白藜芦醇通过SIRT1激活ADAM10促进APPα代谢

目的：探讨白藜芦醇可否通过SIRT1激活解整合素金属蛋白酶10（ADAM10）促进APPα代谢抑制Aβ分泌。方法：选取过表达人瑞典突变淀粉样前体蛋白APP695sw的细胞模型,分别设立DMSO空白对照组、SIRT1激活剂白藜芦醇组和SIRT1抑制剂EX527组。给药后,ELISA法分别检测细胞培养上清中sAPPα和Aβ的含量,免疫印记Western Blot法检测细胞SIRT1和ADAM10的蛋白水平。结果：与对照组比较,白藜芦醇组细胞培养上清sAPPα的含量增高,Aβ含量下降,sAPPα/Aβ比值增大,细胞SIRT1和ADAM10蛋白水平增高;而抑制剂EX527组与对照相比,细胞培养上清sAPPα的含量降低,Aβ含量升高,sAPPα/Aβ比值减小,细胞SIRT1和ADAM10蛋白水平降低;抑制剂下调SIRT1后各项指标与激动剂组呈现相反的趋势（P<0.05）。结论：白藜芦醇可通过SIRT1激活ADAM10,促进APP进行α非淀粉样代谢途径,增强sAPPα分泌并抑制Aβ产生。     

β淀粉样蛋白对大鼠学习记忆功能及tau蛋白异常磷酸化的影响

为了探讨β淀粉样蛋白对大鼠学习记忆功能和tau蛋白异常磷酸化的影响,本文在海马注射Aβ25-35建立阿尔茨海黙病(AD)大鼠模型的基础上,通过行为学检测、HE染色、免疫组化和免疫蛋白印迹技术对动物的学习能力、组织的病理改变和tau(pS202)、tau(pT231)和tau-5的表达情况进行了分析。在行为学检测中,Aβ注射组大鼠在穿梭箱实验中的主动回避次数和被动回避次数减少,失败次数增多,而在Morris水迷宫测试中的逃避潜伏期和游泳距离延长。HE染色显示Aβ注射组大鼠海马CA1、CA3、齿状回的神经细胞数目减少;而免疫组化和免疫印迹结果显示注射组tau(pS202)阳性细胞明显增加,tau(pS202)、tau(pT231)和tau-5蛋白表达增加。以上结果提示海马内注射Aβ25-35可引起大鼠学习记忆功能下降,可能与神经细胞减少,tau蛋白异常磷酸化增多有关。     

Nicotine modifies in vivo and in vitro rat hippocampal amyloid precursor protein processing in young but not old rats

Previous studies have shown that administration of nicotine modifies the expression and secretion of amyloid precursor protein (APP) in various cell lines. The present study investigated the extent to which chronic subcutaneous nicotine administration influences APP levels and processing in cerebral cortex, striatum and hippocampus of young and old rat brains. The results showed that constant nicotine infusion (0.25 or 4.00 mg/kg/day) increased the levels of particulate APP (APPp) but not secreted APP (APPs) in the hippocampus of young rats in vivo. This response to nicotine was not observed in the striatum or cerebral cortex of young rats or in any of the brain regions examined in old animals. Subsequent in vitro analysis demonstrated that nicotine enhanced the release of APPs from hippocampal slice preparations and that this increase was attenuated by mecamylamine, a non-selective nicotinic acetylcholine receptor (nAChR) antagonist. The in vitro effect of nicotine on APPs was age-related, being only detected from hippocampal slices derived from the young but not the older animals. These results suggest that nicotine modulates APP expression and secretion in the hippocampus and that the responses observed to the drug are age-dependent being only detected in younger rats.     

MicroRNA-195 downregulates Alzheimer's disease amyloid-β production by targeting BACE1

Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterized by amyloid-beta (Aβ) deposition and neurofibrillary tangles. Numerous microRNAs have been found to play crucial roles in regulating Aβ production in the process of AD. Previous investigations have reported lower levels of many microRNAs in AD patients and animal models. Here, we examined the role of miR-195 in the process of Aβ formation. Bioinformatics' algorithms predicted miR-195 binding sites within the beta-site APP cleaving enzyme 1 (BACE1) 3'-untranslated region (3'-UTR), and we found the level of miR-195 to be negatively related to the protein level of BACE1 in SAMP8 mice. We confirmed the target site in HEK293 cells by luciferase assay. Overexpression of miR-195 in N2a/WT cells decreased the BACE1 protein level, and inhibition of miR-195 resulted in increase of BACE1 protein level. Furthermore, overexpression of miR-195 in N2a/APP decreased the level of Aβ, while inhibition of miR-195 resulted in an increase of Aβ. Thus, we demonstrated that miR-195 could downregulate the level of Aβ by inhibiting the translation of BACE1. We conclude that miR-195 might provide a therapeutic strategy for AD.     

Glucosamine and Its Analogues as Modulators of Amyloid-β Toxicity

In Alzheimer’s disease (AD), amyloid-β (Aβ) oligomers are considered key mediators of synaptic dysfunction and cognitive impairment. These unstable intermediate Aβ species can interfere with different cellular organelles, leading to neuronal cell death, through the formation of Ca²⁺-permeable membrane pores, impairment in the levels of acetylcholine neurotransmitters, increased insulin resistance, promotion of pro-inflammatory cascades, among others. Based on a series of evidences that indicate the key role of glycosaminoglycans (GAGs) in amyloid plaque formation, we evaluated the capacity of four monosaccharides, i.e., glucosamine (GlcN), N-acetyl glucosamine (GlcNAc), glucosamine-6-sulfate (GlcN6S), and glucosamine-6-phosphate (GlcN6P), to reduce the Aβ-mediated pathological hallmarks. The tested monosaccharides, in particular, GlcN6S and GlcN6P, were able to interact with Aβ aggregates, reducing neuronal cell death, Aβ-mediated damage to the cellular membrane, acetylcholinesterase activity, insulin resistance, and pro-inflammation levels.     

当归芍药散对AD细胞模型铜离子介导的Aβ聚集的影响

目的:以β淀粉样蛋白1-42(Aβ1-42)和铜离子(Cu~(2+))络合物作用的人神经母细胞瘤(SH-SY5Y)细胞为阿尔茨海默病(AD)模型,探讨Cu~(2+)对Aβ聚集方式和神经毒性的影响及当归芍药散对Cu~(2+)介导的Aβ毒性的AD细胞模型的影响。方法:将Aβ1-42(20μmol·L~(-1))与不同浓度硫酸铜(Cu SO4,20,40μmol·L~(-1))反应后用硫黄素T(Th T)染色法检测Aβ聚集状态,再进行Aβ1-42-Cu~(2+)组(20+20μmol·L~(-1)),当归芍药散组(1. 6,3. 2,6. 4 mg·L~(-1)) Th T染色检测Aβ聚集状态。培养SH-SY5Y细胞,分别孵育不同浓度的Aβ1-42(1. 25,2. 5,5,10,20,40μmol·L~(-1))及当归芍药散(1. 6,3. 2,6. 4,12. 8 mg·L~(-1)),24 h后行噻唑蓝(MTT)比色法确定Aβ1-42损伤浓度和当归芍药散保护浓度。随后将SH-SY5Y细胞分为空白组,Aβ1-42-Cu~(2+)组(20+20μmol·L~(-1)),当归芍药散组(20μmol·L~(-1)+20μmol·L~(-1)+1. 6 mg·L~(-1)),空白组加入培养基,共作用24 h后行MTT比色法检测细胞生存率,显微镜拍摄细胞形态,蛋白免疫印迹法(Western blot)检测胞内胞外Aβ1-42聚集状况。结果:与Aβ1-42组比较,Cu~(2+)和Aβ1-42结合成更多更大的Aβ聚集物。与正常组比较,模型组细胞活力明显降低(P 0. 05,P 0. 01),增加细胞内外Aβ1-42聚集(P 0. 05,P 0. 01);与模型组比较,当归芍药散能提高Aβ1-42-Cu~(2+)损伤的SH-SY5Y的细胞生存率(P 0. 05,P 0. 01),减少胞外Aβ1-42蛋白(P 0. 05,P 0. 01)。结论:Cu~(2+)能够增加Aβ的聚集及毒性;当归芍药散能明显减轻Cu~(2+)介导的Aβ聚集导致的SH-SY5Y细胞损伤,使Aβ内吞,减少胞外Aβ聚集,提高细胞生存率。     

五味子醇甲对Aβ1-42诱导SH-SY5Y细胞的RAGE-ROS-凋亡通路的影响

目的探讨五味子醇甲(Schisandrin,SCH)对Aβ1-42诱导SH-SY5Y的RAGE/ROS/凋亡通路的影响。方法MTT法检测control组、SCH高中低剂量组的细胞活力,探讨SCH高中低剂量组对SH-SY5Y细胞的保护作用。MTT法检测control组、Aβ组、Vitamin E组及SCH高中低剂量组细胞活力、双染法观察细胞凋亡和DHE染色法检测ROS含量,探讨SCH对Aβ1-42诱导的SH-SY5Y的细胞损伤的保护作用。Western-blot法检测control组、Aβ组、SCH组RAGE蛋白,探讨SCH对Aβ1-42诱导SH-SY5Y的RAGE影响。结果与control组比较,SCH各剂量组细胞活力均显著增强(P<0.01)。与Aβ组比较,各给药组细胞活力均明显增强(P<0.01)和细胞凋亡率均明显下降(P<0.01);各给药组检测ROS的荧光强度均减弱(P<0.01),SCH(5.0μg/mL)组RAGE蛋白表达降低,有显著性差异(P<0.05)。结论SCH防治阿尔茨海默病的抗氧化作用机制可能与降低ROS含量、细胞凋亡率和下调RAGE蛋白表达有关。     

Impaired autophagy in amyloid-beta pathology: A traditional review of recent Alzheimer's research

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. The major pathological changes in AD progression are the generation and accumulation of amyloid-beta (Aβ) peptides as well as the presence of abnormally hyperphosphorylated tau proteins in the brain. Autophagy is a conserved degradation pathway that eliminates abnormal protein aggregates and damaged organelles. Previous studies have suggested that autophagy plays a key role in the production and clearance of Aβ peptides to maintain a steady-state of Aβ peptides levels. However, a growing body of evidence suggests that autophagy is significantly impaired in the pathogenesis of AD, especially in Aβ metabolism. Therefore, this article reviews the latest studies concerning the mechanisms of autophagy, the metabolism of Aβ peptides, and the defective autophagy in the production and clearance of Aβ peptides. Here, we also summarize the established and new strategies for targeting autophagy in vivo and through clinical AD trials to identify gaps in our knowledge and to generate further questions.     

Protective effects of EUK4010 on beta-amyloid(1-42) induced degeneration of neuronal cells

EUK4010 has been identified to exhibit an inhibitory effect on beta-amyloid (Abeta)(1-42)-induced loss of neuronal cell viability. Further studies demonstrated that EUK4010 attenuated the Abeta(1-42)-induced degeneration in both cultured rat hippocampal neurons and human neuroblastoma cells, as demonstrated by typical morphological changes, cell viability and the chip-based flow cytometric assay. Gene expression analysis using DNA microarray showed that the senescence marker calcium-binding protein, regucalcin (Rgn), GABA-A receptor pi subunit (Gabrp), the huntingtin binding protein, optineurin (Optn) and a semaphorin family plexin A3 similar protein (Plex-similar) changed their expression levels significantly in cultured neurons after Abeta(1-42) treatment. In this report, we have undertaken a chemical genetic approach to study the molecular basis of Abeta(1-42) effects on the neuronal degeneration. Our results demonstrate that EUK4010 completely blocked the Abeta(1-42)-induced up-regulation of GABA-A receptor pi subunit and the semaphorin family plexin A3 similar protein, and partially attenuated the down-regulation of senescence marker calcium-binding protein, regucalcin. These observations suggest that EUK4010 may prevent or reduce the Abeta toxicity by regulating the expression of genes involved in the Abeta induced neuronal degeneration. These genes may represent a promising target for the therapeutic drug development for Alzheimer's disease (AD) and other neurological disorders. Furthermore, EUK4010 and its analogues could potentially be developed as neuronal protective agents for the treatment of these diseases.     

Neuronal failure in Alzheimer’s disease: a view through the oxidative stress looking-glass

Considerable debate and controversy surround the cause(s) of Alzheimer’s disease (AD). To date, several theories have gained notoriety, however none is universally accepted. In this review, we provide evidence for the oxidative stress-induced AD cascade that posits aged mitochondria as the critical origin of neurodegeneration in AD.