Protein phosphatases regulate the growth of developing neurites

The mechanisms underlying morphogenesis of axons and dendrites are critical for understanding both the structure and function of the nervous system. Since a number of kinases have a well-known effect on neurite outgrowth, we tested the hypothesis that specific phosphatases can also play a role in neurite extension and branching. Both protein phosphatase 1 (PP1) and 2A (PP2A) are present in growing processes and can regulate neuronal outgrowth. Loss-, gain- and recovery-of-function analyses in cultured hippocampal neurons tested the role of PP1 and PP2A in neurite growth. siRNA partially knocked down specific phosphatase isoforms and showed that reducing PP2A increased neurite length. Broad spectrum pharmacologic inhibition of PP1 caused the opposite effect from RNAi of specific phosphatases, indicating that two phosphatase pathways likely affect neurite morphogenesis. Over-expression of PP2A resulted in shorter neurites and decreased dendritic branching. Rescue analysis showed that PP2A homologs could restore the longer neurites caused by RNAi, to their normal size, indicating that both reagents target the same pathway. Thus, the well-known effects of specific kinases can be countered by the activity of phosphatases at different times and locations in the growing neurite. By working together, kinases and phosphatases can play a dynamic role in regulating neurite extension during development.     

Alzheimer amyloid beta inhibition of Eg5/kinesin 5 reduces neurotrophin and/or transmitter receptor function

The mechanism by which amyloid beta (Aβ) causes neuronal dysfunction and/or death in Alzheimer's disease (AD) is unclear. Previously, we showed that Aβ inhibits several microtubule-dependent kinesin motors essential for mitosis and also present in mature neurons. Here, we show that inhibition of kinesin 5 (Eg5) by Aβ blocks neuronal function by reducing transport of neurotrophin and neurotransmitter receptors to the cell surface. Specifically, cell-surface NGF/NTR(p75) and NMDA receptors decline in cells treated with Aβ or the kinesin 5 inhibitor monastrol, or expressing APP. Aβ and monastrol also inhibit NGF-dependent neurite outgrowth from PC12 cells and glutamate-dependent Ca++ entry into primary neurons. Like Aβ, monastrol inhibits long-term potentiation, a cellular model of NMDA-dependent learning and memory, and kinesin 5 activity is absent from APP/PS transgenic mice brain or neurons treated with Aβ. These data imply that cognitive deficits in AD may derive in part from inhibition of neuronal Eg5 by Aβ, resulting in impaired neuronal function and/or survival through receptor mislocalization. Preventing inhibition of Eg5 or other motors by Aβ may represent a novel approach to AD therapy.     

Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels

Regeneration of neural tissues will require regrowth of axons lost due to trauma or degeneration to reestablish neuronal connectivity. One approach toward this goal is to provide directional cues to neurons that can promote and guide neurite growth. Our laboratory previously reported the formation of aligned monodomain gels of peptide amphiphile (PA) nanofibers over macroscopic length scales. In this work, we modified these aligned scaffolds specifically to support neural cell growth and function. This was achieved by displaying extracellular matrix (ECM) derived bioactive peptide epitopes on the surface of aligned nanofibers of the monodomain gel. Presentation of IKVAV or RGDS epitopes enhanced the growth of neurites from neurons encapsulated in the scaffold, while the alignment guided these neurites along the direction of the nanofibers. After two weeks of culture in the scaffold, neurons displayed spontaneous electrical activity and established synaptic connections. Scaffolds encapsulating neural progenitor cells were formed in situ within the spinal cord and resulted in the growth of oriented processes in vivo. Moreover, dorsal root ganglion (DRG) cells demonstrated extensive migration inside the scaffold, with the direction of their movement guided by fiber orientation. The bioactive and macroscopically aligned scaffold investigated here and similar variants can potentially be tailored for use in neural tissue regeneration.     

朗飞结处神经突起生长抑制因子

朗飞结以及结侧区是有鞘轴突上的一些极化区域,越来越多的证据表明胶质细胞分泌的某些抑制中枢神经系统损伤后轴突再生过程中神经突起生长的分子如粘蛋白（tenascins）、硫酸软骨素蛋白聚糖（chondroitin sulphate proteoglycans）、髓鞘相关糖蛋白（myelin-associated glycoprotein,MAG）、轴突生长抑制因子（Nogo）以及少突胶质细胞髓鞘糖蛋白（OMGP）等非常特异性的富集于朗飞结区域。这些分子在体外组织培养过程中显示出强烈的神经突起生长抑制作用。在一些基因无义突变的动物模型,能够观察到朗飞结处轴突的生长,表明这些抑制分子能够生理性地保持轴突的完整性并且阻止轴突间随机和错误的联结,然而,大部分的基因无义突变动物模型显示不出明显的中枢神经系统再生改善。这些被称为抑制因子的分子是否是神经再生失败的真正元凶这些抑制因子体内体外实验结果的不一致以及它们特异的定位分布让我们有理由对它们在其他生理作用和功能方面进行重新评价。考虑到轴突-胶质细胞相互作用的双向特性,本综述认为这些抑制因子不仅通过神经元上的受体信号通路调节轴突的极化、离子通道的功能以及轴突的分枝,另一方面轴突产生的化学分子也能反馈性的通过朗飞结区域寡突胶质细胞上的胶质细胞受体信号通路影响寡突胶质细胞的发育。     

The extracellular matrix molecule, laminin, induces Purkinje cell dendritic spine proliferation in granule cell depleted cerebellar cultures

Granule cells and glia were eliminated or reduced in organotypic cerebellar cultures exposed to cytosine arabinoside. Transplantation of such granuloprival cultures with glia or exposure to astrocyte conditioned medium in the absence of parallel fibers (granule cell axons) resulted in proliferation of Purkinje cell dendritic spines. The aim of the present study was to identify specific astrocyte secreted factors that induced dendritic spine proliferation. Known astrocyte secreted, neurite promoting factors were screened by application to granuloprival cultures and assayed for dendritic spine proliferation by electron microscopy. An extracellular matrix molecule, laminin, evoked sprouting of Purkinje cell dendritic spines. Dendritic spine proliferation was not associated with known neurite promoting parts of the laminin molecule, as two laminin-derived peptides with identified neurite promoting domains did not induce dendritic spine sprouting. The purpose of laminin-induced dendritic spine proliferation may be to elaborate postsynaptic membrane, thereby increasing the target area for arriving axon terminals during development or regeneration, both of which have been associated with the presence of laminin secreting astrocytes.     

Sargaquinoic acid promotes neurite outgrowth via protein kinase A and MAP kinases-mediated signaling pathways in PC12D cells

We previously isolated a nerve growth factor (NGF)-dependent neurite outgrowth promoting substance MC14 (sargaquinoic acid) from a marine brown alga, Sargassum macrocarpum. In the present study, the NGF-potentiating activity of MC14 to neural differentiation of PC12D cells was investigated in detail. The treatment of cells with 3 microg/ml MC14 in the presence of 1.25-100 ng/ml NGF markedly enhanced the proportion of neurite-bearing cells compared with the NGF-only controls. In addition, MC14 significantly elevated the NGF-induced specific acetylcholinesterase (AchE) activity in PC12D cells, suggesting that MC14 could morphologically and biochemically promote the differentiation of PC12D cells. The mechanism of action of MC14 was further investigated by pharmacological inhibition of several intracellular signaling molecules. Results indicated that the neurite outgrowth promoting activity of MC14 was almost completely blocked by 10 microM PD98059, suggesting that a TrkA-dependent MAP kinases-mediated signaling pathway may play a crucial role in modulating the effect of MC14. Besides, the MC14-enhanced neurite outgrowth was substantially suppressed by the pretreatment with 10 ng/ml protein kinase A (PKA) inhibitor, demonstrating that the adenylate cyclase-PKA signaling cascade was also involved in the action of MC14. In contrast, a PKC inhibitor chelerythrine chloride did not inhibit the neurite outgrowth promoting activity of MC14. Altogether, these results demonstrate that MC14 enhances the neurite outgrowth by cooperating at least two separated signaling pathways, a TrkA-MAP kinases pathway and an adenylate cyclase-PKA pathway, in PC12D cells.     

Immunohistochemical study of microtubule-associated protein 5 (MAP5) expression in the developing human brain

The expression of microtubule-associated protein 5 (MAP5) in the developing human brain was studied by means of an immunohistochemical method. In the cerebellum, MAP5 immunoreactivity appeared in the molecular layer and subcortical white matter from the early fetal age of 13 gestational weeks (GW), and temporally increased in the outer halves of the molecular layer and subcortical white matter at 36 GW to 2 months of age and 20 to 22 GW, respectively. In the cerebrum, it already appeared in the molecular layer and subcortical white matter from 13 GW, and was marked at 20 to 26 GW and 24 to 32 GW, respectively. Cortical pyramidal neurons gradually became immunoreactive from 28 GW to adolescence. Ependymal cilia were markedly positive in ventricular wall in all ages. In Western blot analyses, MAP5 showed two separate molecular weight bands. In the fetal period 320 kDa protein was prominent, but 300 kDa protein could be detected only at 11 years of age. Thus MAP5 was markedly expressed in growing axon in the fetal period and may be essential for the elongation and maturation as well as the function maintenance of axons and dendrites in developing human brain.     

Development and Elongation of Neurite–like Outgrowth on Small Cell Lung Cancer Cell Lines

One (Lu–134A) of nine human small cell lung cancer cell lines which grow as floating cell aggregates changed its morphology dramatically when cells were cultured on a coverslip coated with poly–ethylenimine or extracellular matrix of human lung adenocarcinoma cell line PC–9 cells. The Lu–134A cells adhered to the substrate and developed elongated cytoplasmic processes which gradually grew into long neuronal–like processes. These processes developed to a length of more than 10 times the cell body length after 20 days of culture. Addition of dibutyryl cyclic adenosine 3', 5 –monophosplmte to the cells on these substrates remarkably promoted the development and elongation of the processes, which grew into a netlike arrangement. The characteristics of these elongated neuronal–like processes were studied using immunocytochemical and electron microscopical methods. The processes reacted intensely with monoclonal antibodies against β –tubulin and microtubule–associated protein–2. The swelling portions of the distal tips of these processes reacted strongly with polyclonal antibody against synaptophysin. Neurosecretory granules and bundles of microtuhules were observed within processes. These findings suggested that this human small cell lung cancer cell line (Lu–134A) differentiated into neuronal cells, and indicated that attachment of cells to a substrate is the key to the development of long neurite–like outgrowths.     

Effects of Growth Factors and Estrogen on the Development of Septal Cholinergic Neurons From the Rat

Cholinergic neurons of the septum are preferentially subject to degeneration in Alzheimer’s disease. There is evidence that nerve growth factor, basic fibroblast growth factor, insulin-like growth factors, and estrogen all have effects on survival of this specific population of neurons at risk. We used a bilaminar culturing method to grow embryonic septal neurons from the rat in the presence of a separate glial plane but in the absence of serum. These neurons were treated with a number of factors, and neurite development of cholinergic neurons was assessed. Basic fibroblast growth factor and estrogen altered the number of primary neurites, number of secondary neurites, and mean total neurite lengths, while none of the other factors affected these end points. This would suggest a mechanism for the effects of these factors on memory.     

RNA干扰CRMP 5抑制大鼠海马神经元突起生长

目的探讨CRMP5对大鼠海马神经元突起生长的影响。方法将带FAM标记的CRMP5的特异性干扰片段及阴性对照转染培养成熟的海马神经元,用免疫荧光的方法验证干扰片段对神经元内源性CRMP5的干扰效果,并利用共聚焦显微镜观察神经元突起以及侧枝的形成。结果携带FAM的si RNA可以成功的进入细胞,分布于神经元的胞体以及树突;免疫荧光证实CRMP5 si RNA可以有效的沉默CRMP5蛋白的表达;沉默CRMP5基因表达后的海马神经元突起短小,而且缺少分支,而对照细胞突起长,分支多;定量分析显示,导入CRMP5 si RNA的细胞突起的长度较对照细胞缩短,差异显著(P0.05);突起的数目比较,一级突起数目无显著差异,而二级及其以上突起的数目明显减少,差异显著(P0.05)。结论沉默CRMP5可抑制海马神经元突起的生长和侧枝形成。