Gualou Guizhi decoction promotes neurological functional recovery and neurogenesis following focal cerebral ischemia/reperfusion

Recovery following stroke involves neurogenesis and axonal remodeling within the ischemic brain. Gualou Guizhi decoction （GLGZD） is a Chinese traditional medicine used for the treatment of post-stroke limb spasm. GLGZD has been reported to have neuroprotective effects in cerebral ischemic injury. However, the effects of GLGZD on neurogenesis and axonal remodeling following cerebral ischemia remain unknown. In this study, a rat model of focal cerebral ischemia/reperfusion was established by middle cerebral artery occlusion. Neurologi- cal function was assessed immediately after reperfusion using Longa＇s 5-point scoring system. The rats were randomly divided into vehicle and GLGZD groups. Rats in the sham group were given sham operation. The rats in the GLGZD group were intragastrically administered GLGZD, once daily, for 14 consecutive days. The rats in the vehicle and sham groups were intragastrically administered distilled water. Modified neurological severity score test, balance beam test and foot fault test were used to assess motor functional changes. Nissl staining was performed to evaluate histopathological changes in the brain. Immunofluorescence staining was used to examine cell proliferation using the marker 5-bromo-2＇-deoxyuridine （BrdU） as well as expression of the neural precursor marker doublecortin （DCX）, the astrocyte marker glial fibrillary acidic protein （GFAP） and the axon regeneration marker growth associated protein-43 （GAP-43）. GLGZD substan- tially mitigated pathological injury, increased the number of BrdU, DCX and GFAP-immunoreactive cells in the subventricular zone of the ischemic hemisphere, increased GAP-43 expression in the cortical peri-infarct region, and improved motor function. These findings suggest that GLGZD promotes neurological functional recovery by increasing cell proliferation, enhancing axonal regeneration, and in- creasing the numbers of neuronal precursors and astrocytes in the peri-infarct area.

Three-dimensional structure of axonal mitochondria reflects the age of drosophila

This study aimed to reconstruct a three-dimensional map of axonal mitochondria using Fiji and Neurolucida software, and to observe directly the morphology and distribution of mitochondria in axons of motor neurons in dorsal longitudinal flight muscles of drosophila aged 5 days and 20 days, using electron microscopy. Results indicated that there was no difference in the total area and volume of mitochondria between 5-day-old drosophila and 20-day-old drosophila in all sections, but the ratio of mitochondrial total areas to axon total areas, as well as mitochondrial density of 20-day-old drosophila, was lower than that of 5-day-old drosophila. The number of mitochondria, whose volume was less than 1 000 000 IJm3, and between 1 000 000 pm3 and 10 000 000 pm3, was higher in 20-day-old drosophila than that in 5-day-old drosophila. The number of mitochondria with a volume between 1 000 000 pm3 and 100 000 000 IJm3 was apparently higher than those with a volume less than 1 000 000 t.lm3 or larger than 100 000 000 IJm3. In addition, the number of mitochondria with a volume more than 100 000 000 tJm3 was small; however, the volume was nearly 70% of the total volume in both 5-day-old and 20-day-old drosophila. In contrast, the number of mitochondria with a volume between 1 000 000 t.Jm3 and 10 000 000 IJm3 was large, but the volume was less than 30% of the total volume. These experimental findings suggest that changes in mitochondrial morphology and number in motor neurons from the dorsal longitudinal muscle of drosophila are present during different ages.

Transient axonal glycoprotein-1 induces apoptosisrelated gene expression without triggering apoptosis in U251 glioma cells

Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor pro- tein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer＇s disease. In this study, we examined the effects of transient axonal glyco- protein-1 on U251 glioma cells. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor recep- tor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.

淀粉样前体蛋白胞内结构域对阿尔茨海默病模型小鼠神经发生和学习记忆的影响

【目的】探讨淀粉样前体蛋白胞内结构域(AICD)对阿尔茨海默病(AD)模型动物神经发生、学习记忆的影响。【方法】本研究使用免疫荧光染色检测AICD转基因小鼠来源的体外培养的神经前体细胞(NPCs)、胚胎大脑皮质、成年海马齿状回(DG)中增殖和分化的细胞数目;水迷宫实验检测老年AICD转基因小鼠对学习记忆能力影响;生物信息学预测和分析潜在的分子机制。【结果】免疫荧光染色结果显示AICD转基因模型体外NPCs、胚胎皮质、海马DG区域的神经干细胞和神经元数量减少(P<0.05),即AICD抑制不同时期AD模型小鼠的神经发生。水迷宫结果显示AICD增加AD模型小鼠逃逸潜伏期,减少其跨越平台次数,并减少DG区域神经元数目(P<0.05)。生物信息学结果显示,AICD参与调节AD发病进程中神经发生和学习记忆的靶点有1723个,关键靶点有TP53、CTNNB1、Akt1、EGFR、SRC、EP300、HDAC1、STAT3、HSP90AA1和MAPK1;另外,KEGG通路注释分析发现PI3KAkt、HIF-1等信号通路在AICD调节神经发生和学习记忆起关键作用。【结论】表明AICD可以抑制AD模型小鼠海马神经发生进而损害学习记忆能力,这可能与PI3K-Akt和HIF-1等信号通路有关。本研究为进一步理解AICD在AD发病进程中作用提供实验依据。

Angiogenesis and neuronal remodeling after ischemic stroke

Increased microvessel density in the peri-infarct region has been reported and has been correlated with longer survival times in ischemic stroke patients and has improved outcomes in ischemic animal models.This raises the possibility that enhancement of angiogenesis is one of the strategies to facilitate functional recovery after ischemic stroke.Blood vessels and neuronal cells communicate with each other using various mediators and contribute to the pathophysiology of cerebral ischemia as a unit.In this mini-review,we discuss how angiogenesis might couple with axonal outgrowth/neurogenesis and work for functional recovery after cerebral ischemia.Angiogenesis occurs within 4 to 7 days after cerebral ischemia in the border of the ischemic core and periphery.Post-ischemic angiogenesis may contribute to neuronal remodeling in at least two ways and is thought to contribute to functional recovery.First,new blood vessels that are formed after ischemia are thought to have a role in the guidance of sprouting axons by vascular endothelial growth factor and laminin/β1-integrin signaling.Second,blood vessels are thought to enhance neurogenesis in three stages:1)Blood vessels enhance proliferation of neural stem/progenitor cells by expression of several extracellular signals,2)microvessels support the migration of neural stem/progenitor cells toward the peri-infarct region by supplying oxygen,nutrients,and soluble factors as well as serving as a scaffold for migration,and 3)oxygenation induced by angiogenesis in the ischemic core is thought to facilitate the differentiation of migrated neural stem/progenitor cells into mature neurons.Thus,the regions of angiogenesis and surrounding tissue may be coupled,representing novel treatment targets.

Neuroprotective role of Noggin in spinal cord injury

Spinal cord injury is one of the leading causes of morbidity and mortality among young adults in many countries including the United States.Difficulty in the regeneration of neurons is one of the main obstacles that leave spinal cord injury patients with permanent paralysis in most instances.Recent research has found that preventing acute and subacute secondary cellular damages to the neurons and supporting glial cells can help slow the progression of spinal cord injury pathogenesis,in part by reactivating endogenous regenerative proteins including Noggin that are normally present during spinal cord development.Noggin is a complex protein and natural inhibitor of the multifunctional bone morphogenetic proteins,and its expression is high during spinal cord development and after induction of spinal cord injury.In this review article,we first discuss the change in expression of Noggin during pathogenesis in spinal cord injury.Second,we discuss the current research knowledge about the neuroprotective role of Noggin in preclinical models of spinal cord injury.Lastly,we explain the gap in the knowledge for the use of Noggin in the treatment of spinal cord injury.The results from extensive in vitro and in vivo research have revealed that the therapeutic efficacy of Noggin treatment remains debatable due to its neuroprotective effects observed only in early phases of spinal cord injury but little to no effect on altering pathogenesis and functional recovery observed in the chronic phase of spinal cord injury.Furthermore,clinical information regarding the role of Noggin in the alleviation of progression of pathogenesis,its therapeutic efficacy,bioavailability,and safety in human spinal cord injury is still lacking and therefore needs further investigation.

极性蛋白与中枢神经系统发育

哺乳动物大脑神经元的形态多样性和突触连接的复杂性是极性细胞的典型例子,形成和维持神经元极性依赖多种极性蛋白的调节。从线虫受精卵发育到哺乳动物神经细胞的极性化通路中,许多极性蛋白存在进化保守机制。中枢神经系统发育的整个过程(包括神经元发生与移行、神经突生长以及突触联系的形成等)都有极性蛋白的直接或间接参与,是各种极性蛋白相互作用/相互制约的动态过程。

Physiological effects of amyloid precursor protein and its derivatives on neural stem cell biology and signaling pathways involved

The pathological implication of amyloid precursor protein(APP)in Alzheimer’s disease has been widely documented due to its involvement in the generation of amyloid-β peptide.However,the physiological functions of APP are still poorly understood.APP is considered a multimodal protein due to its role in a wide variety of processes,both in the embryo and in the adult brain.Specifically,APP seems to play a key role in the proliferation,differentiation and maturation of neural stem cells.In addition,APP can be processed through two canonical processing pathways,generating different functionally active fragments:soluble APP-α,soluble APP-β,amyloid-β peptide and the APP intracellular C-terminal domain.These fragments also appear to modulate various functions in neural stem cells,including the processes of proliferation,neurogenesis,gliogenesis or cell death.However,the molecular mechanisms involved in these effects are still unclear.In this review,we summarize the physiological functions of APP and its main proteolytic derivatives in neural stem cells,as well as the possible signaling pathways that could be implicated in these effects.The knowledge of these functions and signaling pathways involved in the onset or during the development of Alzheimer’s disease is essential to advance the understanding of the pathogenesis of Alzheimer’s disease,and in the search for potential therapeutic targets.

Contactins in the central nervous system: role in health and disease

Contactins are a group of cell adhesion molecules that are mainly expressed in the brain and play pivotal roles in the organization of axonal domains, axonal guidance, neuritogenesis, neuronal development, synapse formation and plasticity, axo-glia interactions and neural regeneration. Contactins comprise a family of six members. Their absence leads to malformed axons and impaired nerve conduction. Contactin mediated protein complex formation is critical for the organization of the axon in early central nervous system development. Mutations and differential expression of contactins have been identified in neuro-developmental or neurological disorders. Taken together, contactins are extensively studied in the context of nervous system development. This review summarizes the physiological roles of all six members of the Contactin family in neurodevelopment as well as their involvement in neurological/neurodevelopmental disorders.

A Disintegrin and Metalloprotease 10 in neuronal maturation and gliogenesis during cortex development

The multiple-layer structure of the cerebral cortex is important for its functions. Such a structure is generated based on the proliferation and differentiation of neural stem/progenitor cells. Notch functions as a molecular switch for neural stem/progenitor cell fate during cortex development but the mechanism remains unclear. Biochemical and cellular studies showed that Notch receptor activation induces several proteases to release the Notch intracellular domain （NICD）. A Disintegrin and Metalloprotease 10 （ADAM10） might be a physiological rate-limiting $2 enzyme for Notch activation. Nestin-driven conditional ADAM10 knockout in mouse cortex showed that ADAM10 is cdtical for maintenance of the neural stem cell population during early embryonic cortex development. However, the expression pattern and function of ADAM10 during later cerebral cortex development remains poorly understood. We performed in situ hybridization for ADAMIO mRNA and immunofluorescent analysis to determine the expression of ADAM10 and NICD in mouse cortex from embryonic day 9 （E14.5） to postnatal day 1 （P1）. ADAM10 and NICD were highly co-localized in the cortex of E16.5 to P1 mice. Comparisons of expression patterns of ADAM10 with Nestin （neural stem cell marker）, Tujl （mature neuron marker）, and S100β （gila marker） showed that ADAM10 expression highly matched that of S10013 and partially matched that of Tujl at later embryonic to early postnatal cortex developmental stages. Such expression patterns indicated that ADAM10-Notch signaling might have a critical function in neuronal maturation and gliogenesis during cortex development.

Nerve bundle formation during the promotion of peripheral nerve regeneration:collagenⅥ-neural cell adhesion molecule 1 interaction

The formation of nerve bundles,which is partially regulated by neural cell adhesion molecule 1(NCAM1),is important for neural network organization during peripheral nerve regeneration.However,little is known about how the extracellular matrix(ECM)microenvironment affects this process.Here,we seeded dorsal root ganglion tissue blocks on different ECM substrates of peripheral nerve ECM-derived matrixgel,Matrigel,laminin 521,collagen I,and collagen IV,and observed well-aligned axon bundles growing in the peripheral nerve ECM-derived environment.We confirmed that NCAM1 is necessary but not sufficient to trigger this phenomenon.A protein interaction assay identified collagen VI as an extracellular partner of NCAM1 in the regulation of axonal fasciculation.Collagen VI interacted with NCAM1 by directly binding to the FNIII domain,thereby increasing the stability of NCAM1 at the axolemma.Our in vivo experiments on a rat sciatic nerve defect model also demonstrated orderly nerve bundle regeneration with improved projection accuracy and functional recovery after treatment with 10 mg/m L Matrigel and 20μg/m L collagen VI.These findings suggest that the collagen VI-NCAM1 pathway plays a regulatory role in nerve bundle formation.This study was approved by the Animal Ethics Committee of Guangzhou Medical University(approval No.GY2019048)on April 30,2019.

Identification of key genes involved in recovery from spinal cord injury in adult zebrafish

Zebrafish are an effective vertebrate model to study the mechanisms underlying recovery after spinal cord injury.The subacute phase after spinal cord injury is critical to the recovery of neurological function,which involves tissue bridging and axon regeneration.In this study,we found that zebrafish spontaneously recovered 44%of their swimming ability within the subacute phase(2 weeks)after spinal cord injury.During this period,we identified 7762 differentially expressed genes in spinal cord tissue:2950 were up-regulated and 4812 were down-regulated.These differentially expressed genes were primarily concentrated in the biological processes of the respiratory chain,axon regeneration,and cell-component morphogenesis.The genes were also mostly involved in the regulation of metabolic pathways,the cell cycle,and gene-regulation pathways.We verified the gene expression of two differentially expressed genes,clasp2 up-regulation and h1m down-regulation,in zebrafish spinal cord tissue in vitro.Pathway enrichment analysis revealed that up-regulated clasp2 functions similarly to microtubule-associated protein,which is responsible for axon extension regulated by microtubules.Down-regulated h1m controls endogenous stem cell differentiation after spinal cord injury.This study provides new candidate genes,clasp2 and h1m,as potential therapeutic intervention targets for spinal cord injury repair by neuroregeneration.All experimental procedures and protocols were approved by the Animal Ethics Committee of Tianjin Institute of Medical&Pharmaceutical Sciences(approval No.IMPS-EAEP-Q-2019-02)on September 24,2019.

Modulating Wnt signaling to improve cell replacement therapy for Parkinson＇s disease

Clinical trials have demonstrated the capacity for dopamine neurons, transplanted ectopicaUy into the striatum, to structurally inte- grate, restore dopamine transmission, and induce long-term functional benefits for Parkinson＇s disease （PD） patients. Despite this proof of principle, a number of limitations have hindered the development of cell replacement therapy over the past 20 years, particu- larly tissue availability, graft survival, and adequate reinnervation of the host brain. With a greater understanding of failure in prior clinical trials, increased knowledge of midbrain dopamine development （now including Wnts）, and the development of pluripotent stem cell technologies, we are better equipped than ever to re-address a number of these challenges. This review summarizes the trials, tribulations, and progress in cell replacement therapy for PD. We discuss the prospects of modulating canonical and non-canon- ical Wnt signalingto improve cell therapy based upon their roles in dopamine neural development and the adult brain. This will include the potential of Wnts to （i） expand fetaUy derived tissue in vitro and foUowing transplantation, （ii） promote the differentiation of pluripotent stem cells, （iii） increase graft integration and restoration of neural circuitry, and finally （iv） enhance graft survival.

TAT-NEP1-40——神经再生的新型药物

在受损的中枢神经系统中,Nogo-A蛋白、髓鞘蛋白和少突髓鞘蛋白是抑制中枢神经轴突再生的主要物质,它们通过一个共同受体——Nogo蛋白受体(Nogo receptor,NgR)介导中枢神经轴突抑制。NEP1-40是NgR受体的竞争性抑制剂,是治疗中枢神经损伤是一个具有潜在性的候选药物。TAT蛋白质转导序列是HIV反式转录激活因子,是目前已知具有有效蛋白质转导功能的序列。利用蛋白质重组技术构建并表达的含有TAT结构域和NEP1-40肽的融合蛋白质TAT-NEP1-40可能成为一种治疗中枢神经系统损伤如中风、脑缺氧、脑出血、脑外伤和脊髓损伤的新颖的候选药物。

Regeneration strategies after the adult mammalian central nervous system injury—biomaterials

The central nervous system(CNS)has very restricted intrinsic regeneration ability under the injury or disease condition.Innovative repair strategies,therefore,are urgently needed to facilitate tissue regeneration and functional recovery.The published tissue repair/regeneration strategies,such as cell and/or drug delivery,has been demonstrated to have some therapeutic effects on experimental animal models,but can hardly find clinical applications due to such methods as the extremely low survival rate of transplanted cells,difficulty in integrating with the host or restriction of blood-brain barriers to administration patterns.Using biomaterials can not only increase the survival rate of grafts and their integration with the host in the injured CNS area,but also sustainably deliver bioproducts to the local injured area,thus improving the microenvironment in that area.This review mainly introduces the advances of various strategies concerning facilitating CNS regeneration.

TAG-1对U251细胞活力的影响及相关基因调节机制研究

目的探讨TAG-1对U251细胞的生长活力和粉样前体蛋白胞内段（AICD）,p53和表皮生长因子受体（EGFR）~因表达变化的影响。方法以MTT法检测不同浓度TAG．1（0、5、10、20μg／mL）对U251细胞活力影响；免疫荧光细胞化学法观察U251细胞8淀粉样前体蛋白（APP）的表达；TUNEL法检测TAG-1对U251细胞凋亡形态学变化的影响：Real-time PCR检测TAG-1对U251细胞AICD,p53和EGFR基因表达的调控。结果TAG．1没有抑制U251细胞的生长．相反表现出一定的促生长效应；APP广泛表达于U251细胞膜；在TAG．1浓度为10μg/mL时，U251细胞形态学检测没有发现明显的凋亡细胞，但AICD,p53和EGFR基因表达增加。结论TAG-1在胶质瘤的增殖中发挥重要作用，但未发现其能通过TAG-1／APP／AICD／p53或TAG-1／APP，AJCD／EGFR信号途径促进U251细胞凋亡。

Neuron:新研究为从根源上治疗神经退行性疾病提供线索

对于许多神经退行性疾病来说。比如帕金森病、肌萎缩侧索硬化和外周神经病变，轴突的损失是一个早期的缺陷。当轴突出现损失。神经细胞就无法正常交流，神经系统功能受到损伤。特别是对外周神经病变来说，受损的轴突会触发自毁程序。在一项新研究中。华盛顿大学医学院的科学家们在自毁的轴突中发现一个特殊分子。深入了解损伤如何发生有助于帮助找到阻止其发生的方法。相关研究结果发表在国际学术期刊Neuron上。