Neurotrophic activity of 2,4,4-trimethyl-3-(15-hydroxypentadecyl)-2-cyclohexen-1-one in cultured central nervous system neurons

Endogenous neurotrophic factors are essential for the development and maintenance of the nervous system. This suggests their potential utilization as therapeutic agents for neurodegenerative diseases. However, the clinical use of these proteic factors is still restricted, and brings about undesirable consequences, including adverse side effects, and bioavailability and stability difficulties. Therefore, the development of low-molecular weight, non-proteic synthetic compounds with neurotrophic properties appears as a promising approach. The aim of this study was to explore the biological activity of 2,4,4-trimethyl-3-(15-hydroxypentadecyl)-2-cyclohexen-1-one (tCFA15), a trimethyl cyclohexenonic long-chain fatty alcohol. To this end, neurons from fetal rat cerebral hemispheres were cultured in the presence of increasing doses of tCFA15 ranging from 0.1 to 1000 nM. Quantification of cell numbers after 48-h culture showed that 100 nM tCFA15 induced a significant increase in the number of surviving cells. Measurement of total neurite length in microtubule-associated protein 2-positive cells also revealed a stimulatory effect in a wider range of concentrations. The extent of this neuritogenic action was similar to that induced by dibutyryl-cyclic AMP, a well-known neurite outgrowth stimulator, but used at much higher concentration (1 mM). Analysis of structure–activity relationships with different tCFA15 analogs and derivatives corroborated the neurotrophic activity. Taken together, these findings provide strong evidence that tCFA15 exhibits neurotrophic properties in vitro.     

远志抽提物DX-1对神经母细胞瘤分化的诱导作用

目的观察神经母细胞瘤细胞分化诱导后形态学改变和端粒酶活性的变化.方法用中药远志抽提物1,7-二羟夹氧蒽酮(DX-1)诱导神经母细胞系Neuro-2A细胞分化,观察分化后瘤细胞的存活率、细胞倍增时间,以及细胞轴突树状突形成、长短和数目,并用TRAP银染法检测分化前后瘤细胞端粒酶活性的改变.结果用DX-1处理后,Neuro-2A细胞生长受明显抑制,其倍增时间延长1.1～13.0倍.在高剂量组(0.1mmol/L)大部分细胞死亡,仅存少量细胞分化成熟.各处理组的瘤细胞呈现神经细胞分化,胞浆具有轴突和树状突样突起,一些突起互相对接,或交织成网状结构.然而,经中、低剂量DX-1作用后,瘤细胞端粒酶活性未见明显的差异,仅在高剂量时端粒酶活性明显下降,甚至完全消失.结论DX-1可诱导体外培养的Neuro-2A细胞分化.在中、低剂量时,瘤细胞出现轴突和树状突突起.在高剂量时,DX-1对瘤细胞有一定的抑制和杀灭作用.在瘤细胞分化过程中,端粒酶活性并不出现明显的变化.     

Substrate effects on the dynamics of neurite growth in vitro: A quantitative multi-parametric analysis

Embryonic chick dorsal root ganglia were cultured in serum-free medium on natural (collagen, fibronectin and hyaluronic acid) and artificial (polylysine and polyornithine) substrata. The movement of individual growth cones was quantified by measuring five parameters using time-lapse cinematography combined with a digitizing-computer system, and the neurite behaviour was compared between the different substrata with multivariate statistical methods. For each substratum, the morphometry of the growth cone was quantified by measuring six morphological parameters. The most discriminative parameters proved to be mean velocity and straightness index for neurite extension, and projected area and cumulated length of filopodia for growth cone morphometry. A good correlation was obtained between behavioural and morphological parameters and the larger the cone area and the filopodia length, the faster and the straighter the neuritic growth. Both quantitative analyses showed highest values for polyornithine and the lowest for hyaluronic acid, and divided the substrata into two opposite groups, artificial and natural. It is concluded that growth cone behaviour and conformation is modulated by substratum properties.     

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on axonal elongation and fasciculation

Summary Ultrastructural examination of neurons treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) confirmed our previous finding that TPA promoted neurite differentiation. At the low concentration of 16 nM TPA, the outgrowth of long neurites was correlated with the increased appearance of membrane-filled varicosities and filopodial extensions along the axons. In contrast, treatment with high concentrations of TPA (160 nM) produced dense outgrowths which were shorter in length and organized as thick fascicles. Increased neurite fasciculation appeared to result from the enhanced side-to-side interactions of neighboring neurites by a neural cell adhesion molecule. Axons within these fascicles were retracted and appeared congested with cytoskeletal and membranous components. Treatment with the antibody to the neural cell adhesion molecule defasciculated the thick outgrowths and permitted further axonal elongation.     

Fasudil protects cultured N1E-115 cells against lysophosphatidic acid-induced neurite retraction through inhibition of Rho-kinase

The aim of this study was to investigate the possible effects of the Rho-kinase inhibitor, fasudil, on the lysophosphatidic acid (LPA)-induced neurite retraction in N1E-115 cells. In cultured N1E-115 cells, LPA produced a marked increase in the population of rounded cells. Fasudil or hydroxyfasudil, an active metabolite of fasudil, blocked cell rounding in a concentration-dependent manner at levels between 1 and 10 μM, with IC₅₀ values of 1.7 or 1.6 μM, respectively. Fasudil or hydroxyfasudil concentration-dependently inhibited phosphorylation of the myosin binding subunit of myosin phosphatase in N1E-115 cells. These results indicate that Rho-kinase is essential for LPA-induced neurite retraction in N1E-115 cells and that inactivation of Rho-kinase by a Rho-kinase inhibitor, such as fasudil, eliminates cell rounding and promotes neurite outgrowth, thus improving neurological function in the brain damage.     

Use of YFP to study amyloid-beta associated neurite alterations in live brain slices

Neuritic plaques are one of the defining neuropathological features of Alzheimer’s disease (AD). These structures are composed of a buildup of fibrils of the amyloid-β (Aβ) peptide (amyloid) surrounded by activated glial cells and degenerating nerve processes (dystrophic neurites). To study neuritic plaques and possible abnormalities associated with dendrites, axons, and synaptic structures, we have developed an acute slice preparation model using PDAPP, yellow fluorescent protein (YFP) double transgenic mice (a mouse model with AD-like pathology that stably expresses YFP in a subset of neurons in the brain). With laser scanning confocal microscopy, we have imaged living brain slices from PDAPP, YFP double transgenic mice as old as 20 months and have been able to visualize axons, dendrites, dendritic spines, and dystrophic neurites for many hours. Our initial studies suggest that dystrophic axons and dendrites within neuritic plaques are fairly stable structures in the absence of exogenous perturbations. This acute slice preparation model should prove to be a useful tool to explore the pathophysiology of Aβ-related axonal, dendritic, and synaptic dysfunction.     

Tenascin-C分子中重组Fibronectin各片段对胎鼠脊髓培养神经元的不同作用

用小鼠重组ｔｅｎａｓｃｉｎ－Ｃ（ＴＮ－Ｃ）结构域中类似Ⅲ型ｆｉｂｒｏｎｅｃｔｉｎ（ＦＮ）的不同重复片段，分别作为基质或培养液成分，研究其对培养的胚胎小鼠脊髓神经突起生长、粘附力及神经元活性的影响。结果显示：无论作为基质还是培养液成分，ＦＮ６－８对神经突起生长及神经元活性均有促进作用；ＦＮ３－５对神经突起生长及神经元活性均有抑制作用。ＴＮ－Ｃ促进神经突起生长的功能区在ＦＮ６－８，ＦＮ３－５介导了神经元与ＴＮ－Ｃ的粘附。     

Effects of Nogo-A and its receptor on the repair of sciatic nerve injury in rats

Regeneration of injured peripheral nerves is an extremely complex process. Nogo-A (neurite outgrowth inhibitor-A) inhibits axonal regeneration by interacting with Nogo receptor in the myelin sheath of the central nervous system (CNS). The aim of this study was to investigate the effects of Nogo-A and its receptor on the repair of sciatic nerve injury in rats. Sprague-Dawley rats (n=96) were randomly divided into 4 groups: control group (control), sciatic nerve transection group (model), immediate repair group (immediate repair), and delayed repair group (delayed repair). The rats were euthanized 1 week and 6 weeks after operation. The injured end tissues of the spinal cord and sciatic nerve were obtained. The protein expressions of Nogo-A and Nogo-66 receptor (NgR) were detected by immunohistochemistry. The protein expressions of Nogo-A, NgR, and Ras homolog family member A (RhoA) were detected by western blot. At 1 week after operation, the pathological changes in the immediate repaired group were less, and the protein expressions of Nogo-A, NgR, and RhoA in the spinal cord and sciatic nerve tissues were decreased (P<0.05) compared with the model group. After 6 weeks, the pathological changes in the immediate repair group and the delayed repair group were alleviated and the protein expressions decreased (P<0.05). The situation of the immediate repair group was better than that of the delayed repair group. Our data suggest that the expression of Nogo-A and its receptor increased after sciatic nerve injury, indicating that Nogo-A and its receptor play an inhibitory role in the repair process of sciatic nerve injury in rats.     

GSK-3β activation mediates Nogo-66-induced inhibition of neurite outgrowth in N2a cells

The axons of the adult mammalian brain and spinal cord fail to regenerate after injury, and it has been suggested that Nogo-66 could prevent CNS axon repair. However, the mechanism of Nogo-66 inhibiting neurite outgrowth remains unknown. Our previous results indicated that protein kinase B (PKB) is involved in the inhibition of the neurite outgrowth by Nogo-66. Glycogen synthase kinase-3β (GSK-3β) is implicated in many processes in the nervous system, including differentiation, specification, polarity, plasticity and axon growth. In addition, GSK-3β is one of the most important molecules downstream of PKB. In the present study, we report on the role of GSK-3β signaling on Nogo-66-treated mouse neuroblastoma N2a cells. Nogo-66 reduced the phosphorylation of GSK-3β at Ser9 in N2a cells. In contrast, pretreatment with SB216763, a specific inhibitor of GSK-3β, resulted in an amelioration of neurite outgrowth by Nogo-66, compared with the Nogo-66 alone group (P < 0.05). Moreover, we performed RNA interference experiments to knock down GSK-3β expression levels in N2a cells via transient transfection of shRNA plasmids. The inhibition of neurite outgrowth by Nogo-66 was subdued in shRNA cells, compared to the non-RNAi cells (P < 0.05). Taken together, these data suggest that GSK-3β is involved in the inhibition by Nogo-66 of neurite outgrowth in N2a cells.     

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。