Conditional Deletion of Foxgl Alleviates Demyelination and Facilitates Remyelination via the Wnt Signaling Pathway in Cuprizone-Induced Demyelinated Mice

The massive loss of oligodendrocytes caused by various pathological factors is a basic feature of many demyelinating diseases of the central nervous system(CNS)...     

Pinocembrin Promotes OPC Differentiation and Remyelination via the mTOR Signaling Pathway

The exacerbation of progressive multiple sclerosis (MS) is closely associated with obstruction of the differentiation of oligodendrocyte progenitor cells (OPCs). To discover novel therapeutic compounds for enhancing remyelination by endogenous OPCs, we screened for myelin basic protein expression using cultured rat OPCs and a library of small-molecule compounds. One of the most effective drugs was pinocembrin, which remarkably promoted OPC differentiation and maturation without affecting cell proliferation and survival. Based on these in vitro effects, we further assessed the therapeutic effects of pinocembrin in animal models of demyelinating diseases. We demonstrated that pinocembrin significantly ameliorated the progression of experimental autoimmune encephalomyelitis (EAE) and enhanced the repair of demyelination in lysolectin-induced lesions. Further studies indicated that pinocembrin increased the phosphorylation level of mammalian target of rapamycin (mTOR). Taken together, our results demonstrated that pinocembrin promotes OPC differentiation and remyelination through the phosphorylated mTOR pathway, and suggest a novel therapeutic prospect for this natural flavonoid product in treating demyelinating diseases.     

CNTF promotes the survival and differentiation of adult spinal cord-derived oligodendrocyte precursor cells in vitro but fails to promote remyelination in vivo

Delivery of factors capable of promoting oligodendrocyte precursor cell (OPC) survival and differentiation in vivo is an important therapeutic strategy for a variety of pathologies in which demyelination is a component, including multiple sclerosis and spinal cord injury. Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine that promotes both survival and maturation of a variety of neuronal and glial cell populations, including oligodendrocytes. Present results suggest that, although CNTF has a potent survival and differentiation promoting effect in vitro on OPCs isolated from the adult spinal cord, CNTF administration in vivo is not sufficient to promote oligodendrocyte remyelination in the glial-depleted environment of unilateral ethidium bromide (EB) lesions.     

局灶性脑缺血模型大鼠转录因子Olig1的表达变化及其在髓鞘修复中的作用

目的探讨Olig1在局灶性脑缺血后不同时间点的表达变化及其参与髓鞘修复的可能机制。方法线栓法建立大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型,使用免疫组化和Western blot的方法观察脑缺血后1d、3d、7d、14d和28d Olig1的表达变化。碱性髓鞘蛋白(myelin basic protein,MBP)是髓鞘的组成成分,用作髓鞘的标志物,检测脑缺血后上述各时间点MBP的表达变化。结果正常大鼠Olig1广泛分布于脑白质和灰质,如皮层、胼胝体和纹状体等区域,典型地沿着神经纤维成束状排列。正常大鼠Olig1主要位于少突胶质细胞的胞浆,脑缺血后Olig1由胞浆移至细胞核。Olig1的表达在脑缺血后1d下降,3d回到正常水平并维持此水平到脑缺血后28d。MBP蛋白表达在脑缺血后1d至28d逐渐下降。结论 Olig1在脑缺血的初期表达下降,抑制了神经的再生,针对Olig1的表达规律进行干预将为脑缺血后髓鞘的修复提供理论依据。     

Remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the MHV model of multiple sclerosis

The behavior and myelinogenic properties of glial cells have been well documented following transplantation into regions of focal experimental demyelination in animal models. However, the ability of glial cell preparations to remyelinate in such models does not necessarily indicate that their transplantation into demyelinated lesions in clinical disease will be successful. One of the precluding factors in this regard is a greater understanding of the environmental conditions that will support transplant-mediated remyelination. In this study, we determined whether the complex and reactive CNS environment of the mouse hepatitis virus (MHV) model of multiple sclerosis (MS) could support transplant-mediated remyelination. Striatal neural precursors derived from postnatal day 1 mice were committed to a glial cell lineage and labeled. Immunohistochemical staining indicated that this population generated >93% glial cells following differentiation in vitro. Transplantation of glial-committed progenitor cells into the T8 spinal cord of MHV-infected mice demonstrating complete hindlimb paralysis resulted in migration of cells up to 12 mm from the implantation site and remyelination of up to 67% of axons. Transplanted-remyelinated animals contained approximately 2x the number of axons within sampled regions of the ventral and lateral columns as compared to non-transplanted animals, suggesting that remyelination is associated with axonal sparing. Furthermore, transplantation resulted in behavioral improvement. This study demonstrates for the first time that transplant-mediated remyelination is possible in the pathogenic environment of the MHV demyelination model and that it is associated with locomotor improvement.     

FOXG1 Directly Suppresses Wnt5a During the Development of the Hippocampus

The Wnt signaling pathway plays key roles in various developmental processes.Wnt5a,which activates the non-canonical pathway,has been shown to be particularly important for axon guidance and outgrowth as well as dendrite morphogenesis.However,the mechanism underlying the regulation of Wnt5a remains unclear.Here,through conditional disruption of Foxg1 in hippocampal progenitors and postmitotic neurons achieved by crossing Foxg1~(fl/fl)with Emx1-Cre and Nex-Cre,respectively,we found that Wnt5a rather than Wnt3a/Wnt2b was markedly upregulated.Overexpression of Foxg1 had the opposite effects along with decreased dendritic complexity and reduced mossy fibers in the hippocampus.We further demonstrated that FOXG1 directly repressed Wnt5a by binding to its promoter and one enhancer site.These results expand our knowledge of the interaction between Foxg1 and Wnt signaling and help elucidate the mechanisms underlying hippocampal development.     

NBQX对大鼠全脑照射后早期脑MyT1阳性细胞的保护作用

目的 探讨大鼠全脑照射后早期大脑皮质少突胶质前体细胞的放射性反应及MK-801和NBQX对其保护作用。方法 以10Gy的剂量时SD大鼠单次全脑照射后即刻分别向其大脑皮质定向注射5mmol／L的MK-801和50mmol／L的NBQX各1μl，然后用免疫荧光组织化学法分别检测各时间点大脑皮质MyT1阳性细胞数量。结果 和假照射组相比，照射组大鼠照射后7d和14d时大脑皮质MyT1阳性细胞数显增加(P＜0．01)；MK-801组大鼠照射后各时间点大脑皮质MyT1阳性细胞数与照射组大鼠无明显差异(P＞0．05)；而NBQX组大鼠照射后1d、7d和14d时的MyT1阳性细胞数明显多于照射组大鼠(P＜0．05)。结论 大鼠全脑照射后早期大脑少突胶质前体细胞反应性增多，并有时程性变化；NBQX对早期的少突胶质前体细胞放射性损伤可能有一定的保护作用。     

Neurotoxicity, Blood-Brain Barrier Breakdown, Demyelination and Remyelination Associated With NMDA-Induced Lesions of the Rat Lateral Hypothalamus

Excitotoxins have been widely used to make lesions in the brains of experimental animals because they have the ability to destroy neurones while sparing fibres of passage. Because loss of fibres of passage can confound the interpretation of lesion effects, this property is of considerable value. Recently, however, there have been reports indicating that excitotoxins acting at different sites within the rat CNS not only destroy neurones but also strip myelin from fibres and compromise the integrity of the blood-brain barrier. However, some reports also indicate that the myelin content of the lesioned area recovers. Excitotoxic lesions of the lateral hypothalamus have been shown to produce local demyelination. The present studies sought to investigate this effect further by (1) defining the time course of demyelination and possible remyelination after excitotoxic lesions of the lateral hypothalamus made with n-methyl-d-aspartate (NMDA); (2) establishing the relationships between neuronal loss, de- and remyelination after various doses of NMDA; and (3) examining the integrity of the blood-brain barrier using an immunohistochemical probe. Our data show that after injection of NMDA into the lateral hypothalamus there was neuronal loss, blood-brain barrier disruption (followed by recovery over approximately 12 days), triggering of reactive gliosis, invasion of the lesioned area by cells from outwith the CNS, demyelination over an area coexistent with but not exceeding the area of neuronal loss, and remyelination. Remyelination occurred over a period of 3 months following the production of the lesion and was associated initially with blood vessels. It occurred across the whole of the lesioned area, not by encroachment from the borders. All doses of NMDA that produced neuronal death also produced demyelination. These data confirm that excitotoxic lesions of the lateral hypothalamus demyelinate fibres, but show for the first time that remyelination occurs here. They are consistent with reports concerning excitotoxin actions at other CNS sites and indicate that de- and remyelination after excitotoxic lesions is a ubiquitous process. Consideration should be given to this when using excitotoxins to make fibre-sparing lesions.     

大鼠局灶性脑缺血后少突胶质前体细胞激活及髓鞘再生的实验研究

目的探讨脑缺血再灌注大鼠少突胶质前体细胞(oligodendrocyte progenitor cells,OPCs)及髓鞘的表达变化。方法线栓法建立大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型,免疫组化方法检测脑缺血再灌注后不同时间点1d、1w和2w不同脑区(梗死中心区、梗死周边区和梗死对侧区)OPCs特异性细胞标志物NG2的阳性细胞数及和髓鞘标志物碱性髓鞘蛋白(myelin basic protein,MBP)的表达变化。结果脑缺血后梗死中心区NG2阳性细胞数和MBP的表达随着再灌注时间延长而逐渐减少;梗死周边区NG2阳性细胞数在1w～2w增加,MBP的表达在24h～1w内降低,2w恢复到正常水平;梗死对侧区NG2阳性细胞数和MBP的表达无明显变化。梗死周边区OPCs细胞呈"单极"或"双极"分裂状,并从梗死灶的外带迁移到内带,提示OPCs细胞激活、增生并发生迁移。结论脑缺血再灌注后梗死周边区NG2细胞增多,使得一度缺失的成熟少突胶质细胞及髓鞘得到补充,提示NG2细胞可能参与缺血损伤的修复过程。     

Up-Regulation of Trem2 Inhibits Hippocampal Neuronal Apoptosis and Alleviates Oxidative Stress in Epilepsy via the PI3K/Akt Pathway in Mice

Epilepsy is a chronic and severe neurological disorder that has negative effects on the autonomous activities of patients. Functionally, Trem2(triggering receptor expressed on myeloid cells-2) is an immunoglobulin receptor that affects neurological and psychiatric genetic diseases. Based on this rationale, we aimed to assess the potential role of Trem2 integration with the PI3 K/Akt pathway in epilepsy. We used microarray-based gene expression profiling to identify epilepsy-related differentially-expressed genes. In a mouse hippocampal neuron model of epilepsy, neurons were treated with lowMg~(2+) extracellular fluid, and the protein and mRNA expression of Trem2 were determined. Using a gain-offunction approach with Trem2, neuronal apoptosis and its related factors were assessed by flow cytometry, RT-qPCR,and Western blot analysis. In a pilocarpine-induced epileptic mouse model, the malondialdehyde(MDA) and8-hydroxy-20-deoxyguanosine(8-OHdG) content and superoxide dismutase(SOD) and glutathione-peroxidase(GSH-Px) activity in the hippocampus were determined,and the protein expression of Trem2 was measured. In addition, the regulatory effect of Trem2 on the PI3 K/Akt pathway was analyzed by inhibiting this pathway in both the cell and mouse models of epilepsy. Trem2 was found to occupy a core position and was correlated with epilepsy.Trem2 was decreased in the hippocampus of epileptic miceand epileptic hippocampal neurons. Of crucial importance,overexpression of Trem2 activated the PI3 K/Akt pathway to inhibit neuronal apoptosis. Moreover, activation of the PI3 K/Akt pathway through over-expression of Trem2 alleviated oxidative stress, as shown by the increased expression of SOD and GSH-Px and the decreased expression of MDA and 8-OHdG. The current study defines the potential role of Trem2 in inhibiting the development of epilepsy, indicating that Trem2 up-regulation alleviates hippocampal neuronal injury and oxidative stress, and inhibits neuronal apoptosis in epilepsy by activating the PI3 K/Akt pathway.     

SphK1/S1P信号通路在脑缺血再灌注神经细胞损伤机制中的研究进展

神经鞘磷脂及其代谢产物--神经酰胺、鞘氨醇（sphingosine,Sph）、鞘氨醇-1-磷酸 （sphingosine-1-phosphate,S1P）作为重要的信号传递分子在细胞增殖、存活、凋亡及炎症免疫等病理生 理过程中发挥重要作用,鞘氨醇激酶（sphingosine kinase,SphK）是催化Sph生成S1P的关键酶。脑缺血再 灌注损伤涉及兴奋性毒性、神经炎症及氧化应激、自由基生成、血脑屏障损伤、细胞死亡等多种机 制。本文主要阐述SphK1及S1P的功能特点,及脑缺血再灌注损伤涉及的机制,并进一步阐明目前关于 SphK1/S1P信号通路在脑缺血再灌注神经细胞损伤中的作用及可能的分子机制。     

Wnt1 expression temporally allocates upper rhombic lip progenitors and defines their terminal cell fate in the cerebellum

The cerebellum (Cb) controls movement related physiology using a diverse array of morphologically and biochemically distinct neurons. During development, the Cb is derived from rhombomere 1 (r1), an embryonic compartment patterned by a signaling center referred to as the isthmus organizer. The secreted glycoprotein WNT1 is expressed in the midbrain primordia (mesencephalon, mes) and at the posterior limit of the mes. WNT1 plays a pivotal role in maintaining the isthmus organizer and mutations in Wnt1 produce severe Cb defects that are generally attributed to aberrant organizer activity. Interestingly, Wnt1 is also expressed at the most posterior limit of dorsal r1, in a region known as the upper rhombic lip (URL). However, the distribution and molecular identity of Wnt1 expressing progenitors have not been carefully described in r1. We used Wnt1-Venus transgenic mice to generate a molecular map of Wnt1 expressing progenitors in relation to other well characterized Cb biomarkers such as MATH1 (ATOH1), LMX1a and OTX2. Our analysis validated Wnt1 expression in the URL and revealed molecularly-defined developmental zones in r1. We then used genetic inducible fate mapping (GIFM) to link transient Wnt1 expression in r1 to terminal cell fates in the mature Cb. Wnt1 expressing progenitors primarily contributed to neurons in deep cerebellar nuclei, granule cells, and unipolar brush cells in distinct but overlapping temporal windows and sparsely contributed to inhibitory neurons and Bergmann glia. We further demonstrate that the Wnt1 lineage does not follow a competency model of progressive lineage restriction to generate the Cb or the functionally related precerebellar system. Instead, progenitors initiate Wnt1 expression de novo to give rise to each Cb cell type and precerebellar nuclei. We also used GIFM to determine how the temporal control of Wnt1 expression is related to molecular identity and cell migration in Cb development. Our findings provide new insight into how lineage and timing establish cell diversity within the Cb system.     

Effects of TNFα on immature and mature oligodendrocytes and their progenitors in vitro

Tumor necrosis factor α (TNFα) appears to take part in the pathogenesis of multiple sclerosis and to contribute to the degeneration of oligodendrocytes as well as neurons. TNFα is produced by microglia and astrocytes, which also produce hormones and cytokines that influence its biological activity. Thus, in mixed cultures the effects of exogenous TNFα might be modified by products of astrocytes and microglia. The effects of TNFα in oligodendrocyte-enriched cultures are reported below. We prepared the cultures by shaking oligodendrocytes off primary mixed glial-cell cultures from brains of 2-day-old rats at 7 days in vitro and plating them (0 days post-shake, DPS). Platelet-derived growth factor and fibroblast growth factor were included in the media at 1–5 DPS in order to encourage proliferation. At 2 DPS media were added with no TNFα (controls) or 1000, 2000 or 5000 U/ml of TNFα, and at 5 DPS media were replaced with fresh serum-free media. Cultures were fixed with 4% paraformaldehyde at 5, 7, 9 and 12 DPS and immunostained. Oligodendrocyte progenitors were not reduced in numbers immediately after the incubation with TNFα (i.e. at 5 DPS). However, after an additional 4 days in culture fewer progenitors remained in the cultures that had been treated with TNFα than in the untreated cultures. In the absence of the growth factors there were fewer progenitors, but their numbers also were reduced by TNFα. Maturation to the myelin basic protein (MBP)-positive stage was inhibited by about 36% at 9 DPS by 1000–2000 U/ml of TNFα, while numbers of O4+/MBP− precursors were unaffected. It is interesting that the steady-state number of O4-positive precursors was unchanged by TNFα at 9 DPS, when there were reductions in the numbers of A2B5-positive progenitors and MBP-positive mature oligodendrocytes. That observation suggests that the rates of proliferation, death and maturation are controlled by multiple factors, with a particularly vulnerable time at the maturation to the MBP-positive stage. At 5000 U/ml TNFα the specific effect on maturation was overtaken cytotoxicity. These data and a summary of the literature suggest that inhibition of MBP expression is sensitive to lower TNFα concentrations and incubation times than is cell survival. Specific effects on numbers of MBP-positive cells, morphology and MBP expression occur at 1000–2000 U/ml for 48–72 h or at up to 10 000 U/ml for≤24 h, and the deficits remain after removal of the TNFα.     

Overexpression of Wnt3a facilitates the proliferation and neural differentiation of neural stem cells in vitro and after transplantation into an injured rat retina

Neural stem cell‐based therapy is a promising option for repair after injury. However, poor stem cell proliferation and insufficient differentiation of the stem cells into neurons are still difficult problems. The present study investigated whether transplantation of neural stem cells (NSCs) genetically modified to express Wnt3a is a promising approach to overcome these difficulties. We explored the possibility that Wnt3a might contribute to the therapeutic effect of NSC transplantation in retinal repair. The relative promotion of proliferation and neural differentiation by modified NSCs was investigated in a rat model of optic nerve crush. A recombinant lentivirus (Lenti‐Wnt3a) was engineered to express Wnt3a. NSCs infected with control lentivirus (Lenti‐GFP) or Lenti‐Wnt3a were transplanted into the subretinal space immediately after the optic nerve crush. The proliferation and neural differentiation activity of the NSCs were assessed in vitro and in vivo. Overexpression of Wnt3a in NSCs induced activation of Wnt signaling, promoted proliferation, and directed the differentiation of the NSCs into neurons both in vitro and in vivo. Our study suggests that Wnt3a can potentiate the therapeutic benefits of NSC‐based therapy in the injured retina. © 2013 Wiley Periodicals, Inc.     

GSK3β regulates oligodendrogenesis in the dorsal microdomain of the subventricular zone via Wnt‐β‐catenin signaling

Oligodendrocytes, the myelinating cells of the CNS, are derived postnatally from oligodendrocyte precursors (OPs) of the subventricular zone (SVZ). However, the mechanisms that regulate their generation from SVZ neural stem cells (NSC) are poorly understood. Here, we have examined the role of glycogen synthase kinase 3β (GSK3β), an effector of multiple converging signaling pathways in postnatal mice. The expression of GSK3β by rt‐qPCR was most prominent in the SVZ and in the developing white matter, around the first 1–2 weeks of postnatal life, coinciding with the peak periods of OP differentiation. Intraventricular infusion of the GSK3β inhibitor ARA‐014418 in mice aged postnatal day (P) 8–11 significantly increased generation of OPs in the dorsal microdomain of the SVZ, as shown by expression of cell specific markers using rt‐qPCR and immunolabelling. Analysis of stage specific markers revealed that the augmentation of OPs occurred via increased specification from earlier SVZ cell types. These effects of GSK3β inhibition on the dorsal SVZ were largely attributable to stimulation of the canonical Wnt/β‐catenin signaling pathway over other pathways. The results indicate GSK3β is a key endogenous factor for specifically regulating oligodendrogenesis from the dorsal SVZ microdomain under the control of Wnt‐signaling. GLIA 2014;62:778–789