N-methyladenosine (mA), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However, the roles and precise mechanisms of mA modification in regulating neuronal development and adult neurogenesis remain unclear. Here, we examined the function of Mettl3, the key component of the complex, in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced mA levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neuronal development and skewed the differentiation of aNSCs more toward glial lineage, but also affected the morphological maturation of newborn neurons in the adult brain. mA immunoprecipitation combined with deep sequencing (MeRIP-seq) revealed that mA was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically, mA was present on the transcripts of histone methyltransferase Ezh2, and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively, our results uncover a crosstalk between RNA and histone modifications and indicate that Mettl3-mediated mA modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2.
Adenosine ; analogs & derivatives ; metabolism ; Adult Stem Cells ; cytology ; metabolism ; Animals ; Brain ; metabolism ; Cell Differentiation ; genetics ; Cell Proliferation ; Enhancer of Zeste Homolog 2 Protein ; metabolism ; Gene Expression Regulation ; Methyltransferases ; metabolism ; Mice, Inbred C57BL ; Neural Stem Cells ; cytology ; metabolism ; Neurogenesis ; genetics ; Neurons ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism

The complex spatial and temporal organization of neural activity in the brain is important for information-processing that guides behavior. Hence, revealing the real-time neural dynamics in freely-moving animals is fundamental to elucidating brain function. Miniature fluorescence microscopes have been developed to fulfil this requirement. With the help of GRadient INdex (GRIN) lenses that relay optical images from deep brain regions to the surface, investigators can visualize neural activity during behavioral tasks in freely-moving animals. However, the application of GRIN lenses to deep brain imaging is severely limited by their availability. Here, we describe a protocol for GRIN lens coating that ensures successful long-term intravital imaging with commercially-available GRIN lenses.
Animals ; Biocompatible Materials ; Brain ; physiology ; Hippocampus ; cytology ; Lenses ; Mice, Inbred C57BL ; Mice, Transgenic ; Microscopy, Fluorescence ; methods ; Neuroimaging ; instrumentation ; methods ; Neurons ; physiology

The aim of this study was to investigate whether G protein-coupled estrogen receptor (GPER) could alleviate hippocampal neuron injury under cerebral ischemia-reperfusion injury (CIRI) by acting on endoplasmic reticulum stress (ERS). The CIRI animal model was established by middle cerebral artery occlusion (MCAO). Female ovariectomized (OVX) Sprague-Dawley (SD) female rats were randomly divided into 4 groups: control, ischemia-reperfusion injury (MCAO), vehicle (MCAO+DMSO), and GPER-specific agonist G1 (MCAO+G1) groups. The neurobehavioral score was assessed by the Longa score method, the morphological changes of the neurons were observed by the Nissl staining, the cerebral infarction was detected by the TTC staining, and the neural apoptosis in the hippocampal CA1 region was detected by TUNEL staining. The distribution and expression of GRP78 (78 kDa glucose-regulated protein 78) in the hippocampal CA1 region were observed by immunofluorescent staining. The protein expression levels of GRP78, Caspase-12, CHOP and Caspase-3 were detected by Western blot, and the mRNA expression levels of GRP78, Caspase-12, and CHOP were detected by the real-time PCR. The results showed that the neurobehavioral score, cerebral infarct volume, cellular apoptosis index, as well as GRP78, Caspase-12 and CHOP protein and mRNA expression levels in the MCAO group were significantly higher than those of control group. And G1 reversed the above-mentioned changes in the MCAO+G1 group. These results suggest that the activation of GPER can decrease the apoptosis of hippocampal neurons and relieve CIRI, and its mechanism may involve the inhibition of ERS.
Animals ; Apoptosis ; Brain Ischemia ; CA1 Region, Hippocampal ; cytology ; Caspase 12 ; metabolism ; Caspase 3 ; metabolism ; Endoplasmic Reticulum Stress ; Female ; Heat-Shock Proteins ; metabolism ; Neurons ; cytology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Estrogen ; physiology ; Receptors, G-Protein-Coupled ; agonists ; Reperfusion Injury ; Transcription Factor CHOP ; metabolism

Neural stem cell therapy, as a new therapeutic method for neural diseases, has aroused a wide concern for over 20 years since neural stem cells were first found in 1992. Ischemic stroke is highly concerned because of its high incidence, mortality and disability rates. Because the brain has a limited ability to repair itself, to improve neural function and promote neural regeneration may help to prevent occurrence and development of neurological diseases. It is noteworthy that some stroke patients showed an ability to repair brain several months after the stroke happened, suggesting an existence of endogenous nerve repair in these patients. The research advances in functions of endogenous neural stem cells in neural regeneration and the related regulators after ischemic stroke are summarized in this review to provide new views of the mechanism of neural functional recovery after ischemic stroke.
Brain Ischemia ; therapy ; Humans ; Nerve Regeneration ; Neural Stem Cells ; cytology ; Stroke ; therapy

BackgroundGlehnia littoralis has been used for traditional Asian medicine, which has diverse therapeutic activities. However, studies regarding neurogenic effects of G. littoralis have not yet been considered. Therefore, in this study, we examined effects of G. littoralis extract on cell proliferation, neuroblast differentiation, and the maturation of newborn neurons in the hippocampus of adult mice.

MethodsA total of 39 male ICR mice (12 weeks old) were randomly assigned to vehicle-treated and 100 and 200 mg/kg G. littoralis extract-treated groups (n = 13 in each group). Vehicle and G. littoralis extract were orally administrated for 28 days. To examine neurogenic effects of G. littoralis extract, we performed immunohistochemistry for 5-bromo-2-deoxyuridine (BrdU, an indicator for cell proliferation) and doublecortin (DCX, an immature neuronal marker) and double immunofluorescence staining for BrdU and neuronal nuclear antigen (NeuN, a mature neuronal marker). In addition, we examined expressional changes of brain-derived neurotrophic factor (BDNF) and its major receptor tropomyosin-related kinase B (TrkB) using Western blotting analysis.

ResultsTreatment with 200 mg/kg, not 100 mg/kg, significantly increased number of BrdU-immunoreactive () and DCX cells (48.0 ± 3.1 and 72.0 ± 3.8 cells/section, respectively) in the subgranular zone (SGZ) of the dentate gyrus (DG) and BrdU/NeuN cells (17.0 ± 1.5 cells/section) in the granule cell layer as well as in the SGZ. In addition, protein levels of BDNF and TrkB (about 232% and 244% of the vehicle-treated group, respectively) were significantly increased in the DG of the mice treated with 200 mg/kg of G. littoralis extract.

ConclusionG. littoralis extract promots cell proliferation, neuroblast differentiation, and neuronal maturation in the hippocampal DG, and neurogenic effects might be closely related to increases of BDNF and TrkB proteins by G. littoralis extract treatment.

Animals ; Apiaceae ; chemistry ; Blotting, Western ; Brain-Derived Neurotrophic Factor ; metabolism ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Dentate Gyrus ; cytology ; drug effects ; Hippocampus ; cytology ; drug effects ; Immunohistochemistry ; Male ; Mice ; Microtubule-Associated Proteins ; metabolism ; Neurogenesis ; drug effects ; Neuropeptides ; metabolism ; Plant Extracts ; pharmacology ; Receptor, trkB ; metabolism

The blood-brain barrier (BBB) is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein 1 (DMP1)-proteoglycan (PG), which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMP1-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 (S89) in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine (S89G), which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that regulated BBB formation, but also assigned a new function to DMP1-PG.
Animals ; Astrocytes ; cytology ; metabolism ; Blood-Brain Barrier ; cytology ; metabolism ; Cells, Cultured ; Extracellular Matrix Proteins ; genetics ; metabolism ; Female ; Glycosylation ; Male ; Mice ; Proteoglycans ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Aging associated cognitive decline has been linked to dampened neural stem/progenitor cells (NSC/NPCs) activities manifested by decreased proliferation, reduced propensity to produce neurons, and increased differentiation into astrocytes. While gene transcription changes objectively reveal molecular alterations of cells undergoing various biological processes, the search for molecular mechanisms underlying aging of NSC/NPCs has been confronted by the enormous heterogeneity in cellular compositions of the brain and the complex cellular microenvironment where NSC/NPCs reside. Moreover, brain NSC/NPCs themselves are not a homogenous population, making it even more difficult to uncover NSC/NPC sub-type specific aging mechanisms. Here, using both population-based and single cell transcriptome analyses of young and aged mouse forebrain ependymal and subependymal regions and comprehensive "big-data" processing, we report that NSC/NPCs reside in a rather inflammatory environment in aged brain, which likely contributes to the differentiation bias towards astrocytes versus neurons. Moreover, single cell transcriptome analyses revealed that different aged NSC/NPC subpopulations, while all have reduced cell proliferation, use different gene transcription programs to regulate age-dependent decline in cell cycle. Interestingly, changes in cell proliferation capacity are not influenced by inflammatory cytokines, but likely result from cell intrinsic mechanisms. The Erk/Mapk pathway appears to be critically involved in regulating age-dependent changes in the capacity for NSC/NPCs to undergo clonal expansion. Together this study is the first example of using population and single cell based transcriptome analyses to unveil the molecular interplay between different NSC/NPCs and their microenvironment in the context of the aging brain.
Aging ; genetics ; Animals ; Astrocytes ; cytology ; metabolism ; Brain ; cytology ; metabolism ; Cell Differentiation ; genetics ; Cell Division ; genetics ; Cell Proliferation ; genetics ; Gene Expression Regulation ; genetics ; Mice ; Neural Stem Cells ; metabolism ; Single-Cell Analysis ; Stem Cells ; cytology ; metabolism ; Transcriptome ; genetics

This paper aimed to observe the protective effect of catalpol on the high glucose induced destruction of tight junctions of rat primary brain microvascular endothelial cells (BMECs). Catalpol co-administrated with high glucose increased BMECs survival, decreased its ET-1 secretion, and improved transmembrane electrical resistance in a time-dependent manner. Furthermore, transmission electron microscopy was used to observe catalpol's protective effect on tight junction. Fluorescence staining displayed that catalpol reversed the rearrangement of the cytoskeleton protein F-actin and up-regulated the tight junction proteins claudin-5 and ZO-1, which were further demonstrated by the mRNA expression levels of claudin-5, occludin, ZO-1, ZO-2, ZO-3, -actintin, vinculin and cateinins. This study indicated that catalpol reverses the disaggregation of cytoskeleton actin in BMECs and up-regulates the expression of tight junction proteins, such as claudin-5, occludin, and ZO-1, and finally alleviates the increase in high glucose-induced BMECs injury.
Actin Cytoskeleton ; drug effects ; Actins ; metabolism ; Animals ; Brain ; cytology ; Cells, Cultured ; Claudin-5 ; metabolism ; Endothelial Cells ; drug effects ; Glucose ; Iridoid Glucosides ; pharmacology ; Phosphoproteins ; Rats ; Tight Junctions ; drug effects ; Zonula Occludens-1 Protein ; metabolism

Animals choose among sleep, courtship, and feeding behaviors based on the integration of both external sensory cues and internal states; such choices are essential for survival and reproduction. These competing behaviors are closely related and controlled by distinct neural circuits, but whether they are also regulated by shared neural nodes is unclear. Here, we investigated how a set of male-specific P1 neurons controls sleep, courtship, and feeding behaviors in Drosophila males. We found that mild activation of P1 neurons was sufficient to affect sleep, but not courtship or feeding, while stronger activation of P1 neurons labeled by four out of five independent drivers induced courtship, but only the driver that targeted the largest number of P1 neurons affected feeding. These results reveal a common neural node that affects sleep, courtship, and feeding in a threshold-dependent manner, and provide insights into how competing behaviors can be regulated by a shared neural node.
Animals ; Animals, Genetically Modified ; Brain ; cytology ; Courtship ; Drosophila ; Drosophila Proteins ; genetics ; metabolism ; Feeding Behavior ; physiology ; Locomotion ; Male ; Neural Inhibition ; physiology ; Neural Pathways ; physiology ; Neurons ; physiology ; Sex Factors ; Sleep ; physiology

The brain has very high energy requirements and consumes 20% of the oxygen and 25% of the glucose in the human body. Therefore, the molecular mechanism underlying how the brain metabolizes substances to support neural activity is a fundamental issue for neuroscience studies. A well-known model in the brain, the astrocyte-neuron lactate shuttle, postulates that glucose uptake and glycolytic activity are enhanced in astrocytes upon neuronal activation and that astrocytes transport lactate into neurons to fulfill their energy requirements. Current evidence for this hypothesis has yet to reach a clear consensus, and new concepts beyond the shuttle hypothesis are emerging. The discrepancy is largely attributed to the lack of a critical method for real-time monitoring of metabolic dynamics at cellular resolution. Recent advances in fluorescent protein-based sensors allow the generation of a sensitive, specific, real-time readout of subcellular metabolites and fill the current technological gap. Here, we summarize the development of genetically encoded metabolite sensors and their applications in assessing cell metabolism in living cells and in vivo, and we believe that these tools will help to address the issue of elucidating neural energy metabolism.
Animals ; Biosensing Techniques ; Brain ; cytology ; metabolism ; Cytological Techniques ; Energy Metabolism ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Time Factors