5-Bromo-2'-deoxyuridine labeling:historical perspectives,factors infiuencing the detection,toxicity,and its implications in the neurogenesis

The halopyrimidine 5-bromo-2′-deoxyuridine(BrdU)is an exogenous marker of DNA synthesis.Since the introduction of monoclonal antibodies against BrdU,an increasing number of methodologies have been used for the immunodetection of this synthesized bromine-tagged base analogue into replicating DNA.BrdU labeling is widely used for identifying neuron precursors and following their fate during the embryonic,perinatal,and adult neurogenesis in a variety of vertebrate species including birds,reptiles,and mammals.Due to BrdU toxicity,its incorporation into replicating DNA presents adverse consequences on the generation,survival,and settled patterns of cells.This may lead to false results and misinterpretation in the identification of proliferative neuroblasts.In this review,I will indicate the detrimental effects of this nucleoside during the development of the central nervous system,as well as the reliability of BrdU labeling to detect proliferating neuroblasts.Moreover,it will show factors influencing BrdU immunodetection and the contribution of this nucleoside to the study of prenatal,perinatal,and adult neurogenesis.Human adult neurogenesis will also be discussed.It is my hope that this review serves as a reference for those researchers who focused on detecting cells that are in the synthetic phase of the cell cycle.

Astrocytic endothelin-1 overexpression impairs learning and memory ability in ischemic stroke via altered hippocampal neurogenesis and lipid metabolism

Vascular etiology is the second most prevalent cause of cognitive impairment globally.Endothelin-1,which is produced and secreted by endothelial cells and astrocytes,is implicated in the pathogenesis of stroke.However,the way in which changes in astrocytic endothelin-1 lead to poststroke cognitive deficits following transient middle cerebral artery occlusion is not well understood.Here,using mice in which astrocytic endothelin-1 was overexpressed,we found that the selective overexpression of endothelin-1 by astrocytic cells led to ischemic stroke-related dementia(1 hour of ischemia;7 days,28 days,or 3 months of reperfusion).We also revealed that astrocytic endothelin-1 overexpression contributed to the role of neural stem cell proliferation but impaired neurogenesis in the dentate gyrus of the hippocampus after middle cerebral artery occlusion.Comprehensive proteome profiles and western blot analysis confirmed that levels of glial fibrillary acidic protein and peroxiredoxin 6,which were differentially expressed in the brain,were significantly increased in mice with astrocytic endothelin-1 overexpression in comparison with wild-type mice 28 days after ischemic stroke.Moreover,the levels of the enriched differentially expressed proteins were closely related to lipid metabolism,as indicated by Kyoto Encyclopedia of Genes and Genomes pathway analysis.Liquid chromatography-mass spectrometry nontargeted metabolite profiling of brain tissues showed that astrocytic endothelin-1 overexpression altered lipid metabolism products such as glycerol phosphatidylcholine,sphingomyelin,and phosphatidic acid.Overall,this study demonstrates that astrocytic endothelin-1 overexpression can impair hippocampal neurogenesis and that it is correlated with lipid metabolism in poststroke cognitive dysfunction.

Activation of endogenous neurogenesis and angiogenesis by basic fibroblast growth factor-chitosan gel in an adult rat model of ischemic stroke

Attempts have been made to use cell transplantation and biomaterials to promote cell proliferation,differentiation,migration,and survival,as well as angiogenesis,in the context of brain injury.However,whether bioactive materials can repair the damage caused by ischemic stroke by activating endogenous neurogenesis and angiogenesis is still unknown.In this study,we applied chitosan gel loaded with basic fibroblast growth factor to the stroke cavity 7 days after ischemic stroke in rats.The gel slowly released basic fibroblast growth factor,which improved the local microenvironment,activated endogenous neural stem/progenitor cells,and recruited these cells to migrate toward the penumbra and stroke cavity and subsequently differentiate into neurons,while enhancing angiogenesis in the penumbra and stroke cavity and ultimately leading to partial functional recovery.This study revealed the mechanism by which bioactive materials repair ischemic strokes,thus providing a new strategy for the clinical application of bioactive materials in the treatment of ischemic stroke.

Adult neurogenesis:a real hope or a delusion?

Adult neurogenesis,the process of creating new neurons,involves the coordinated division,migration,and differentiation of neural stem cells.This process is restricted to neurogenic niches located in two distinct areas of the brain:the subgranular zone of the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle,where new neurons are generated and then migrate to the olfactory bulb.Neurogenesis has been thought to occur only during the embryonic and early postnatal stages and to decline with age due to a continuous depletion of neural stem cells.Interestingly,recent years have seen tremendous progress in our understanding of adult brain neurogenesis,bridging the knowledge gap between embryonic and adult neurogenesis.Here,we discuss the current status of adult brain neurogenesis in light of what we know about neural stem cells.In this notion,we talk about the importance of intra cellular signaling molecules in mobilizing endogenous neural stem cell prolife ration.Based on the current understanding,we can declare that these molecules play a role in targeting neurogenesis in the mature brain.However,to achieve this goal,we need to avoid the undesired proliferation of neural stem cells by controlling the necessary checkpoints,which can lead to tumorigenesis and prove to be a curse instead of a blessing or hope.

The role of exosomes in adult neurogenesis:implications for neurodegenerative diseases

Exosomes are cup-shaped extracellular vesicles with a lipid bilayer that is approximately 30 to 200 nm in thickness.Exosomes are widely distributed in a range of body fluids,including urine,blood,milk,and saliva.Exosomes exert biological function by transporting factors between different cells and by regulating biological pathways in recipient cells.As an important form of intercellular communication,exosomes are increasingly being investigated due to their ability to transfer bioactive molecules such as lipids,proteins,mRNAs,and microRNAs between cells,and because they can regulate physiological and pathological processes in the central nervous system.Adult neurogenesis is a multistage process by which new neurons are generated and migrate to be integrated into existing neuronal circuits.In the adult brain,neurogenesis is mainly localized in two specialized niches:the subventricular zone adjacent to the lateral ventricles and the subgranular zone of the dentate gyrus.An increasing body of evidence indicates that adult neurogenesis is tightly controlled by environmental conditions with the niches.In recent studies,exosomes released from different sources of cells were shown to play an active role in regulating neurogenesis both in vitro and in vivo,thereby participating in the progression of neurodegenerative disorders in patients and in various disease models.Here,we provide a state-of-the-art synopsis of existing research that aimed to identify the diverse components of exosome cargoes and elucidate the therapeutic potential of exosomal contents in the regulation of neurogenesis in several neurodegenerative diseases.We emphasize that exosomal cargoes could serve as a potential biomarker to monitor functional neurogenesis in adults.In addition,exosomes can also be considered as a novel therapeutic approach to treat various neurodegenerative disorders by improving endogenous neurogenesis to mitigate neuronal loss in the central nervous system.

Long non-coding RNA H19 regulates neurogenesis of induced neural stem cells in a mouse model of closed head injury

Stem cell-based therapies have been proposed as a potential treatment for neural regeneration following closed head injury.We previously reported that induced neural stem cells exert beneficial effects on neural regeneration via cell replacement.However,the neural regeneration efficiency of induced neural stem cells remains limited.In this study,we explored differentially expressed genes and long non-coding RNAs to clarify the mechanism underlying the neurogenesis of induced neural stem cells.We found that H19 was the most downregulated neurogenesis-associated lnc RNA in induced neural stem cells compared with induced pluripotent stem cells.Additionally,we demonstrated that H19 levels in induced neural stem cells were markedly lower than those in induced pluripotent stem cells and were substantially higher than those in induced neural stem cell-derived neurons.We predicted the target genes of H19 and discovered that H19 directly interacts with mi R-325-3p,which directly interacts with Ctbp2 in induced pluripotent stem cells and induced neural stem cells.Silencing H19 or Ctbp2 impaired induced neural stem cell proliferation,and mi R-325-3p suppression restored the effect of H19 inhibition but not the effect of Ctbp2 inhibition.Furthermore,H19 silencing substantially promoted the neural differentiation of induced neural stem cells and did not induce apoptosis of induced neural stem cells.Notably,silencing H19 in induced neural stem cell grafts markedly accelerated the neurological recovery of closed head injury mice.Our results reveal that H19 regulates the neurogenesis of induced neural stem cells.H19 inhibition may promote the neural differentiation of induced neural stem cells,which is closely associated with neurological recovery following closed head injury.

Versatile strategies for adult neurogenesis:avenues to repair the injured brain

Brain injuries due to trauma or stroke are major causes of adult death and disability.Unfortunately,few interventions are effective for post-injury repair of brain tissue.After a long debate on whether endogenous neurogenesis actually happens in the adult human brain,there is now substantial evidence to support its occurrence.Although neurogenesis is usually significantly stimulated by injury,the reparative potential of endogenous differentiation from neural stem/progenitor cells is usually insufficient.Alternatively,exogenous stem cell transplantation has shown promising results in animal models,but limitations such as poor long-term survival and inefficient neuronal differentiation make it still challenging for clinical use.Recently,a high focus was placed on glia-to-neuron conversion under single-factor regulation.Despite some inspiring results,the validity of this strategy is still controversial.In this review,we summarize historical findings and recent advances on neurogenesis strategies for neurorepair after brain injury.We also discuss their advantages and drawbacks,as to provide a comprehensive account of their potentials for further studies.

Potential role of hippocampal neurogenesis in spinal cord injury induced post-trauma depression

It has been reported both in clinic and rodent models that beyond spinal cord injury directly induced symptoms, such as paralysis, neuropathic pain, bladder/bowel dysfunction, and loss of sexual function, there are a variety of secondary complications, including memory loss, cognitive decline, depression, and Alzheimer's disease. The largescale longitudinal population-based studies indicate that post-trauma depression is highly prevalent in spinal cord injury patients. Yet, few basic studies have been conducted to address the potential molecular mechanisms. One of possible factors underlying the depression is the reduction of adult hippocampal neurogenesis which may come from less physical activity, social isolation, chronic pain, and elevated neuroinflammation after spinal cord injury. However, there is no clear consensus yet. In this review, we will first summarize the alteration of hippocampal neurogenesis post-spinal cord injury. Then, we will discuss possible mechanisms underlie this important spinal cord injury consequence. Finally, we will outline the potential therapeutic options aimed at enhancing hippocampal neurogenesis to ameliorate depression.

Post-transcriptional mechanisms controlling neurogenesis and direct neuronal reprogramming

Neurogenesis is a tightly regulated process in time and space both in the developing embryo and in adult neurogenic niches.A drastic change in the transcriptome and proteome of radial glial cells or neural stem cells towards the neuronal state is achieved due to sophisticated mechanisms of epigenetic,transcriptional,and post-transcriptional regulation.Understanding these neurogenic mechanisms is of major importance,not only for shedding light on very complex and crucial developmental processes,but also for the identification of putative reprogramming factors,that harbor hierarchically central regulatory roles in the course of neurogenesis and bare thus the capacity to drive direct reprogramming towards the neuronal fate.The major transcriptional programs that orchestrate the neurogenic process have been the focus of research for many years and key neurogenic transcription factors,as well as repressor complexes,have been identified and employed in direct reprogramming protocols to convert non-neuronal cells,into functional neurons.The post-transcriptional regulation of gene expression during nervous system development has emerged as another important and intricate regulatory layer,strongly contributing to the complexity of the mechanisms controlling neurogenesis and neuronal function.In particular,recent advances are highlighting the importance of specific RNA binding proteins that control major steps of mRNA life cycle during neurogenesis,such as alternative splicing,polyadenylation,stability,and translation.Apart from the RNA binding proteins,microRNAs,a class of small non-coding RNAs that block the translation of their target mRNAs,have also been shown to play crucial roles in all the stages of the neurogenic process,from neural stem/progenitor cell proliferation,neuronal differentiation and migration,to functional maturation.Here,we provide an overview of the most prominent post-transcriptional mechanisms mediated by RNA binding proteins and microRNAs during the neurogenic process,giving particular emphasis on the interplay of specific RNA binding proteins with neurogenic microRNAs.Taking under consideration that the molecular mechanisms of neurogenesis exert high similarity to the ones driving direct neuronal reprogramming,we also discuss the current advances in in vitro and in vivo direct neuronal reprogramming approaches that have employed microRNAs or RNA binding proteins as reprogramming factors,highlighting the so far known mechanisms of their reprogramming action.

Repetitive traumatic brain injury–induced complement C1–related inflammation impairs long-term hippocampal neurogenesis

Repetitive traumatic brain injury impacts adult neurogenesis in the hippocampal dentate gyrus,leading to long-term cognitive impairment.However,the mechanism underlying this neurogenesis impairment remains unknown.In this study,we established a male mouse model of repetitive traumatic brain injury and performed long-term evaluation of neurogenesis of the hippocampal dentate gyrus after repetitive traumatic brain injury.Our results showed that repetitive traumatic brain injury inhibited neural stem cell proliferation and development,delayed neuronal maturation,and reduced the complexity of neuronal dendrites and spines.Mice with repetitive traumatic brain injuryalso showed deficits in spatial memory retrieval.Moreover,following repetitive traumatic brain injury,neuroinflammation was enhanced in the neurogenesis microenvironment where C1q levels were increased,C1q binding protein levels were decreased,and canonical Wnt/β-catenin signaling was downregulated.An inhibitor of C1 reversed the long-term impairment of neurogenesis induced by repetitive traumatic brain injury and improved neurological function.These findings suggest that repetitive traumatic brain injury–induced C1-related inflammation impairs long-term neurogenesis in the dentate gyrus and contributes to spatial memory retrieval dysfunction.

Rbm8a regulates neurogenesis and reduces Alzheimer's disease-associated pathology in the dentate gyrus of 5×FAD mice

Alzheimer’s disease is a prevalent and debilitating neurodegenerative condition that profoundly affects a patient’s daily functioning with progressive cognitive decline,which can be partly attributed to impaired hippocampal neurogenesis.Neurogenesis in the hippocampal dentate gyrus is likely to persist throughout life but declines with aging,especially in Alzheimer’s disease.Recent evidence indicated that RNA-binding protein 8A(Rbm8a)promotes the proliferation of neural progenitor cells,with lower expression levels observed in Alzheimer’s disease patients compared with healthy people.This study investigated the hypothesis that Rbm8a overexpression may enhance neurogenesis by promoting the proliferation of neural progenitor cells to improve memory impairment in Alzheimer’s disease.Therefore,Rbm8a overexpression was induced in the dentate gyrus of 5×FAD mice to validate this hypothesis.Elevated Rbm8a levels in the dentate gyrus triggered neurogenesis and abated pathological phenotypes(such as plaque formation,gliosis reaction,and dystrophic neurites),leading to ameliorated memory performance in 5×FAD mice.RNA sequencing data further substantiated these findings,showing the enrichment of differentially expressed genes involved in biological processes including neurogenesis,cell proliferation,and amyloid protein formation.In conclusion,overexpressing Rbm8a in the dentate gyrus of 5×FAD mouse brains improved cognitive function by ameliorating amyloid-beta-associated pathological phenotypes and enhancing neurogenesis.

Role of the histone methyltransferases Ezh2 and Suv4-20h1/Suv4-20h2 in neurogenesis

Mechanisms regulating neurogenesis involve broad and complex processes that represent intriguing therapeutic targets in the field of regenerative medicine.One influential factor guiding neural stem cell proliferation and cellular differentiation during neurogenesis are epigenetic mechanisms.We present an overview of epigenetic mechanisms including chromatin structure and histone modifications;and discuss novel roles of two histone modifiers,Ezh2 and Suv4-20h1/Suv4-20h2(collectively referred to as Suv4-20h),in neurodevelopment and neurogenesis.This review will focus on broadly reviewing epigenetic regulatory components,the roles of epigenetic components during neurogenesis,and potential applications in regenerative medicine.

Melatonin:a multitasking indoleamine to modulate hippocampal neurogenesis

Neurodegeneration affects a large number of cell types including neurons,astrocytes or oligodendrocytes,and neural stem cells.Neural stem cells can generate new neuronal populations through proliferation,migration,and differentiation.This neurogenic potential may be a relevant factor to fight neurodegeneration and aging.In the last years,we can find growing evidence suggesting that melatonin may be a potential modulator of adult hippocampal neurogenesis.The lack of therapeutic strategies targeting neurogenesis led researchers to explore new molecules.Numerous preclinical studies with melatonin observed how melatonin can modulate and enhance molecular and signaling pathways involved in neurogenesis.We made a special focus on the connection between these modulation mechanisms and their implication in neurodegeneration,to summarize the current knowledge and highlight the therapeutic potential of melatonin.

Inhibition of Notch 1 signaling in the subacute stage after stroke promotes striatal astrocyte-derived neurogenesis

Inhibition of Notch1 signaling has been shown to promote astrocyte-derived neurogenesis after stroke.To investigate the regulatory role of Notch1 signaling in this process,in this study,we used a rat model of stroke based on middle cerebral artery occlusion and assessed the behavior of reactive astrocytes post-stroke.We used theγ-secretase inhibitor N-[N-(3,5-diuorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester(DAPT)to block Notch1 signaling at 1,4,and 7 days after injury.Our results showed that only administration of DAPT at 4 days after stroke promoted astrocyte-derived neurogenesis,as manifested by recovery of white matter fiber bundle integrity on magnetic resonance imaging,which is consistent with recovery of neurologic function.These findings suggest that inhibition of Notch1 signaling at the subacute stage post-stroke mediates neural repair by promoting astrocyte-derived neurogenesis.

Oscillating field stimulation promotes neurogenesis of neural stem cells through miR-124/Tal1 axis to repair spinal cord injury in rats

Spinal cord injury often leads to severe motor and sensory deficits,and prognosis using the currently available therapies remains poor.Therefore,we aimed to explore a novel therapeutic approach for improving the prognosis of spinal cord injury.In this study,we implanted oscillating field stimulation devices and transplanted neural stem cells into the thoracic region(T9–T10)of rats with a spinal cord contusion.Basso-Beattie-Bresnahan scoring revealed that oscillating field stimulation combined with neural stem cells transplantation promoted motor function recovery following spinal cord injury.In addition,we investigated the regulation of oscillating field stimulation on the miR-124/Tal1 axis in neural stem cells.Transfection of lentivirus was performed to investigate the role of Tal1 in neurogenesis of neural stem cells induced by oscillating field stimulation.Quantitative reverse transcription-polymerase chain reaction,immunofluorescence and western blotting showed that oscillating field stimulation promoted neurogenesis of neural stem cells in vitro and in vivo.Hematoxylin and eosin staining showed that oscillating field stimulation combined with neural stem cells transplantation alleviated cavities formation after spinal cord injury.Taking the results together,we concluded that oscillating field stimulation decreased miR-124 expression and increased Tal1 content,thereby promoting the neurogenesis of neural stem cells.The combination of oscillating field stimulation and neural stem cells transplantation improved neurogenesis,and thereby promoted structural and functional recovery after spinal cord injury.

Transient neurogenesis in ischemic cortex from Sox2^(+)astrocytes

The adult cortex has long been regarded as non-neurogenic.Whether injury can induce neurogenesis in the adult cortex is still controversial.Here,we report that focal ischemia stimulates a transient wave of local neurogenesis.Using 5′-bromo-2′-deoxyuridine labeling,we demonstrated a rapid generation of doublecortin-positive neuroblasts that died quickly in mouse cerebral cortex following ischemia.Nestin-Cre^(ER)-based cell ablation and fate mapping showed a small contribution of neuroblasts by subventricular zone neural stem cells.Using a mini-photothrombotic ischemia mouse model and retrovirus expressing green fluorescent protein labeling,we observed maturation of locally generated new neurons.Furthermore,fate tracing analyses using PDGFRα-,GFAP-,and Sox2-Cre^(ER) mice showed a transient wave of neuroblast generation in mild ischemic cortex and identified that Sox2-positive astrocytes were the major neurogenic cells in adult cortex.In addition,a similar upregulation of Sox2 and appearance of neuroblasts were observed in the focal ischemic cortex of Macaca mulatta.Our findings demonstrated a transient neurogenic response of Sox2-positive astrocytes in ischemic cortex,which suggests the possibility of inducing neuronal regeneration by amplifying this intrinsic response in the future.

Dysregulation of neurogenesis by neuroinflammation:key differences in neurodevelopmental and neurological disorders

Embryonic neurogenesis is the process of generating neurons,the functional units of the brain.Because of its sensitivity to adverse intrauterine environment such as infection,dysregulation of this process has emerged as a key mechanism underlying many neurodevelopmental disorders such as autism spectrum disorders（ASD）.Adult neurogenesis,although is restricted to a few neurogenic niches,plays pivotal roles in brain plasticity and repair.Increasing evidence suggests that impairments in adult neurogenesis are involved in major neurodegenerative disorders such as Alzheimer＇s disease.A hallmark feature of these brain disorders is neuroinflammation,which can either promote or inhibit neurogenesis depending upon the context of brain microenvironment.In this review paper,we present evidence from both experimental and human studies to show a complex picture of relationship between these two events,and discussed potential factors contributing to different or even opposing actions of neuroinflammation on neurogenesis in neurodevelopmental and neurological disorders.

Features of adult neurogenesis and neurochemical signaling in the Cherry salmon Oncorhynchus masou brain

We investigated the distribution of gamma aminobutyric acid, tyrosine hydroxylase and nitric oxide-producing elements in a cherry salmon Oncorhynchus masou brain at various stages of postnatal ontogenesis by immunohistochemical staining and histochemical staining. The periventricular region cells exhibited the morphology of neurons and glia including radial glia-like cells and contained several neurochemical substances. Heterogeneous populations of tyrosine hydroxylase-, gamma aminobutyric acid-immunoreactive, as well as nicotinamide adenine dinucleotide phosphate diaphorase-positive cells were observed in proliferating cell nuclear antigen-immunoreactive proliferative zones in periventricular area of diencephalon, central grey layer of dorsomedial tegmentum, medulla and spinal cord. Immunolocalization of Pax6 in the cherry salmon brain revealed a neuromeric construction of the brain at various stages of postnatal ontogenesis, and this was confirmed by tyrosine hydroxylase and gamma aminobutyric acid labeling.

Mesenchymal stem cell-derived exosomes promote neurogenesis and cognitive function recovery in a mouse model of Alzheimer’s disease

Studies have shown that mesenchymal stem cell-derived exosomes can enhance neural plasticity and improve cognitive impairment.The purpose of this study was to investigate the effects of mesenchymal stem cell-derived exosomes on neurogenesis and cognitive capacity in a mouse model of Alzheimer’s disease.Alzheimer’s disease mouse models were established by injection of beta amyloid 1?42 aggregates into dentate gyrus bilaterally.Morris water maze and novel object recognition tests were performed to evaluate mouse cognitive deficits at 14 and 28 days after administration.Afterwards,neurogenesis in the subventricular zone was determined by immunofluorescence using doublecortin and PSA-NCAM antibodies.Results showed that mesenchymal stem cells-derived exosomes stimulated neurogenesis in the subventricular zone and alleviated beta amyloid 1?42-induced cognitive impairment,and these effects are similar to those shown in the mesenchymal stem cells.These findings provide evidence to validate the possibility of developing cell-free therapeutic strategies for Alzheimer’s disease.All procedures and experiments were approved by Institutional Animal Care and Use Committee(CICUAL)(approval No.CICUAL 2016-011)on April 25,2016.

Adipose-derived mesenchymal stem cell transplantation promotes adult neurogenesis in the brains of Alzheimer's disease mice

In the present study, we transplanted adipose-derived mesenchymal stem cells into the hippo-campi of APP/PS1 transgenic Alzheimer＇s disease model mice. Immunofluorescence staining revealed that the number of newly generated （BrdU＋） cells in the subgranular zone of the dentate gyrus in the hippocampus was signiifcantly higher in Alzheimer＇s disease mice after adipose-de-rived mesenchymal stem cell transplantation, and there was also a significant increase in the number of BrdU＋/DCX＋neuroblasts in these animals. Adipose-derived mesenchymal stem cell transplantation enhanced neurogenic activity in the subventricular zone as well. Furthermore, adipose-derived mesenchymal stem cell transplantation reduced oxidative stress and alleviated cognitive impairment in the mice. Based on these ifndings, we propose that adipose-derived mes-enchymal stem cell transplantation enhances endogenous neurogenesis in both the subgranular and subventricular zones in APP/PS1 transgenic Alzheimer＇s disease mice, thereby facilitating functional recovery.