduced NSC proliferation. Overexpression of Tet1 activates Notch signaling, offsets the decline in neurogenesis, and enhances learning and memory abilities in FGR offspring. Our data indicate that a long-term decrease in Tet1/Notch signaling in hippocampal NSCs contributes to impaired neurogenesis following FGR and could serve as potential targets for the intervention of FGR-related cognitive disorders.

PAUPAR and PAX6 sequentially regulate human embryonic stem cell cortical differentiation.

Long noncoding RNAs (lncRNAs) play a wide range of roles in the epigenetic regulation of crucial biological processes, but the functions of lncRNAs in cortical development are poorly understood. Using human embryonic stem cell (hESC)-based 2D neural differentiation approach and 3D cerebral organoid system, we identified that the lncRNA PAUPAR, which is adjacent to PAX6, plays essential roles in cortical differentiation by interacting with PAX6 to regulate the expression of a large number of neural genes. Mechanistic studies showed that PAUPAR confers PAX6 proper binding sites on the target neural genes by directly binding the genomic regions of these genes. Moreover, PAX6 recruits the histone methyltransferase NSD1 through its C-terminal PST enrichment domain, then regulate H3K36 methylation and the expression of target genes. Collectively, our data reveal that the PAUPAR/PAX6/NSD1 complex plays a critical role in the epigenetic regulation of hESC cortical differentiation and highlight the importance of PAUPAR as an intrinsic regulator of cortical differentiation.

Human Stem Cell-Derived Neurons Repair Circuits and Restore Neural Function.

Although cell transplantation can rescue motor defects in Parkinson's disease (PD) models, whether and how grafts functionally repair damaged neural circuitry in the adult brain is not known. We transplanted hESC-derived midbrain dopamine (mDA) or cortical glutamate neurons into the substantia nigra or striatum of a mouse PD model and found extensive graft integration with host circuitry. Axonal pathfinding toward the dorsal striatum was determined by the identity of the grafted neurons, and anatomical presynaptic inputs were largely dependent on graft location, whereas inhibitory versus excitatory input was dictated by the identity of grafted neurons. hESC-derived mDA neurons display A9 characteristics and restore functionality of the reconstructed nigrostriatal circuit to mediate improvements in motor function. These results indicate similarity in cell-type-specific pre- and post-synaptic integration between transplant-reconstructed circuit and endogenous neural networks, highlighting the capacity of hPSC-derived neuron subtypes for specific circuit repair and functional restoration in the adult brain.

Critical regulation of a NDIME/MEF2C axis in embryonic stem cell neural differentiation and autism.

A microdeletion within human chromosome 5q14.3 has been associated with the occurrence of neurodevelopmental disorders, such as autism and intellectual disability, and MEF2C haploinsufficiency was identified as main cause. Here, we report that a brain-enriched long non-coding RNA, NDIME, is located near the MEF2C locus and is required for normal neural differentiation of mouse embryonic stem cells (mESCs). NDIME interacts with EZH2, the major component of polycomb repressive complex 2 (PRC2), and blocks EZH2-mediated trimethylation of histone H3 lysine 27 (H3K27me3) at the Mef2c promoter, promoting MEF2C transcription. Moreover, the expression levels of both NDIME and MEF2C were strongly downregulated in the hippocampus of a mouse model of autism, and the adeno-associated virus (AAV)-mediated expression of NDIME in the hippocampus of these mice significantly increased MEF2C expression and ameliorated autism-like behaviors. The results of this study reveal an epigenetic mechanism by which NDIME regulates MEF2C transcription and neural differentiation and suggest potential effects and therapeutic approaches of the NDIME/MEF2C axis in autism.

Fetal growth restriction mice are more likely to exhibit depression-like behaviors due to stress-induced loss of dopaminergic neurons in the VTA.

Fetal growth restriction (FGR) is a severe perinatal complication that can increase risk for mental illness. To investigate the mechanism by which FGR mice develop mental illness in adulthood, we established the FGR mouse model and the FGR mice did not display obvious depression-like behaviors, but after environmental stress exposure, FGR mice were more likely to exhibit depression-like behaviors than control mice. Moreover, FGR mice had significantly fewer dopaminergic neurons in the ventral tegmental area but no difference in serotoninergic neurons in the dorsal raphe. RNA-seq analysis showed that the downregulated genes in the midbrain of FGR mice were associated with many mental diseases and were especially involved in the regulation of NMDA-selective glutamate receptor (NMDAR) activity. Furthermore, the NMDAR antagonist memantine can relieve the stress-induced depression-like behaviors of FGR mice. In summary, our findings provide a theoretical basis for future research and treatment of FGR-related depression.

MicroRNA-153 improves the neurogenesis of neural stem cells and enhances the cognitive ability of aged mice through the notch signaling pathway.

Aging-related cognitive ability impairments are one of the main threats to public health, and impaired hippocampal neurogenesis is a major cause of cognitive decline during aging. However, the regulation of adult neurogenesis in the hippocampus requires further study. Here, we investigated the role of microRNA-153 (miR-153), a highly conserved microRNA in mice and humans, in adult neurogenesis. During the passaging of neural stem cells (NSCs) in vitro, endogenous miR-153 expression was downregulated, with a decrease in neuronal differentiation ability. In addition, miR-153 overexpression increased the neurogenesis of NSCs. Further studies showed that miR-153 regulated neurogenesis by precisely targeting the Notch signaling pathway through inhibition of Jagged1 and Hey2 translation. In vivo analysis demonstrated that miR-153 expression was decreased in the hippocampi of aged mice with impaired cognitive ability, and that miR-153 overexpression in the hippocampus promoted neurogenesis and markedly increased the cognitive abilities of the aged mice. Overall, our findings revealed that miR-153 affected neurogenesis by regulating the Notch signaling pathway and elucidated the function of miR-153 in aging-related, hippocampus-dependent cognitive ability impairments, and neurodegenerative diseases.

Linc1557 is critical for the initiation of embryonic stem cell differentiation by directly targeting the LIF/STAT3 signaling pathway.

Embryonic stem cells (ESCs) have self-renewal and multi-lineage differentiation potential and perform critical functions in development and biomedicine. Several long noncoding RNAs (lncRNAs) have been reported as key regulators of stem cell pluripotency and differentiation. However, the function and regulatory mechanism of lncRNAs during the initiation of ESC differentiation remains unclear. Here, we found that linc1557 was highly expressed in mouse ESCs and required for the initiation of ESC differentiation. Knockdown of linc1557 increased the expression and phosphorylation levels of signal transducer and activator of transcription 3 (STAT3), a key factor in the leukemia inhibitory factor (LIF)/STAT3 signaling pathway. Furthermore, we found that linc1557 directly bound to Stat3 mRNA and affected its stability. The differentially expressed transcriptome after linc1557 knockdown in ESCs was involved primarily in multicellular organism development and cell differentiation as similar to that after Stat3 knockdown. Moreover, either knockdown of Stat3 or addition of a LIF/STAT3 signaling inhibitor rescued the suppressive effects of linc1557 knockdown on the initiation of mouse ESC differentiation. These findings not only elucidated the critical function of linc1557 in the initiation of mouse ESC differentiation but also clarified that its specific mechanism as directly affecting Stat3 mRNA stability, which enhanced the understanding of the lncRNA-mediated regulatory mechanism for mRNA stability and key signaling pathways in ESC pluripotency and differentiation.

Pwp1 regulates telomere length by stabilizing shelterin complex and maintaining histone H4K20 trimethylation.

Telomere maintenance is critical for chromosome stability. Here we report that periodic tryptophan protein 1 (PWP1) is involved in regulating telomere length homeostasis. Pwp1 appears to be essential for mouse development and embryonic stem cell (ESC) survival, as homozygous Pwp1-knockout mice and ESCs have never been obtained. Heterozygous Pwp1-knockout mice had shorter telomeres and decreased reproductive capacity. Pwp1 depletion induced rapid telomere shortening accompanied by reduced shelterin complex and increased DNA damage in telomeric regions. Mechanistically, PWP1 bound and stabilized the shelterin complex via its WD40 domains and regulated the overall level of H4K20me3. The rescue of telomere length in Pwp1-deficient cells by PWP1 overexpression depended on SUV4-20H2 co-expression and increased H4K20me3. Therefore, our study revealed a novel protein involved in telomere homeostasis in both mouse and human cells. This knowledge will improve our understanding of how chromatin structure and histone modifications are involved in maintaining telomere integrity.

Structurally Tunable Reduced Graphene Oxide Substrate Maintains Mouse Embryonic Stem Cell Pluripotency.

Culturing embryonic stem cells (ESCs) in vitro usually requires animal-derived trophoblast cells, which may cause pathogenic and immune reactions; moreover, the poor repeatability between batches hinders the clinical application of ESCs. Therefore, it is essential to synthesize a xenogeneic-free and chemically well-defined biomaterial substrate for maintaining ESC pluripotency. Herein, the effects of structurally tunable reduced graphene oxide (RGO) substrates with different physicochemical properties on ESC pluripotency are studied. Colony formation and CCK-8 assays show that the RGO substrate with an average 30 µm pore size promotes cell survival and proliferation. The unannealed RGO substrate promotes ESC proliferation significantly better than the annealed substrate due to the interfacial hydrophilic groups. The RGO substrate can also maintain ESC for a long time. Additionally, immunofluorescence staining shows that ESCs cultured on an RGO substrate highly express E-cadherin and ß-catenin, whereas after being modified by Dickkopf-related protein 1, the RGO substrate is unable to sustain ESC pluripotency. Furthermore, the cell line that interferes with E-cadherin is also unable to maintain pluripotency. These results confirm that the RGO substrate maintains ESC pluripotency by promoting E-cadherin-mediated cell-cell interaction and Wnt signaling.

A Linc1405/Eomes Complex Promotes Cardiac Mesoderm Specification and Cardiogenesis.

Large intergenic non-coding RNAs (lincRNAs) play widespread roles in epigenetic regulation during multiple differentiation processes, but little is known about their mode of action in cardiac differentiation. Here, we identified the key roles of a lincRNA, termed linc1405, in modulating the core network of cardiac differentiation by functionally interacting with Eomes. Chromatin- and RNA-immunoprecipitation assays showed that exon 2 of linc1405 physically mediates a complex consisting of Eomes, trithorax group (TrxG) subunit WDR5, and histone acetyltransferase GCN5 binding at the enhancer region of Mesp1 gene and activates its expression during cardiac mesoderm specification of embryonic stem cells. Importantly, linc1405 co-localizes with Eomes, WDR5, and GCN5 at the primitive streak, and linc1405 depletion impairs heart development and function in vivo. In summary, linc1405 mediates a Eomes/WDR5/GCN5 complex that contributes to cardiogenesis, highlighting the critical roles of lincRNA-based complexes in the epigenetic regulation of cardiogenesis in vitro and in vivo.

N6-Methyladenosine modification of lincRNA 1281 is critically required for mESC differentiation potential.

Previous studies have revealed the critical roles of N6-methyladenosine (m6A) modification of mRNA in embryonic stem cells (ESCs), but the biological function of m6A in large intergenic noncoding RNA (lincRNA) is unknown. Here, we showed that the internal m6A modification of linc1281 mediates a competing endogenous RNA (ceRNA) model to regulate mouse ESC (mESC) differentiation. We demonstrated that loss of linc1281 compromises mESC differentiation and that m6A is highly enriched within linc1281 transcripts. Linc1281 with RRACU m6A sequence motifs, but not an m6A-deficient mutant, restored the phenotype in linc1281-depleted mESCs. Mechanistic analyses revealed that linc1281 ensures mESC identity by sequestering pluripotency-related let-7 family microRNAs (miRNAs), and this RNA-RNA interaction is m6A dependent. Collectively, these findings elucidated the functional roles of linc1281 and its m6A modification in mESCs and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns.

Long non-coding RNAs in glioma progression.

Glioma is one of most malignant primary tumors of the brain. However, due to a lack of effective means for diagnosing and treating glioma, the prognosis of glioma patients remains poor. Therefore, understanding the molecular mechanism of glioma progression is essential for effective treatment. Long non-coding RNAs (lncRNAs) are novel regulators of gene expression at the transcriptional, post-transcriptional and epigenetic levels. Recent evidence indicates that lncRNAs may play important roles in regulating the progression of glioma. In this article, we review the expression profile of lncRNAs in glioma and discuss the functions and known mechanisms of several representative lncRNAs in detail, as well as the prospects of lncRNAs as diagnostic and prognostic biomarkers and therapeutic targets.

RTL1 promotes melanoma proliferation by regulating Wnt/ß-catenin signalling.

Cutaneous melanoma is a highly malignant and metastatic skin cancer with high mortality. However, its underlying mechanisms remain largely unclear. Here, we found that retrotransposon-like 1 (RTL1) is highly enriched in melanoma tissue, especially in early and horizontal growth tissues. Knockdown of RTL1 in melanoma cells resulted in cell proliferation suppression; cell cycle arrest at G1 phase; and down-regulation of E2F1, CYCLIN D1, cyclin-dependent kinase 6 (CDK6) and c-MYC. Moreover, overexpression of RTL1 in melanoma cells accelerated cell proliferation, promoted passage of the cell cycle beyond G1 phase, and increased the expression of cell cycle related genes. Mechanistically, we found that knockdown of RTL1 inhibited the Wnt/ß-Catenin pathway by regulating the expression of genes specifically involved in ß-CATENIN stabilization. Furthermore, the overexpression and knockdown of ß-CATENIN rescued the effects of RTL1 on melanoma cell proliferation and the cell cycle. These findings were also confirmed via tumour xenografts in nude mice. Together, our results demonstrated that RTL1 promotes melanoma cell proliferation by regulating the Wnt/ß-Catenin signalling pathway.

Mir-29b Mediates the Neural Tube versus Neural Crest Fate Decision during Embryonic Stem Cell Neural Differentiation.

During gastrulation, the neuroectoderm cells form the neural tube and neural crest. The nervous system contains significantly more microRNAs than other tissues, but the role of microRNAs in controlling the differentiation of neuroectodermal cells into neural tube epithelial (NTE) cells and neural crest cells (NCCs) remains unknown. Using embryonic stem cell (ESC) neural differentiation systems, we found that miR-29b was upregulated in NTE cells and downregulated in NCCs. MiR-29b promoted the differentiation of ESCs into NTE cells and inhibited their differentiation into NCCs. Accordingly, the inhibition of miR-29b significantly inhibited the differentiation of NTE cells. A mechanistic study revealed that miR-29b targets DNA methyltransferase 3a (Dnmt3a) to regulate neural differentiation. Moreover, miR-29b mediated the function of Pou3f1, a critical neural transcription factor. Therefore, our study showed that the Pou3f1-miR-29b-Dnmt3a regulatory axis was active at the initial stage of neural differentiation and regulated the determination of cell fate.

Motifs in the amino-terminus of CENP-A are required for its accumulation within the nucleus and at the centromere.

Centromere protein A (CENP-A) is a variant of core histone H3 that marks the centromere's location on the chromosome. The mechanisms that target the protein to the nucleus and the centromere have not been defined. In this study, we found that deletion of the first 53 but not the first 29 residues of CENP-A from the amino-terminus, resulted in its cytoplasmic localization. Two motifs, R42R43R44 and K49R52K53K56, which are reported to be required for DNA contact in the centromere nucleosome, were found to be critical for CENP-A nuclear accumulation. These two motifs potentially mediated its interaction with Importin-ß but were not involved in CENP-A centromeric localization. A third novel motif, L60L61I62R63K64, was found to be essential for the centromeric accumulation of CENP-A. The nonpolar hydrophobic residues L60L61I62, but not the basic residues R63K64, were found to be the most important residues. A protein interaction assay suggested that this motif is not involved in the interaction of CENP-A with its deposition factors but potentially mediates its interaction with core histone H4 and CENP-B. Our study uncovered the role of the amino-terminus of CENP-A in localization.

Dysregulation of the SIRT1/OCT6 Axis Contributes to Environmental Stress-Induced Neural Induction Defects.

Environmental stresses are increasingly acknowledged as core causes of abnormal neural induction leading to neural tube defects (NTDs). However, the mechanism responsible for environmental stress-triggered neural induction defects remains unknown. Here, we report that a spectrum of environmental stresses, including oxidative stress, starvation, and DNA damage, profoundly activate SIRT1, an NAD+-dependent lysine deacetylase. Both mouse embryos and in vitro differentiated embryonic stem cells (ESCs) demonstrated a negative correlation between the expression of SIRT1 and that of OCT6, a key neural fate inducer. Activated SIRT1 radically deacetylates OCT6, triggers an OCT6 ubiquitination/degradation cascade, and consequently increases the incidence of NTD-like phenotypes in mice or hinders neural induction in both human and mouse ESCs. Together, our results suggest that early exposure to environmental stresses results in the dysregulation of the SIRT1/OCT6 axis and increases the risk of NTDs.