Neuronal microtubule-associated proteins in the embryonic avian spinal cord

We have used monoclonal antibodies to study the distribution of three developmentally regulated microtubule-associated proteins-MAP2, MAP5, and tau-during the morphogenesis of the thoracic spinal cord and peripheral nervous system in the quail. MAP5 is the only one of the three that is present in growing motor neuron processes in the day 3 embryo. The low-molecular weight form of MAP2, MAP2c, is found in motor neuron cell bodies at embryonic day 3. At later stages MAP2c appears in axons and in glia; it decreases in abundance between embryonic days 5 and 7. High-molecular weight MAP2 appears in motor neuron cell bodies and spinal cord gray matter at embryonic day 4, and is never encountered in axons. Tau is found in axons, but only at embryonic day 3.5, after they have commenced active extension. The molecular form and patterns of intracellular compartmentalization of each of the microtubule-associated proteins studied is conserved in mammalian and avian neurons. We conclude that MAP5 may be involved in the active growth of neuronal processes, whereas MAP2 and tau are not, and that high-molecular weight MAP2 and tau may stabilize dendritic and axonal processes, respectively.     

Boundaries and inhibitory molecules in developing neural tissues

Numerous studies of the past decade have illuminated the importance of intercellular adhesion events for neural pattern formation. It has been documented that members of the Ig and cadherin gene superfamilies, that glycoproteins and, probably to some extent, proteoglycans of the extracellular matrix play a role in this context. Recent observations suggest that, in addition to adhesive interactions, repulsive and/or inhibitory phenoma are also of importance in regulating neural pattern formation. Several molecules are under study which are cosidered possible mediators of inhibitory interactions in the nervous system. The hypothesis has been advanced that some of these might be partially responsible for restrictive, boundary-like properties ascribed to glial cells in developing and regenerating tissues. The current review summarizes these studies and focusses on molecular aspects of boundary and compartmentation phenomena. © 1995 Wiley-Liss, Inc.     

COMPARISON OF NEURITE OUTGROWTH WITH NEUROFILAMENT PROTEIN SUBUNIT LEVELS IN NEUROBLASTOMA CELLS FOLLOWING MERCURIC OXIDE EXPOSURE

1. The objectives of the study were to establish that inhibition of neuronal differentiation in culture (assessed by neurite outgrowth) can be used as a broad spectrum in vitro measure of neurotoxicity. 2. To establish whether a rapid measure of neurite outgrowth could be used. Thus the study examined the relationship between the degree of neurite outgrowth assessed directly by image analysis and neurofilament protein subunit levels measured by an ELISA. 3. SKNSH neuroblastoma cells, exposed for up to 6 days to mercuric chloride during initiation and continuation of differentiation, had lower levels of neurofilament proteins than unexposed cells. 4. Preliminary data from parallel examinations of neurite outgrowth assessed by image analysis and neurofilament protein subunit levels assessed by ELISA support a correlation when neurofilament protein levels are decreased by sub-cytotoxic doses of mercuric chloride in SKNSH cells.     

SOCS3通过抑制STAT3抑制受损伤大鼠初级感觉神经元生长

目的通过慢病毒转导SOCS3和作用相反的突变型SOCS3(mSOCS3),体外研究SOCS3在成年鼠的初级感觉神经元再生中的作用。方法将慢病毒载体质粒pRRL-SOCS3-IRES-GFP,pRRL-mSOCS3-IRES-GFP和pRRL-STAT3ER-IRES-GFP分别转染293T细胞,包装慢病毒载体并测定滴度。完全切断大鼠左侧坐骨神经,术后饲养8-128h,在不同时间点摘取双侧L5背根神经节。通过实时PCR和原位杂交检测DRGs中SOCS3 mRNA的存在;通过背根神经节分离神经元培养,分别将三种慢病毒载体感染神经元细胞,采用免疫荧光染色法观察神经元细胞核质反应和突起的长度。结果大鼠左侧坐骨神经损伤后,SOCS3 mRNA的表达在背根神经节神经元中明显增加,外源性SOCS3能阻止神经元中STAT3的磷酸化及核移位,mSOCS3增强了突起生长。结论 SOCS3可通过抑制STAT3抑制轴突生长。     

Activation of Src/kinase/phospholipase C/mitogen-activated protein kinase and induction of neurite expression by ATP, independent of nerve growth factor

Extracellular ATP has been reported to potentiate the neurite outgrowth induced by nerve growth factor. In the present study the neurotrophic effect of ATP and other nucleotides was examined in mouse neuroblastoma neuro2a cells which lack nerve growth factor receptor. Exposure of neuro2a cells to ATP resulted in a dramatic increase in neurite bearing cells as compared with untreated control cells. Experiments performed with purinergic receptor agonists and antagonists suggest that the ATP stimulates neurite outgrowth via P2 receptors. Neurite outgrowth was completely blocked by P2 receptor antagonist suramin whereas the P1 receptor antagonist CGS15943 was ineffective. P1 receptor agonist 5'-(N-ethylcarboxamido)adenosine failed to induce neurite outgrowth. The potency order of different P2 receptor agonists was ATP=ATPgammaS>ADP>2Me-S-ATP. It was insensitive to UTP and antagonist pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) suggesting the involvement of P2Y11 receptor in the observed neuritogenic effect. The signaling pathway leading to ATP-induced neuritogenesis was investigated. The neuritogenic effect of ATP is independent of rise in intracellular Ca(2+) as pharmacological profile of neuritogenic P2Y receptor does not match with that of P2Y2 receptor associated with [Ca(2+)](i) signaling cascade. Exposure of cells to ATP caused activation of Src kinase, phospholipase Cgamma and extracellular signal-regulated kinases ERK1/2. Mitogen-activated protein kinase (MAPK) inhibitor U0126 drastically reduced the number of neurite bearing cells in ATP-treated cultures implying that the neurotrophic effect of ATP is mediated by MAPK. Our results demonstrate that ATP can stimulate neurite outgrowth independent of other neurotrophic factors and can be an effective trophic agent.     

Staurosporin induces neurite outgrowth through ROS generation in HN33 hippocampal cell lines

Summary. Staurosporin, a specific inhibitor of PKC, is widely used in studies of signal transduction pathways. Previous studies have shown that staurosporin induces neurite outgrowth, but the underlying mechanisms remain unclear. Here we report that staurosporin induces neurite outgrowth in HN33 hippocampal cells. Two other PKC inhibitors, Go 6976 (specific for α- and β-isoforms) and rotterlin (a selective inhibitor of PKC δ), have no neuritogenic effect. In addition, staurosporin specifically increases ROS generation. NAC, which inhibits the generation of ROS, suppresses the staurosporin-induced neurite outgrowth in HN33 cells. Further, H₂O₂ causes neurite outgrowth. Taken together, these results confirm a neuritogenic effect of staurosporin and point to ROS as the signal mediator of staurosporin-induced neurite outgrowth in HN33 hippocampal cells. Theme: Development and regeneration Topic: Neurotrophic factors: receptors and cellular mechanisms     

Two new dendrocandins with neurite outgrowth-promoting activity from Dendrobium officinale

Two new bibenzyl derivatives, dendrocandin T (1) and dendrocandin U (2), together with eight known bibenzyls, were isolated from the stems of Dendrobium officinale. Those compounds were sent for the first time for central nervous system-related bioassay and the results indicated that compounds 3, 4, and 5 have a certain degree of neurite outgrowth-promoting activity, and compounds 1, 2, 6, and 7 also have weak activity. The results indicated that D. officinale used as health food and traditional Chinese medicine “Tiepi Shihu” has a health function of neurotrophic effects.     

阿托伐他汀通过激活PI3K/Akt/mTOR信号转导而促进神经元突起生长

目的探讨阿托伐他汀(Ato)对体外培养大鼠皮质神经元突起生长促进作用的信号转导机制。方法 取培养7 d大脑皮质神经元,分为Ato 10μmo.lL-1作用48 h组和阻断剂+Ato组,先分别加入阻断剂PD98059 50μmo.l L-1、LY294002 30μmol.L-1、曲西立滨(TCBN)2.5μmol.L-1和西罗莫司(雷帕霉素,Rapa)100 nmo.l L-1作用1 h,再加入Ato共同作用48 h。应用倒置相差显微镜观察神经元突起生长状况;Western印迹法检测磷酸化的磷酸肌醇依赖激酶1(PDK1)、磷酸化蛋白激酶B(Akt)、磷酸化西罗莫司靶蛋白(mTOR)、磷酸化的核糖体S6激酶(p70S6K)和磷酸化的真核翻译起始因子4E结合蛋白1(p-4E-BP1)的表达。结果 形态学观察结果显示,Ato 10μmo.lL-1组可明显促进突起生长,表现为突起总长度增加、一级突起数目增多、末端分支数增多及胞体面积增大。PD98059,LY294002,TCBN和Rapa均可阻断Ato对神经元突起生长的促进作用。Western印迹结果显示,Ato 10μmo.lL-1可显著上调p-PDK1,p-Akt(Ser473),p-mTOR,p-p70S6K和p-4E-BP1蛋白表达水平(P<0.01)。LY294002可显著阻断Ato引起的p-PDK1,p-Akt(Ser473)蛋白表达水平增加(P<0.01)。TCBN可显著阻断Ato引起的p-mTOR蛋白表达水平增加(P<0.01)。Rapa可明显阻断Ato引起的p-p70S6K和p-4E-BP1蛋白表达水平增加(P<0.01)。结论 Ato对体外培养皮质神经元突起发育的促进作用可能与激动MEK/ERK信号转导通路有一定的关系,主要可能与通过激活PI3K/Akt/mTOR信号转导通路有关。     

The interaction of Nogo-66 receptor with Nogo-p4 inhibits the neuronal differentiation of neural stem cells

The Nogo-66 receptor (NgR) has been found throughout axons in the adult and maturing CNS. An interaction of Nogo on the oligodendrocyte surface with NgR on axons has been suggested to play an important role in limiting axonal growth. In our study, we found that neural stem cells (NSCs) derived from the spinal cords of rats expressed NgR significantly. After normal NSCs differentiation, the average neuronal neurite length was 97.80+/-6.97 microm and the percentage of differentiated neurons was 34.73+/-5.21% 3 days after the differentiation was initiated in vitro. If NSCs were allowed to differentiate in the presence of Nogo-p4 (the active segment of Nogo-66), the average neurite length and the percentage of differentiated neurons were decreased, respectively, to 60.31+/-6.58 microm and 10.26+/-1.22%. An siRNA-mediated knockdown of NgR on NSCs could reverse the inhibitory effect of Nogo-p4 and restore the average neuronal neurite length as well as the percentage of differentiated neurons to 94.01+/-8.37 microm and 31.84+/-4.03%. These results deepen our knowledge about the distribution of NgR and provide a possible strategy of treating NSCs to ameliorate neuronal differentiation after transplantation.     

The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of Rho and ROCK inhibitors on neurite outgrowth and myelination

It is currently thought that treatment for spinal cord injury (SCI) will involve a combined pharmacological and biological approach; however, testing their efficacy in animal models of SCI is time-consuming and requires large animal cohorts. For this reason we have modified our myelinating cultures as an in vitro model of SCI and studied its potential as a prescreen for combined therapeutics. This culture comprises dissociated rat embryonic spinal cord cells plated onto a monolayer of astrocytes, which form myelinated axons interspaced with nodes of Ranvier. After cutting the culture, an initial cell-free area appears persistently devoid of neurites, accompanied over time by many features of SCI, including demyelination and reduced neurite density adjacent to the lesion, and infiltration of microglia and reactive astrocytes into the lesioned area. We tested a range of concentrations of the Rho inhibitor C3 transferase (C3) and ROCK inhibitor Y27632 that have been shown to promote SCI repair in vivo. C3 promoted neurite extension into the lesion and enhanced neurite density in surrounding areas but failed to induce remyelination. In contrast, while Y27632 did not induce significant neurite outgrowth, myelination adjacent to the lesion was dramatically enhanced. The effects of the inhibitors were concentration-dependent. Combined treatment with C3 and Y27632 had additive affects with an enhancement of neurite outgrowth and increased myelination adjacent to the lesion, demonstrating neither conflicting nor synergistic effects when coadministered. Overall, these results demonstrate that this culture serves as a useful tool to study combined strategies that promote CNS repair.