Adult neural stem cells in the mammalian central nervous system

Neural stem cells （NSCs） are present not only during the embryonic development but also in the adult brain of all mammalian species, including humans. Stem cell niche architecture in vivo enables adult NSCs to continuously generate functional neurons in specific brain regions throughout life. The adult neurogenesis process is subject to dynamic regulation by various physiological, pathological and pharmacological stimuli. Multipotent adult NSCs also appear to be intrinsically plastic, amenable to genetic programing during normal differentiation, and to epigenetic reprograming during de-differentiation into pluripotency. Increasing evidence suggests that adult NSCs significantly contribute to specialized neural functions under physiological and pathological conditions. Fully understanding the biology of adult NSCs will provide crucial insights into both the etiology and potential therapeutic interventions of major brain disorders. Here, we review recent progress on adult NSCs of the mammalian central nervous system, including topics on their identity, niche, function, plasticity, and emerging roles in cancer and regenerative medicine.

S6B-2 Matrine Inhibits Itching by Lowering the Activity of Calcium Channel

Sophorae Flavescentis Radix(SFR)is a medicinal herb with many functions that are involved in anti-inflammation,antinociception,and anticancer.SFR is also used to treat a variety of itching diseases.Matrine(MT)is one of the main constituents in SFR and also has the effect of relieving itching,but the antipruritic mechanism is still unclear.Here,we investigated the effect of MT on antipruritus.In acute and chronic itch models,MT significantly inhibited the scratching behavior not only in acute itching induced by histamine(His),chloroquine(CQ)and compound 48/80 with a dose-depended manner,but also in the chronic pruritus models of Atopic dermatitis(AD)and Acetone-ether-water(AEW)in mice.Furthermore,MT can be detected in the blood after intraperitoneal injection(i.p.)and subcutaneous injection(s.c.).Finally,electrophysiological and calcium image results show that MT inhibits the excitatory synaptic transmission from dorsal root ganglion(DRG)to the dorsal horn of the spinal cord by suppressing presynaptic N-type calcium channels.Taken together,we believe that MT is a novel drug candidate in treating pruritus diseases,especially for histamine-independent and chronic pruritus,which might be attributed to inhibition of presynaptic N-type calcium channels.

Locus coeruleus-norepinephrine: basic functions and insights into Parkinson’s disease

The locus coeruleus is a pontine nucleus that produces much of the brain's norepinephrine.Despite its small size,the locus coeruleus is critical for a myriad of functions and is involved in many neurodegenerative and neuropsychiatric disorders.In this review,we discuss the physiology and anatomy of the locus coeruleus system and focus on norepinephrine's role in synaptic plasticity.We highlight Parkinson's disease as a disorder with motor and neuropsychiatric symptoms that may be understood as aberrations in the normal functions of locus coeruleus.

Sialylation of TLR2 initiates osteoclast fusion

The molecular control of osteoclast formation is still not clearly elucidated. Here, we show that a process of cell recognition mediated by Siglec15-TLR2 binding is indispensable and occurs prior to cell fusion in RANKL-mediated osteoclastogenesis. Siglec15 has been shown to regulate osteoclastic bone resorption. However, the receptor for Siglec15 has not been identified, and the signaling mechanism involving Siglec15 in osteoclast function remains unclear. We found that Siglec15 bound sialylated TLR2 as its receptor and that the binding of sialylated TLR2 to Siglec15 in macrophages committed to the osteoclast-lineage initiated cell fusion for osteoclast formation, in which sialic acid was transferred by the sialyltransferase ST3 Gal1. Interestingly, the expression of Siglec15 in macrophages was activated by M-CSF, whereas ST3 Gal1 expression was induced by RANKL. Both Siglec15-specific deletion in macrophages and intrafemoral injection of sialidase abrogated cell recognition and reduced subsequent cell fusion for the formation of osteoclasts, resulting in increased bone formation in mice. Thus, our results reveal that cell recognition mediated by the binding of sialylated TLR2 to Siglec15 initiates cell fusion for osteoclast formation.

Engineering human pluripotent stem cell-derived 3D brain tissues for drug discovery

The quest to find novel therapeutics for mental and neurological disorders has been hindered by the lack of access to l ive human brain samples and relevant experimental models. Conventional 2D human pluripotent stem cell-derived neuronal cultures and animal models do not ful ly recapitulate many endogenous human biochemical processes and disease phenotypes. Currently, the majority of candidate drugs obtained from preclinical testing in conventional systems does not usually translate into success and have a high failure rate in clinical trials. Recent advancements in bioengineering and stem cell technologies have resulted in three-dimensional brain-like tissues, such as oragnoids, which better resemble endogenous tissue and are more physiologically relevant than monolayer cultures. These brain-like tissues can bridge the gap between existing models and the patient, and may revolutionize the field of translational neuroscience. Here, we discuss utilities and challenges of using stem cell-derived human brain tissues in basic research and pharmacotherapy.

Tbr2-expressing intermediate progenitor cells in the adult mouse hippocampus are unipotent neuronal precursors with limited amplification capacity under homeostasis

Neurogenesis persists in two locations of the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus. In the adult subgranular zone, radial glial- like cells （RGLs） are multipotent stem cells that can give rise to both astrocytes and neurons. In the process of generating neurons, RGLs divide asymmetrically to give rise to one RGL and one intermediate progenitor cell （IPC）. IPCs are considered to be a population of transit amplifying cells that proliferate and eventually give rise to mature granule neurons. The properties of individual IPCs at the clonai level are not well understood. Furthermore, it is not clear whether IPCs can generate astrocytes or revert back to RGLs, besides generating neurons. Here we developed a genetic marking strategy for clonal analysis and lineage-tracing of individual Tbr2-expressing IPCs in the adult hippocampus in vivo using Tbr2-CreERT2 mice. Using this technique we identified Tbr2-CreERT2 labeled IPCs as unipotent neuronal precursors that do not generate astrocytes or RGLs under homeostasis. Additionally, we showed that these labeled IPCs rapidly generate immature neurons in a synchronous manner and do not undergo a significant amount of amplification under homeostasis, in animals subjected to an enriched environment/running, or in animals with different age. In summary, our study suggests that Tbr2-expressing IPCs in the adult mouse hippocampus are unipotent precursors and rapidly give rise to immature neurons without major amplification.

Genome-wide antagonism between 5-hydroxymethylcytosine and DNA methylation in the adult mouse brain

Mounting evidence points to critical roles for DNA modifications, including 5-methylcytosine （5mC） and its oxidized forms, in the development, plasticity and disorders of the mammalian nervous system. The novel DNA base 5- hydroxymethylcytosine （5hmC） is known to be capable of initiating passive or active DNA demethylation, but whether and how extensively 5hmC functions in shaping the post-mitotic neuronal DNA methylome is unclear. Here we report the genome-wide distribution of 5hmC in dentate granule neurons from adult mouse hippocampus in vivo. 5hmC in the neuronal genome is highly enriched in gene bodies, especially in exons, and correlates with gene expression. Direct genome-wide comparison of 5hmC distribution between embryonic stem cells and neurons reveals extensive differences, reflecting the functional disparity between these two cell types. Importantly, integrative analysis of 5hmC, overall DNA methylation and gene expression profiles of dentate granule neurons in vivo reveals the genome-wide antagonism between these two states of cytosine modifications, supporting a role for 5hmC in shaping the neuronal DNA methylome by promoting active DNA demethylation.

Application of reprogrammed patient cells to investigate the etiology of neurological and psychiatric disorders

Cellular reprogramming allows for the de novo generation of human neurons and glial cells from patients with neurological and psychiatric disorders. Crucially, this technology preserves the genome of the donor individual and thus provides a unique opportunity for systematic investigation of genetic influences on neuronal pathophysiology. Although direct reprogramming of adult somatic cells to neurons is now possible, the majority of recent studies have used induced pluripotent stem cells （iPSCs） derived from patient fibroblasts to generate neural progenitors that can be differentiated to specific neural cell types. Investigations of monogenic diseases have established proof-of-principle for many aspects of cellular disease modeling, including targeted differentiation of neuronal populations and rescue of phenotypes in patient iPSC lines. Refinement of protocols to allow for efficient generation of iPSC lines from large patient cohorts may reveal common functional pathology and genetic interactions in diseases with a polygenic basis. We review several recent studies that illustrate the utility of iPSC-based cellular models of neurodevelopmental and neurodegenerative disorders to identify novel phenotypes and therapeutic approaches.

Identification of Persister Drug Combination Clinafloxacin+Cefuroxime+Gentamicin That Eradicates Persistent Pseudomonas aeruginosa Infection in a Murine Cystic Fibrosis Model

Pseudomonas aeruginosa can cause persistent infections,such as biofilm infections,in cystic fibrosis patients,which are difficult to cure due to non-growing persister bacteria that are not effectively killed by the current treatments.While antibiotic activity against growing P.aeruginosa is well documented,their activity against non-growing stationary phase cultures is less clear.Here,we evaluated six major classes of antibiotics,including cell wall and cell membrane inhibitors,protein synthesis inhibitors,DNA synthesis inhibitors,RNA synthesis inhibitors,sulfa drugs and nitrofurantoin,for their activity against growing and non-growing P.aeruginosa.We foundthat cell wall and cell membrane inhibitors(cefuroxime and colistin),DNA synthesis inhibitors(clinafloxacin)and sulfa drugs(sulfamethoxazole)had good activity against stationary-phase bacteria,while protein synthesis inhibitors(gentamicin),RNA synthesis inhibitor(rifampin)and nitrofurantoin showed relatively poor activity.Clinafloxacin was the only drug able to completely eradicate stationary-phase bacteria within four days.The cefuroxime+gentamicin+clinafloxacin combination was able to kill all bacteria from a biofilm within two days,whereas the clinically used drug combination cefuroxime+gentamicin/colistin only partially killed the biofilmbacteria.In amurine persistent cystic fibrosis lung infectionmodel,only the cefuroxime+gentamicin+clinafloxacin drug combination eradicated all bacteria from the lungs,whereas clinafloxacin alone,cefuroxime+clinafloxacin or the currently recommended drug combination cefuroxime+gentamicin failed to do so.The complete eradication is a property of the clinafloxacin combination,as the otherwise identical levofloxacin combination did not clear the bacterial loads from the lungs.Our findings offer new therapeutic options for more effective treatment of persistent P.aeruginosa infections,with possible implications for treating other persistent infections.

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.

Investigation of Pain Mechanisms by Calcium Imaging Approaches

Due to the complex circuitry and plethora of cell types involved in somatosensation, it is becoming increasingly important to be able to observe cellular activity at the population level. In addition, since cells rely on an intricate variety of extracellular factors, it is important to strive to maintain the physiological environment. Many electrophysiological techniques require the implementation of artificially-produced physiological environments and it can be difficult to assess the activity of many cells simultane- ously. Moreover, imaging Ca^2＋ transients using Ca^2＋- sensitive dyes often requires in vitro preparations or in vivo injections, which can lead to variable expression levels. With the development of more sensitive geneticallyencoded Ca^2＋ indicators （GECIs） it is now possible to observe changes in Ca^2＋ transients in large populations of cells at the same time. Recently, groups have used a GECI called GCaMP to address fundamental questions in somatosensation. Researchers can now induce GCaMP expression in the mouse genome using viral or gene knock- in approaches and observe the activity of populations of cells in the pain pathway such as dorsal root ganglia （DRG）, spinal neurons, or glia. This approach can be used in vivo and thus maintains the organism＇s biological integrity. The implementation of GCaMP imaging has led to many advances in our understanding of somatosensation. Here, we review the current findings in pain research using GCaMP imaging as well as discussing potential method- ological considerations.

Signaling pathways that regulate axon regeneration

Neurons in the mammalian central nervous system （CNS） cannot regenerate axons after injury. In contrast, neurons in the mammalian peripheral nervous system and in some non-mammalian models, such as C. elegans and Drosophila, are able to regrow axons. Understanding the molecular mechanisms by which these neurons support axon regeneration will help us find ways to enhance mammalian CNS axon regeneration. Here, recent studies in which signaling pathways regulating naturally-occurring axon regeneration that have been identified are reviewed, focusing on how these pathways control gene expression and growth-cone function during axon regeneration.

Peripheral mechanisms of itch

Detection of environmental stimuli that provoke an aversive response has been shown to involve many receptors in the periphery. Probably the least-studied of these stimuli are those that induce the perception of itch （pruritus）, an often-experienced unpleasant stimulus. This review covers the ligands and their receptors which are known to cause primary sensory neuron activation and initiate itch sensation. Also covered are several itch-inducing substances which may act indirectly by activating other cell types in the periphery which then signal to primary neurons. Finally, progress in identifying candidate neurotransmitters that sensory neurons use to propagate the itch signal is discussed.

BDNF rescues prefrontal dysfunction elicited by pyramidal neuron-specific DTNBP1 deletion in vivo

Dystrobrevin-binding protein 1 （Dtnbp1） is one of the earliest identified schizophrenia susceptibility genes. Reduced expression of DTNBP1 is commonly found in brain areas of schizophrenic patients. Dtnbp1-nuU mutant mice exhibit abnormalities in beha- viors and impairments in neuronal activities. However, how diminished DTNBP1 expression contributes to clinical relevant fea- tures of schizophrenia remains to be illustrated. Here, using a conditional Dtnbp1 knockout mouse line, we identified an in vivo schizophrenia-relevant function of DTNBP1 in pyramidal neurons of the medial prefrontal cortex （mPFC）. We demonstrated that DTNBP1 elimination specifically in pyramidal neurons of the mPFC impaired mouse pre-pu[se inhibition （PPI） behavior and reduced perisomatic GABAergic synapses. We further revealed that loss of DTNBP1 in pyramidal neurons diminished activity- dependent secretion of brain-derived neurotrophic factor （BDNF）. Finally, we showed that chronic BDNF infusion in the mPFC fully rescued both GABAergic synaptic dysfunction and PPI behavioral deficit induced by DTNBP1 elimination from pyramidal neurons. Our findings highlight brain region- and cell type-specific functions of DTNBP1 in the pathogenesis of schizophrenia, and under- score BDNF restoration as a potential therapeutic strategy for schizophrenia.

Peripheral mechanisms of chronic pain

Acutely,pain serves to protect us from potentially harmful stimuli,however damage to the somatosensory system can cause maladaptive changes in neurons leading to chronic pain.Although acute pain is fairly well controlled,chronic pain remains difficult to treat.Chronic pain is primarily a neuropathic condition,but studies examining the mechanisms underlying chronic pain are now looking beyond afferent nerve lesions and exploring new receptor targets,immune cells,and the role of the autonomic nervous system in contributing chronic pain conditions.The studies outlined in this review reveal how chronic pain is not only confined to alterations in the nervous system and presents findings on new treatment targets and for this debilitating disease.

N6-methyladenine DNA Demethylase ALKBH1 Regulates Mammalian Axon Regeneration

Dear Editor,DNA methylation is a key epigenetic regulatory approach for many biological processes,such as genomic imprinting,epigenetic memory maintenance,aging,and neural development.In addition to 5-methylcytosine,DNA methylation at N6-deoxyadenosine(N6-mA)is the most prevalent DNA modification in prokaryotes[1].

Transcription Factor MAFA Regulates Mechanical Sensation by Modulating Piezo2 Expression

Dear Editor,Essential for survival,mechanical sensation is detected and transmited to the spinal cord by primary sensory afferents and their cell bodies located in the dorsal root ganglia(DRG).Multiple types of mechanically sensitive DRG neurons have been identified that mediate various mechanosensation modalities(e.g.non-painful versus painful).