Probing the Secrets of Alzheimer’s Disease Using Human-induced Pluripotent Stem Cell Technology

Our understanding of Alzheimer’s disease (AD) is still incomplete and, as a result, we lack effective therapies. Reprogramming to generate human-induced pluripotent stem cells provides a new approach to the generation of human neurons that carry the genomes of people with familial or sporadic AD. Differentiation of such stem cells to human neurons is already providing new insights into AD and molecular pathways that may provide new targets for effective therapy. These pathways include typical amyloid response pathways, as well as pathways leading from altered behavior of amyloid precursor protein to the elevated phosphorylation of tau protein. There is also a need for standardization of models so that isogenic lines differing only in the familial AD mutation can be compared.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0326-6) contains supplementary material, which is available to authorized users.     

The role of genetic variation across IL-1β, IL-2, IL-6, and BDNF in antipsychotic-induced weight gain

Objectives. Antipsychotics with high weight gain-inducing propensities influence the expression of immune and neurotrophin genes, which have been independently related to obesity indices. Thus, we investigated whether variants in the genes encoding interleukin (IL)-1β, IL-2, and IL-6 and brain-derived neurotrophic factor (BDNF) Val66Met are associated with antipsychotic-induced weight gain (AIWG). Methods. Nineteen polymorphisms were genotyped using Taqman^® assays in 188 schizophrenia patients on antipsychotic treatment for up to 14 weeks. Mean weight change (%) from baseline was compared across genotypic groups using analysis of covariance (ANCOVA). Epistatic effects between cytokine polymorphisms and BDNF Val66Met were tested using Model-Based Multifactor Dimensionality Reduction. Results. In European patients, IL-1β rs16944*GA (P = 0.013, P_corrected = 0.182), IL-1β rs1143634*G (P = 0.001, P_corrected = 0.014), and BDNF Val66Met (Val/Val, P = 0.004, P_corrected = 0.056) were associated with greater AIWG, as were IL-1β rs4849127*A (P = 0.049, P_corrected = 0.784), and IL-1β rs16944*GA (P = 0.012, P_corrected = 0.192) in African Americans. BDNF Val66Met interacted with both IL-1β rs13032029 (Val/Met+ TT, P_Perm = 0.029), and IL-6 rs2069837 (Val/Val+ AA, P_Perm = 0.021) in Europeans, in addition to IL-1β rs16944 (Val/Val+ GA, P_Perm = 0.006) in African Americans. Conclusions. SNPs across IL-1β and BDNF Val66Met may influence AIWG. Replication of these findings in larger, independent samples is warranted.     

钙激活中性蛋白酶-2对整合素β4水解的影响

目的:观察乳腺癌细胞系MCF7细胞中,钙激活中性蛋白酶2(calpain-2)对整合素(integrin)β4水解的影响。方法:利用"短发夹"RNA(shRNA)和微小RNA(mirRNA)技术结合的calpain-2基因沉默(gene silencing)慢病毒质粒(GIPZ lentiviral shRNAmir)转染乳腺癌细胞株MCF7,嘌呤霉素筛选稳定沉默calpain-2基因的细胞株,细胞株传4代,用荧光显微镜观察转染效率,用蛋白质印迹(Western blot)实验检测calpain-2基因沉默效率及integrinβ4水解的情况。结果:嘌呤霉素筛选、细胞传4代,荧光显微镜下观察显示转染和筛选后,EGFP表达阳性的MCF7细胞的比例分别达到(95.6±2.6)%(空shRNAmir载体)、(97.1±1.7)%(Calpain-2 shRNAmir1)和(97.7±0.2)%(Calpain-2 shRNAmir 2),差异无统计学意义(P>0.05);Western blot结果显示,基因沉默的MCF7细胞中calpain-2的表达被明显抑制,相对于空shRNAmir载体转染的MCF7细胞,差异有统计学意义(P<0.01),calpain-2的表达下调到21.5%(Calpain-2 shRNAmir 1)和18.8%(Calpain-2 shRNAmir 2),空shRNAmir载体转染的MCF7细胞中calpain-2的表达与未转染的MCF7细胞比较,差异无统计学意义(P>0.05);比较calpain-2基因沉默和空shRNAmir载体转染的MCF7细胞,发现integrinβ4均被水解,水解片段的分子量主要有200 k D、130和95 k D;calpain-2基因沉默后,calpain-2基因沉默MCF7细胞中200 k D的水解片段比空shRNAmir载体转染的MCF7细胞减少(P<0.01)。结论:在乳腺癌细胞MCF7中,calpain-2可能参与integrinβ4的200 k D片段的形成,从而参与调整integrinβ4的构象变化。     

人羊膜上皮细胞移植改善AD样病变模型大鼠学习记忆能力

目的:观察人羊膜上皮细胞(h AECs)对阿尔茨海默病(AD)样病变大鼠模型的治疗效应。方法:采用胰蛋白酶消化法分离h AECs,流式细胞术分析表型。48只雄性SD大鼠随机分为假手术组、模型组、培养基组和h AECs移植组,每组12只。采用双侧脑室注入脂多糖(LPS)复制AD样病变大鼠模型。AD样病变大鼠海马区移植5×105个h AECs。细胞移植后2周,Morris水迷宫试验观察行为学变化,HE和硫磺素S染色观察海马病理变化,免疫组化染色检测β-淀粉样蛋白42(Aβ42)、Tau蛋白和乙酰胆碱(ACh)的变化,流式细胞术检测外周血T淋巴细胞亚群的变化,流式微球阵列捕获技术(cytometric bead array,CBA)检测血清细胞因子含量,免疫荧光染色检测海马区人细胞核抗原阳性细胞及其神经元特异性核蛋白(Neu N)的表达。结果:与模型组和培养基组比较,h AECs移植组大鼠逃避潜伏期明显缩短(P0.01),跨域平台次数明显增加(P0.05);海马神经元病损减轻,Aβ沉积减轻(P0.05),磷酸化Tau蛋白水平下降(P0.05),ACh增加(P0.05);外周血Th1和Th17细胞百分比下降(P0.05),而Th2和Treg细胞升高(P0.05);IL-2和IFN-γ水平下调(P0.05),而IL-4上调(P0.05);移植区可见h AECs,并表达Neu N。结论:h AECs可明显改善AD样病变模型大鼠空间辨别性学习记忆能力,减轻海马病理损伤,其免疫调节效应可能发挥重要作用。     

电针对SAMP8小鼠行为学及APP mRNA和BACE-1 mRNA影响

目的　探讨电针对SAMP8 小鼠行为学及淀粉样前体蛋白( amyloid precursor protein, APP) 和APP切割酶1(B site APP cleaving enzyme1, BACE-1)的影响,为临床治疗阿尔茨海默病病提供新的途径。 方法　将快速老化模型小鼠(SAMP8) 60只随机分成模型组、电针组和西药组,每组20只,正常老化小鼠 (SAMR1) 20只作为正常组。电针组电针刺激“百会”、 “肾俞”、“内关”、“大椎”穴;西药物组给予石杉碱甲 0.02 mg/kg剂量灌胃;正常组和模型组不给予治疗。治疗 20 d 后,用 Morris 水迷宫检测小鼠学习记忆能力,免疫组化定性检测小鼠海马APP和 BACE-1蛋白的表达,进一步实时荧光定量 PCR法测定APP mRNA和 BACE-1 mRNA 的表达。 结果　与模型组比较,西药组与电针组逃避潜伏期缩短,有效区停留时间和及跨越平台次数均显著增加(P＜0.05),电针组APP mRNA 和 BACE-1 mRNA 相对表达量显著减少(P<0.05) 。 结论　电针可能抑制 SAMP8 小鼠脑内APP mRNA and BACE-1 mRNA表达,减少Aβ 生成,改善其学习记忆障碍。     

IL-8对肺癌NCI-H157细胞增殖和迁移的影响

目的 研究IL-8对肺癌细胞增殖和迁移的影响,初步探讨IL-8调控肺癌细胞的分子机制.方法 体外培养肺癌NCI-H157细胞,用不同浓度的IL-8刺激肺癌细胞,分别用MTT法检测IL-8对肺癌细胞的增殖作用;划痕损伤实验和Transwdl小室两种方法检测肺癌细胞的迁移能力;Western blot检测Rac1和Cdc 42蛋白的表达变化.结果 IL-8促进NCI-H157细胞增殖,但随浓度增加,细胞增殖活性差异无统计学意义;细胞划痕损伤和Transwell实验均表明IL-8可诱导NCI-H157细胞迁移,且具有浓度依赖性;Western blot结果显示,随着IL-8浓度增加,Rac1和Cdc 42的表达水平逐渐升高,以Cdc42变化最为显著.结论 IL-8可促进肺癌细胞增殖和迁移,可能与Rac1和Cdc42表达有关.     

Myr-RKEFAK肽选择性调控血小板外向内信号转导相关功能

目的:研究踝蛋白杆部整合素结合位点2与整合素β3相互作用对于血小板信号转导的影响。方法:设计合成模拟整合素β3胞浆近膜端α螺旋上6个氨基酸序列(R724KEFAK729)的寡肽,并通过十四烷酰化修饰以使其具有穿细胞膜性(myr-RKEFAK肽)。观察myr-RKEFAK肽对经典血小板外向内信号转导事件(固相纤维蛋白原上的稳定黏附和伸展、二相聚集、纤维蛋白凝块回缩),以及内向外信号转导事件(一相聚集、游离纤维蛋白原的结合)的影响。结果:myr-RKEFAK肽可以浓度依赖性地抑制血小板固相化纤维蛋白原上稳定黏附和伸展、二相聚集以及纤维蛋白凝块回缩等外向内信号转导功能;但不影响游离纤维蛋白原结合和一相聚集等内向外信号转导功能。结论:穿膜肽myr-RKEFAK对血小板的外向内信号转导相应功能产生抑制,但不影响血小板内向外信号转导相关功能。     

Cdc42: Role in Cancer Management

Contribution of Cdc42, a member of Rho family, has been characterized for the beginning of variety of cellular responses including cellular transformation, cell division, cell invasion, migration, invadopodia formation, enzyme activity, filopodia formation, and cell polarity in cells. Deregulation of Cdc42 can alter the normal functioning of the cells, responsible for the initiation of signaling pathways and is correlated with several pathogenic processes such as cancer. Therefore, maintaining the level of Cdc42 and its effectors in cells, tumor progression can be controlled. Therefore, it can be suggested that deeper understanding about the Cdc42 contribution in cancer cell progression at molecular level can approach to the development of Cdc42 inhibitors in cancer management.     

电针对局灶性脑缺血再灌注大鼠皮质Slit-Robo三磷酸鸟苷酶激活蛋白-1和细胞分裂周期蛋白42表达的影响

目的:观察电针对大脑中动脉闭塞(MCAO)模型大鼠皮质Slit-Robo三磷酸鸟苷酶激活蛋白-1(Slit-Robo GTPase-activating protein-1,srGAP 1)和细胞分裂周期蛋白42(cell division-cycle 42,Cdc 42)表达的影响,探讨srGAP 1和Cdc 42在电针治疗MCAO后神经恢复中的作用。方法:雄性SD大鼠随机分为正常组、模型组、非经穴组和电针组,每组12只。利用改进的Longa法复制MCAO模型。电针组取"曲池""足三里"穴,非经穴组取手足阳明经与少阳经之间非经穴处,每日1次,每次30min,共14d。分别在术后1、3、7、14d时间点对各组大鼠进行神经功能评分(mNSS),并利用免疫荧光法检测缺血侧大脑皮质中srGAP 1和Cdc 42的荧光强度及分布,用免疫印迹(Western blot)法检测大鼠梗死区周围大脑皮质中srGAP1和Cdc 42蛋白表达。结果:神经功能评分显示,在术后7、14d时电针组较模型组、非经穴组同时间段的mNSS评分显著降低(P0.01)。免疫荧光检测结果显示,srGAP 1和Cdc 42主要在细胞质表达;模型组srGAP 1高于正常组(P0.01),而电针组显著低于模型组和非经穴组(P0.01);模型组Cdc 42显著低于正常组(P0.01),而电针组明显高于模型组和非经穴组(P0.01)。Western blot检测结果显示,模型组srGAP 1蛋白表达量较正常组明显升高(P0.01),电针组srGAP 1蛋白表达显著低于模型组及非经穴组(P0.01);模型组Cdc 42蛋白表达量与正常组的差异无统计学意义(P0.05),而电针组Cdc 42蛋白表达显著高于模型组和非经穴组(P0.01)。结论:局灶性脑缺血再灌注后大鼠脑损区周围皮质srGAP 1明显增高,而Cdc 42明显减少,这可能与srGAP 1使Cdc 42失活有关;电针治疗后srGAP 1显著下调,而Cdc42上调,可能是电针促进脑梗死后神经功能恢复的机制之一。     

Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin

BACKGROUND & AIMS: Obesity is one of the risk factors for liver fibrosis, in which plasma adiponectin, an adipocytokine, levels are decreased. Hepatic stellate cells play central roles in liver fibrosis. When they are activated, they undergo transformation to myofibroblast-like cells. Adiponectin suppresses the proliferation and migration of vascular smooth muscle cells, whose characteristics are similar to those of hepatic stellate cells. Adiponectin could have biological significances in liver fibrosis. METHODS: The role of adiponectin on liver fibrosis induced by the administration of carbon tetrachloride twice a week for 12 weeks was tested by using adiponectin-knockout mice and an adenovirus-mediated adiponectin-expression system. We also investigated the effect of adiponectin in activated hepatic stellate cells. RESULTS: When mice were administered carbon tetrachloride (300 microL/kg body weight) twice a week for 12 weeks, knockout mice showed extensive liver fibrosis with an enhanced expression of transforming growth factor-beta 1 and connective tissue growth factor compared with wild-type mice (P < 0.05). Injection of adenovirus producing adiponectin (AdADN) before carbon tetrachloride (1000 microL/kg body weight) treatment prevented liver fibrosis in wild-type mice (P < 0.001). Injection of AdADN at 6 weeks attenuated liver fibrosis even though carbon tetrachloride was given for an additional 6 weeks (total of 12 weeks). In cultured hepatic stellate cells, adiponectin suppressed platelet-derived growth factor-induced proliferation and migration and attenuated the effect of transforming growth factor-beta 1 on the gene expression of transforming growth factor-beta 1 and connective tissue growth factor and on nuclear translocation of Smad2. CONCLUSIONS: The findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention.