Collapsin response‐mediator protein 5 (CRMP5) phosphorylation at threonine 516 regulates neurite outgrowth inhibition

The collapsin response‐mediator proteins (CRMPs) are multifunctional proteins highly expressed during brain development but down‐regulated in the adult brain. They are involved in axon guidance and neurite outgrowth signalling. Among these, the intensively studied CRMP2 has been identified as an important actor in axon outgrowth, this activity being correlated with the reorganisation of cytoskeletal proteins via the phosphorylation state of CRMP2. Another member, CRMP5, restricts the growth‐promotional effects of CRMP2 by inhibiting dendrite outgrowth at early developmental stages. This inhibition occurs when CRMP5 binds to tubulin and the microtubule‐associated protein MAP2, but the role of CRMP5 phosphorylation is still unknown. Here, we have studied the role of CRMP5 phosphorylation by mutational analysis. Using non‐phosphorylatable truncated constructs of CRMP5 we have demonstrated that, among the four previously identified CRMP5 phosphorylation sites (T509, T514, T516 and S534), only the phosphorylation at T516 residue was needed for neurite outgrowth inhibition in PC12 cells and in cultured C57BL/6J mouse hippocampal neurons. Indeed, the expression of the CRMP5 non‐phosphorylated form induced a loss of function of CRMP5 and the mutant mimicking the phosphorylated form induced the growth inhibition function seen in wildtype CRMP5. The T516 phosphorylation was achieved by the glycogen synthase kinase‐3β (GSK‐3β), which can phosphorylate the wildtype protein but not the non‐phosphorylatable mutant. Furthermore, we have shown that T516 phosphorylation is essential for the tubulin‐binding property of CRMP5. Therefore, CRMP5‐induced growth inhibition is dependent on T516 phosphorylation through the GSK‐3β pathway. The findings provide new insights into the mechanisms underlying neurite outgrowth.     

早期运动训练对脑梗死后缺血半暗带GAP-43、Nogo-A及GFAP表达的影响*

目的： 探讨早期运动训练对脑梗死缺血半暗带区轴突生长相关蛋白43 （ GAP-43）、神经突起生长抑制因 子A（ Nogo-A）和神经胶质纤维酸性蛋白（ GFAP）表达的调控作用,及其对大鼠神经功能恢复的影响。方法 ： 健康成年雄性Wistar 大鼠,按随机数字表法分为假手术组、模型组、训练组。采用线栓法建立大脑中动脉闭塞 （ MCAO）大鼠模型,术后24 h 进行强度渐增的跑台运动训练14 d 和28 d。采用改良神经功能缺损评分（ mNSS） 评估神经功能;平衡木行走试验评估大鼠神经行为学改变;H-E 染色观察各组缺血半暗带区神经元病理形态学变 化;免疫印迹检测大鼠脑缺血半暗带GAP-43、Nogo-A 和GFAP蛋白表达变化;结果： 与假手术组相比,模型组 大鼠mNSS评分和平衡木试验评分均显著升高,脑缺血半暗带区GAP-43 和GFAP蛋白表达上调,而Nogo-A 的表 达下调。与模型组比较,跑台训练组大鼠mNSS评分与平衡木试验评分均显著降低,脑缺血半暗带区GAP-43 表 达上调,而Nogo-A 和GFAP表达下调。结论： 早期跑台训练可以促进轴突再生,抑制胶质瘢痕形成,最终改善 脑梗死大鼠神经功能障碍。     

牛膝活性提取物对大鼠海马神经元生长的促进作用

目的:研究牛膝活性提取物(ABPPk)对体外培养的大鼠海马神经元生长的促进作用.方法:以体外原代培养的胎鼠海马神经元为模型,通过微管蛋白(β-tubulinⅢ)免疫荧光染色,采用Leica Qwin软件测量不同浓度ABPPk处理24 h对海马神经元突起延伸和突起分支的影响;通过突触结合蛋白(SYN)和突触后致密蛋白(PSD95)双标免疫荧光染色,采用LeicaQwin软件测量ABPPk(250 ng/ml)处理7d对海马神经元突触形成的影响;通过免疫印迹定量分析不同浓度ABPPk作用24 h对海马神经元生长相关蛋白(GAP-43)表达水平的影响,作用7d对PSD-95表达水平的影响,以及ABPPk(250 ng/ml)作用不同时间对磷酸化胞外信号调节激酶(ERK1/2)水平的调节.分析ABPPk对海马神经元的作用与ERK通路的关系.结果:ABPPk可有效促进海马神经元的突起延伸和突触形成,免疫印迹结果显示,ABPPk能显著增加海马神经元GAP-43和PSD-95蛋白表达,作用5 min即能显著上调ERK磷酸化水平,作用15 min ERK磷酸化水平达到峰值,PD98059可抑制该过程.结论:ABPPk能促进体外培养的海马神经元突起生长和突触形成,上调GAP-43和PSD-95蛋白水平,其作用可能与ERK通路的激活有关.     

不同组织膜蛋白及二甲基亚砜对小脑颗粒细胞突起生长的影响

目的探讨不同组织膜蛋白提取物和二甲基亚砜对大鼠小脑颗粒细胞（CGC）突起生长的影响。方法提取成年大鼠肝、坐骨神经和脑白质的组织膜蛋白包被盖片,接种CGC于盖片上,同时将二甲基亚砜（DMSO）作为添加剂加入培养液,观察CGC突起的生长。结果①脑白质膜蛋白能明显抑制CGC突起的生长,随浓度增加抑制效应更加明显;坐骨神经膜蛋白对CGC突起生长抑制不明显,肝组织膜蛋白则能促进突起生长;②随DMSO浓度升高CGC突起生长逐渐受到抑制,当DMSO浓度小于1％时,CGC突起长度与对照对照差别不大。结论脑白质膜蛋白对CGC突起生长有明显抑制作用,而DMSO添加到培养液中在低浓度时不会显著影响神经元突起生长,可用于检测药物及疏水性分子对体外培养的神经细胞突起生长的作用。     

Neurocognitive and psychiatric disorders-related axonal degeneration in Parkinson's disease

Neurocognitive and psychiatric disorders have significant consequences for quality of life in patients with Parkinson's disease (PD). In the current study, we evaluated microstructural white matter (WM) alterations associated with neurocognitive and psychiatric disorders in PD using neurite orientation dispersion and density imaging (NODDI) and linked independent component analysis (LICA). The indices of NODDI were compared between 20 and 19 patients with PD with and without neurocognitive and psychiatric disorders, respectively, and 25 healthy controls using tract-based spatial statistics and tract-of-interest analyses. LICA was applied to model inter-subject variability across measures. A widespread reduction in axonal density (indexed by intracellular volume fraction [ICVF]) was demonstrated in PD patients with and without neurocognitive and psychiatric disorders, as compared with healthy controls. Compared with patients without neurocognitive and psychiatric disorders, patients with neurocognitive and psychiatric disorders exhibited more extensive (posterior predominant) decreases in axonal density. Using LICA, ICVF demonstrated the highest contribution (59% weight) to the main effects of diagnosis that reflected widespread decreases in axonal density. These findings suggest that axonal loss is a major factor underlying WM pathology related to neurocognitive and psychiatric disorders in PD, whereas patients with neurocognitive and psychiatric disorders had broader axonal pathology, as compared with those without. LICA suggested that the ICVF can be used as a useful biomarker of microstructural changes in the WM related to neurocognitive and psychiatric disorders in PD.     

Advanced diffusion magnetic resonance imaging in patients with Alzheimer’s and Parkinson’s diseases

The prevalence of neurodegenerative diseases is increasing as human longevity increases. The objective biomarkers that enable the staging and early diagnosis of neurodegenerative diseases are eagerly anticipated. It has recently become possible to determine pathological changes in the brain without autopsy with the advancement of diffusion magnetic resonance imaging techniques. Diffusion magnetic resonance imaging is a robust tool used to evaluate brain microstructural complexity and integrity, axonal order, density, and myelination via the micron-scale displacement of water molecules diffusing in tissues. Diffusion tensor imaging, a type of diffusion magnetic resonance imaging technique is widely utilized in clinical and research settings;however, it has several limitations. To overcome these limitations, cutting-edge diffusion magnetic resonance imaging techniques, such as diffusional kurtosis imaging, neurite orientation dispersion and density imaging, and free water imaging, have been recently proposed and applied to evaluate the pathology of neurodegenerative diseases. This review focused on the main applications, findings, and future directions of advanced diffusion magnetic resonance imaging techniques in patients with Alzheimer's and Parkinson's diseases, the first and second most common neurodegenerative diseases, respectively.     

The interaction between F3 immunoglobulin domains and protein tyrosine phosphatases zeta/beta triggers bidirectional signalling between neurons and glial cells

F3, a mouse glycosyl-phosphatidylinositol anchored molecule of the immunoglobulin superfamily, is known to influence axonal growth and fasciculation via multiple interactions of its modular immunoglobulin-like domains. We prepared an Fc chimeric molecule (F3IgFc) to identify molecules interacting with these domains and characterize the functional impact of the interactions. We affinity-isolated tenascin-C and isoforms of the proteoglycan-type protein tyrosine phosphatases zeta/beta (PTPzeta/RPTPbeta) from extracts of developing mouse brain. We showed that both PTPzeta/RPTPbeta and tenascin-C can bind directly to F3, possibly in an exclusive manner, with the highest affinity for the F3-PTPzeta/RPTPbeta interaction. We observed a strong binding of F3IgFc-coated fluorospheres to astrocytes in neural primary cultures and to C6 astrocytoma cells, and demonstrated, in antibody perturbation experiments, that F3-Ig binding on astrocytes depends on its interaction with PTPzeta/RPTPbeta. We also found by confocal analysis that tenascin-C and PTPzeta/RPTPbeta were colocalized on astrocytes which suggests a complex interplay of interactions between PTPzeta/RPTPbeta, tenascin-C and F3. We showed that the interaction between PTPzeta/RPTPbeta and F3-Ig-like domains can trigger bidirectional signalling. C6 glia-expressed PTPzeta/RPTPbeta stimulated neurite outgrowth by cortical and cerebellar neurons, whereas preclustered F3IgFc specifically modified the distribution of phosphotyrosine labelling in these glial cells. Both effects could be prevented and/or mimicked by anti-F3 and anti-6B4PG antibodies. These results identify F3 and PTPzeta/RPTPbeta as potential mediators of a reciprocal exchange of information between glia and neurons.     

蛋白激酶C调节脊髓神经元突起的生长(英文)

目的关于蛋白激酶C(PKC)在神经元突起生长和神经再生中的作用,目前仍存有争议。本研究主要观察PKC对离体培养的脊髓神经元生长的调节作用,旨在阐明PKC对突起生长的调节作用。方法分离纯化胎龄14天(E14)的SD胎鼠的脊髓前角神经元,进行原代培养,并检测不同时相点膜/浆PKC活性(m/c-PKCactivity)的比值。结果神经元培养3-11d期间,神经元内m/c-PKC比值以及PKC-βII在突起中的表达水平均与突起生长呈显著相关关系(r=0.95,P<0.01;r=0.73,P<0.01)。此外,PKC激动剂PMA能显著提高m/c-PKC比值,且与神经突起的生长一致(r=0.99,P<0.01)。而PKC抑制剂GF109203X则能显著抑制突起生长,且不被PMA作用所逆转。结论PKC的活性在脊髓神经元突起生长调节中具有重要作用,其中βII亚型可能扮演重要角色。     

Primary culture of mouse embryonic spinal cord neurons: cell composition and suitability for axonal regeneration studies

Objective: Primary culture is an effective experimental model to study molecular mechanisms that drive axonal regeneration after central nervous system injury. However, the culture of spinal cord (SC) cells remains poorly characterized. Here, we have analyzed the cell composition of a primary SC culture during its maturation.

Methods: Primary cell culture was prepared from mouse embryo spinal cords. After 2, 7, and 14 days of cultivation, the cells were fixed and stained with antibodies against β3-tubulin, nestin, crmp1, SMI-32, DCC or GFAP. We counted percentage of cells positive for the mentioned markers and measured the length of cell processes.

Results: We found that β3-tubulin and nestin were both expressed at day 2 of culture in vitro. Surprisingly (given the use of differentiation-supporting culture medium), the number of nestin+ cells has significantly increased during the first week of cultivation. The GFAP+ cells appeared only at the seventh day in vitro, and their fraction increased during the following cultivation. At 14 day in vitro, SC culture contained cells that expressed the markers typical of commissural and motor neurons. At this age, the neurons had the ability to repair injured neurites after mechanical damage.

Conclusion: Primary culture of SC cells is a dynamically developing cell population that contains all main types of SC cells and is capable of self-repair. Therefore, the culture of mouse embryonic SC cells represents an adequate experimental model for studying cellular and molecular processes taking place in SC neurons after axonal damage in the absence of external inhibitors.     

脑源性神经营养因子基因转染可促进弥漫性轴突损伤神经元修复和轴突再生

应用脂质体将外源脑源性神经营养因子基因导入弥漫性轴突损伤模型大鼠脑内,力图通过脑源性神经营养因子促进神经元再生及修复的作用,促进损伤大鼠的形态功能恢复。结果显示基因转染后弥漫性轴突损伤额叶皮质神经元的形态得到改善,额叶皮质组织神经丝蛋白表达增加,证实脑源性神经营养因子可促进弥漫性轴突损伤后神经元的修复及轴突的再生。