Exogenous brain-derived neurotrophic factor attenuates cognitive impairment induced by okadaic acid in a rat model of Alzheimer's disease

Decreased expression of brain-derived neurotrophic factor(BDNF) plays an important role in the pathogenesis of Alzheimer's disease, and a typical pathological change in Alzheimer's disease is neurofibrillary tangles caused by hyperphosphorylation of tau. An in vivo model of Alzheimer's disease was developed by injecting okadaic acid(2 μL) and exogenous BDNF(2 μL) into the hippocampi of adult male Wister rats. Spatial learning and memory abilities were assessed using the Morris water maze. The expression levels of protein phosphatase 2 A(PP2 A), PP2 Ac-Yp307, p-tau(Thr231), and p-tau(Ser396/404) were detected by western blot assay. The expression levels of BDNF, TrkB, and synaptophysin mRNA were measured by quantitative real-time polymerase chain reaction. Our results indicated that BDNF expression was suppressed in the hippocampus of OA-treated rats, which resulted in learning and memory deficits. Intra-hippocampal injection of BDNF attenuated this OA-induced cognitive impairment. Finally, our findings indicated an involvement of the PI3 K/GSK-3β/AKT pathway in the mechanism of BDNF in regulating cognitive function. These results indicate that BDNF has beneficial effect on Alzheimer's disease, and highlight the potential of BDNF as a drug target for treatment of Alzheimer's disease.     

Mutations of beta-amyloid precursor protein alter the consequence of Alzheimer's disease pathogenesis

Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β(Aβ). Approximately 25 mutations in β-amyloid precursor protein(APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations(A673T, A673 V, E682 K, E693 G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ_(1–40) and Aβ_(1–42) levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673 T mutation decreases Aβ_(42)/Aβ_(40) rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673 V, E682 K, and E693 Q mutations promote Aβ_(42)/Aβ_(40) rate by increasing levels of CTF99, Aβ_(42), Aβ_(40), and IAT, and decreasing VVIA levels. Pathogenic E693 G mutation shows no significant change in Aβ_(42)/Aβ_(40) ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ_(42/40) rate, thereby resulting in Alzheimer's disease.     

MicroRNA-98 induces an Alzheimer’s disease-like disturbance by targeting insulin-like growth factor 1

Alzheimer ’s disease（ A D） is a progressive neurodegenerative disorder characterized by extracellular senile plaques and intracellular neurofibrillary tangles.Many microRNAs（miRs） participate in regulating amyloid β（Aβ） formation and the metabolism of tau protein in the process of AD,and some are up-regulated in AD patients or transgenic models of AD.However,the role of miR-98 in AD remains unclear.Here,we showed that the expression of miR-98 was negatively correlated with the insulin-like growth factor 1（IGF-1） protein level in APP/PS1 mice.MiR-98 target sites in IGF-1 were confirmed by luciferase assay in HEK293 cells.Overexpression of miR-98 in N2a/APP cells down-regulated the IGF-1 protein level and promoted Aβ production,whereas inhibition of miR-98 in N2a/APP cells up-regulated the IGF-1 protein level and suppressed Aβ production.Furthermore,overexpression of miR-98 in N2a/WT cells increased the phosphorylation of tau,whereas inhibition of miR-98 reduced it.These results suggest that miR-98 increases Aβ formation and tau phosphorylation by inhibiting the translation of IGF-1,which might provide a therapeutic strategy for AD.     

Tau Modulates Neurovascular Coupling

The microtubule-associated protein tau was identified in the neurofibrillary tangles(NFTs)of Alzheimer's disease(AD)more than 30 years ago,and its mutation can directly cause neurodegeneration.As implied by its purification from microtubules,tau was thought to stabilize microtubules and therefore to be of crucial importance in the brain.However,the physiological function of tau is still in debate since there is no detectable deficit in tau-knockout mice until they are 12 months old[1].Recently.     

A viral vector expressing hypoxia-inducible factor 1 alpha inhibits hippocampal neuronal apoptosis

Hypoxia-inducible factor 1(HIF-1) attenuates amyloid-beta protein neurotoxicity and decreases apoptosis induced by oxidative stress or hypoxia in cortical neurons. In this study, we constructed a recombinant adeno-associated virus(rAAV) vector expressing the human HIF-1α gene(rAAV-HIF-1α), and tested the assumption that rAAV-HIF-1α represses hippocampal neuronal apoptosis induced by amyloid-beta protein. Our results confirmed that rAAV-HIF-1α significantly reduces apoptosis induced by amyloid-beta protein in primary cultured hippocampal neurons. Direct intracerebral rAAV-HIF-1α administration also induced robust and prolonged HIF-1α production in rat hippocampus. Single rAAV-HIF-1α administration resulted in decreased apoptosis of hippocampal neurons in an Alzheimer's disease rat model established by intracerebroventricular injection of aggregated amyloid-beta protein(25–35). Our in vitro and in vivo findings demonstrate that HIF-1 has potential for attenuating hippocampal neuronal apoptosis induced by amyloid-beta protein, and provides experimental support for treatment of neurodegenerative diseases using gene therapy.     

MPP+活化GSK-3β诱导PC12细胞内质网的应激

1-methyl-4-phenylpyridinium ion (MPP+) induces endoplasmic reticulum stress and activates caspase-12 in PC12 cells, leading to neuronal apoptosis. However, the underlying molecular mechanism remains unknown. The present study investigated the regulatory effects of nerve growth factor (Akt activator) and lithium chloride (glycogen synthase kinase-3β inhibitor) on the endoplasmic reticulum stress signaling pathway. The results revealed that MPP+ induced expression of Bip and C/EBP homologous protein. The upregulation of Bip and C/EBP homologous protein, as well as the decreased pro-caspase-12 level induced by MPP+ were inhibited by pretreatment of the nerve growth factor or lithium chloride. These results suggest that the phosphatidylinositol 3 kinase-Akt- glycogen synthase kinase-3β pathway is involved in MPP+-induced endoplasmic reticulum stress.     

Mannotriose regulates learning and memory signal transduction in the hippocampus

Rehmannia is a commonly used Chinese herb, which improves ieaming and memory. However, the crucial components of the signal transduction pathway associated with this effect remain elusive. Pri- mary hippocampal neurons were cultured in vitro, insulted with high-concentration （1 × 10-4 mol/L） cor- ticosterone, and treated with 1 × 104 mol/L mannotriose. Thiazolyl blue tetrazolium bromide assay and western blot analysis showed that hippocampal neuron survival rates and protein levels of glucocorti- cold receptor, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor were all dramatically decreased after high-concentration corticosterone-induced injury. This effect was reversed by mannotriose, to a similar level as RU38486 and donepezil. Our findings indicate that mannotriose could protect hippocampal neurons from high-concentration corticosterone-induced injury. The mechanism by which this occurred was associated with levels of glucocorticoid receptor protein, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor.     

Down-regulation of insulin-degrading enzyme by presenilin 1 V97L mutant potentially underlies increased levels of amyloid beta 42

Amyloid beta (Abeta)42 plays a pivotal role in Alzheimer's disease. We previously reported a novel presenilin (PS)1 mutant (V97L) that was expressed in related patients with early onset Alzheimer's disease. We found that patients with the V97L mutation had increased levels of extracellular and intracellular Abeta42. Here we found that the increased extracellular level of Abeta42 was always accompanied by a reduction of insulin-degrading enzyme (IDE) activity on the plasma membranes. However, increase of intracellular Abeta42 was associated with decreased expression and activity of IDE in the cytosol and endoplasmic reticulum in the PS1 V97L mutant-transfected human SH-SY5Y cell line. These studies indicate that pathological levels of Abeta42 may be caused by the negative effects of PS1 (V97L) on IDE expression and activity. Our findings provide evidence for the molecular basis of familial Alzheimer's disease pathogenesis.     

雌激素阻抑OA诱导的SH-SY5Y细胞tau蛋白磷酸化

目的研究雌激素对冈田酸(OA)诱导的人神经母细胞瘤系(SH-SY5Y)细胞tau蛋白磷酸化的影响。方法 MTT观察OA对SH-SY5Y细胞活力的影响,制备AD时tau蛋白过度磷酸化的细胞模型;Western blot检测OA及雌激素对Thr231位点tau蛋白磷酸化的影响。结果 MTT结果表明:当OA浓度大于40nmol/L时,细胞活力受到明显抑制,低于此浓度的OA对细胞活力的影响不明显;Western blot结果表明,SH-SY5Y细胞经OA(40nmol/L 12h)处理后,tau蛋白磷酸化水平明显增加,这种作用可被雌激素所抑制。结论雌激素抑制了OA诱导的SH-SY5Y细胞的tau蛋白过度磷酸化。     

LRRK2干扰对MPP+诱导PD细胞模型线粒体功能的影响

目的 探讨富亮氨酸重复激酶2(LRRK2)干扰对1-甲基-4-苯基吡啶离子(MPP+)诱导的帕金森病(PD)细胞模型线粒体功能及CaMKK-β/AMPK通路的影响.方法 采用MPP+诱导传代培养的SH-SY5Y细胞构建PD细胞模型.将造模的SH-SY5Y细胞随机分PD模型组、空载转染组(空载转染PD模型细胞)和siRN...     

丙酮酸乙酯对鱼藤酮诱导的多巴胺能神经细胞损伤的保护作用

目的 建立鱼藤酮(Rotenone, Rot)诱导的SH-SY5Y细胞模型,探讨丙酮酸乙酯(Ethyl pyruvate,EP)对多巴胺能神经细胞的保护作用及机制。方法 Rot作用于SH-SY5Y细胞,构建Rot诱导的PD细胞模型,使用不同浓度的EP作用于PD细胞模型,MTT法检测细胞活力,Annexin V/PI染色检测细胞凋亡,免疫印迹法检测LC3 Ⅱ/Ⅰ、P62蛋白的表达,免疫荧光染色观察HMGB1的表达及定位。结果 EP减轻了Rot引起的多巴胺能神经细胞凋亡,并提高了细胞活力; EP减少了Rot诱导的LC3 Ⅱ/Ⅰ蛋白表达比例增高,增加P62蛋白的表达,且与EP的水平呈正相关。激光共聚焦显微镜观察到EP抑制了HMGB1的胞浆转位。结论 EP可能通过抑制HMGB1的转位来减少Rot诱导的自噬性损伤,从而对多巴胺能神经细胞起到保护作用。     

亨廷顿舞蹈病的IT15基因片段真核表达载体构建及其在SH-SY5Y细胞中的表达定位

目的 构建亨廷顿舞蹈病的致病基因IT15基因片段的真核表达载体,观察其在SH-SY5Y细胞内的表达分布情况.方法 以PCR方法扩增出健康人和亨廷顿舞蹈病患者的IT15基因1号外显子片段,应用基因重组技术与绿色荧光蛋白(GFP)基因融合构建以GFP为报告基因的重组真核表达质粒GFP-Htt-19Q与GFP-mHtt-70Q,并经酶切分析和测序证实.利用脂质体瞬时转染人神经母细胞瘤细胞SH-SY5Y细胞;Western blot证实亨廷顿蛋白氨基末端片段在SH-SY5Y细胞内表达;荧光显微镜观察正常与变异亨廷顿蛋白氨基末端片段在细胞内的表达定位情况.结果 健康人和亨廷顿舞蹈病患者的IT15基因1号外显子的PCR产物分别为170、310 bp的特异性片段;重组体GFP-Htt-199与GFP-mHtt-70Q经测序分析读码框正确,转染SH-SY5Y细胞后发现正常亨廷顿蛋白氨基末端片段弥散分布于胞质内,胞核内少见,而变异亨廷顿蛋白氨基末端片段在核周及核内形成聚集物.结论 构建IT15基因片段真核表达载体是研究亨廷顿舞蹈病的发病机制的有效方法之一;变异亨廷顿蛋白氨基末端片段在核周及核内形成聚集物可能是亨廷顿舞蹈病致病的关键.     

Notch-1 activation by familial Alzheimer's disease (FAD)-linked mutant forms of presenilin-1

We prepared a cleavage site-directed antibody against Notch-1, that specifically recognized the cleaved Notch-1 intracellular domain (NICD). To assess Notch-1 processing and its nuclear localization in familial Alzheimer's disease (FAD)-linked presenilin-1 (PS-1) mutants, we overexpressed wild type, M146V, A246E, C410Y, or deltaE9 PS-1 mutant with a membrane-bound Notch-1 in a PS-1-deficient cell line. On Western blot and immunocytochemical analyses using the NICD specific antibody, M146V and A246E mutants showed the comparable levels of Notch-1 processing and nuclear localizing activities to wild type PS-1 whereas C410Y and deltaE9 mutants failed to show these activities. These results suggest that the loss or partial loss of PS-1 activities in Notch-1 proteolysis and its nuclear translocation may be irrelevant for the neuropathology of Alzheimer's disease.     

SH-SY5Y神经细胞中α7神经型尼古丁受体基因过表达对CaMK Ⅱ和CREB的影响

目的研究SH-SY5Y神经细胞中α7 nAChR基因过表达对CaMKⅡ和CREB的影响。方法复苏稳定转染α7 nAChR-pc DNA3.1质粒及空载质粒的SH-SY5Y神经细胞后,用含G418的培养液进行筛选培养;应用实时荧光定量PCR法和蛋白印迹法(Western blot)检测α7 nAChR基因过表达组、空载质粒组和正常对照组细胞中CaMKⅡ、CREB mRNA及蛋白表达水平的变化。结果与对照组相比,α7 nAChR基因过表达细胞组的CaMKⅡ、CREB mRNA表达水平分别增加了116.8%和114.7%(P0.01);及CaMKⅡ、CREB蛋白表达水平分别增加了8.7%(P0.05)和41.4%(P0.05)。结论α7 nAChR的神经保护作用可能与上调细胞CaMKⅡ、CREB水平有关。