爬梯运动提高Presenilin1/Presenilin2双敲鼠的学习记忆能力

目的 Presenilin1/Presenilin2双基因敲除(PS1/PS2DKO)可导致小鼠学习记忆能力受损,该文主要研究跑台运动和爬梯运动对PS1/PS2 DKO小鼠认知能力的影响,并通过检测运动前后各组小鼠体内神经胶质酸性蛋白(GFAP)、丙二醛(MDA)、超氧化物歧化酶(SOD)等含量的变化,探讨2种运动方式对DKO小鼠认知能力受损的干预效果及其可能的分子机制。方法将6个月龄雄性DKO小鼠及其同窝对照小鼠(CON小鼠)分为4组,分别为CON不运动组、DKO不运动组、DKO跑台组和DKO爬梯组。对DKO跑台组小鼠进行10周的跑台训练,对DKO爬梯组小鼠进行10周的爬梯训练。运动结束后以开场实验、新异物体识别实验和恐惧条件反射实验测试各组小鼠的运动能力和学习记忆能力;用Western blotting、硫代巴比妥酸法、黄嘌呤氧化酶法检测各组小鼠GFAP、MDA、SOD含量的变化。结果爬梯运动可明显改善DKO小鼠的恐惧学习记忆能力;跑台运动和爬梯运动均可降低DKO小鼠前脑中GFAP的含量,但前者也会增加前脑中脂质过氧化程度;而跑台运动和爬梯运动对DKO小鼠前脑中的超氧化物歧化酶活性无明显影响。结论爬梯运动可以改善DKO小鼠的恐惧学习记忆能力,前脑中星型胶质细胞活化程度的降低可能是其分子机制之一。跑台运动对DKO小鼠的学习记忆能力无影响,究其原因可能是跑台运动后DKO小鼠前脑产生大量的氧自由基,其导致的脂质过氧化引起神经细胞损伤,从而抵消了跑台运动对DKO小鼠学习记忆能力的改善作用。

回医烙灸疗法对脊髓损伤大鼠Notch信号通路中Presenilin1、Hes1蛋白表达的影响

目的回医烙灸疗法对脊髓损伤大鼠Notch信号通路中Presenilin1、Hes1蛋白表达的影响。方法采用Allen’s法制备急性脊髓损伤模型。实验动物分为假手术组(A组)、模型组(B组)、西药组(C组)、2 h烙灸组(D组)、6 h烙灸组(E组),于第1、7、14、28 d取损伤大鼠脊髓组织,免疫组化法检测大鼠脊髓组织中Presenilin1、Hes1蛋白表达。结果 Presenilin1、Hes1免疫组化染色统计结果表明D、E组小于B、C组,且D组表达率最显著减弱,差异有统计学意义(P <0.01)。结论回医烙灸疗法通过抑制Notch信号通路的表达,促进内源性神经干细胞向神经元细胞和少突胶质细胞的分化,以促进其局部神经的修复,且前期烙灸干预的效果好于后期干预。

Presenilin1基因对心肌细胞肌浆网钙容量的影响

目的:探讨Presenilin1基因对心肌细胞肌浆网钙容量的影响。方法:本实验以AAV-9为载体构建心肌特异性PS1-shRNA重组腺相关病毒,通过尾静脉注射PS1-shRNA重组腺相关病毒干扰大鼠心肌组织中Presenilin1基因的表达,并利用激光扫描共聚焦显微镜检测并记录心肌细胞肌浆网钙容量及舒张期自发钙释放事件的变化;同时,采用western blot技术分析各组大鼠心肌组织中雷诺丁受体及钙泵表达水平的改变。结果:与对照组相比,PS1-shRNA重组腺相关病毒干预组大鼠心肌细胞肌浆网钙容量显著降低,自发钙释放事件明显增加;雷诺丁受体表达水平略降低,钙泵表达水平无显著性变化。结论:Presenilin1基因表达水平降低会引起心肌细胞钙泄漏,从而造成心肌细胞肌浆网钙容量降低。

Treadmill exercise in combination with acousto-optic and olfactory stimulation improves cognitive function in APP/PS1 mice through the brain-derived neurotrophic factor-and Cygb-associated signaling pathways

A reduction in adult neurogenesis is associated with behavioral abnormalities in patients with Alzheimer's disease.Consequently,enhancing adult neurogenesis represents a promising therapeutic approach for mitigating disease symptoms and progression.Nonetheless,nonpharmacological interventions aimed at inducing adult neurogenesis are currently limited.Although individual non-pharmacological interventions,such as aerobic exercise,acousto-optic stimulation,and olfactory stimulation,have shown limited capacity to improve neurogenesis and cognitive function in patients with Alzheimer's disease,the therapeutic effect of a strategy that combines these interventions has not been fully explored.In this study,we observed an age-dependent decrease in adult neurogenesis and a concurrent increase in amyloid-beta accumulation in the hippocampus of amyloid precursor protein/presenilin 1 mice aged 2-8 months.Amyloid deposition became evident at 4 months,while neurogenesis declined by 6 months,further deteriorating as the disease progressed.However,following a 4-week multifactor stimulation protocol,which encompassed treadmill running(46 min/d,10 m/min,6 days per week),40 Hz acousto-optic stimulation(1 hour/day,6 days/week),and olfactory stimulation(1 hour/day,6 days/week),we found a significant increase in the number of newborn cells(5'-bromo-2'-deoxyuridine-positive cells),immature neurons(doublecortin-positive cells),newborn immature neurons(5'-bromo-2'-deoxyuridine-positive/doublecortin-positive cells),and newborn astrocytes(5'-bromo-2'-deoxyuridine-positive/glial fibrillary acidic protein-positive cells).Additionally,the amyloid-beta load in the hippocampus decreased.These findings suggest that multifactor stimulation can enhance adult hippocampal neurogenesis and mitigate amyloid-beta neuropathology in amyloid precursor protein/presenilin 1 mice.Furthermore,cognitive abilities were improved,and depressive symptoms were alleviated in amyloid precursor protein/presenilin 1 mice following multifactor stimulation,as evidenced by Morris water maze,novel object recognition,forced swimming test,and tail suspension test results.Notably,the efficacy of multifactor stimulation in consolidating immature neurons persisted for at least 2weeks after treatment cessation.At the molecular level,multifactor stimulation upregulated the expression of neuron-related proteins(NeuN,doublecortin,postsynaptic density protein-95,and synaptophysin),anti-apoptosis-related proteins(Bcl-2 and PARP),and an autophagyassociated protein(LC3B),while decreasing the expression of apoptosis-related proteins(BAX and caspase-9),in the hippocampus of amyloid precursor protein/presenilin 1 mice.These observations might be attributable to both the brain-derived neurotrophic factor-mediated signaling pathway and antioxidant pathways.Furthermore,serum metabolomics analysis indicated that multifactor stimulation regulated differentially expressed metabolites associated with cell apoptosis,oxidative damage,and cognition.Collectively,these findings suggest that multifactor stimulation is a novel non-invasive approach for the prevention and treatment of Alzheimer's disease.

Investigational treatments for neurodegenerative diseases caused by inheritance of gene mutations:lessons from recent clinical trials

We reviewed recent major clinical trials with investigational drugs for the treatment of subjects with neurodegenerative diseases caused by inheritance of gene mutations or associated with genetic risk factors.Specifically,we discussed randomized clinical trials in subjects with Alzheimer's disease,Huntington's disease and amyotrophic lateral sclerosis bearing pathogenic gene mutations,and glucocerebrosidase-associated Parkinson's disease.Learning potential lessons to improve future therapeutic approaches is the aim of this review.Two long-term,controlled trials on three anti-β-amyloid monoclonal antibodies(solanezumab,gantenerumab and crenezumab)in subjects carrying Alzheimer's disease-linked mutated genes encoding for amyloid precursor protein or presenilin 1 or presenilin 2 failed to show cognitive or functional benefits.A major trial on tominersen,an antisense oligonucleotide designed to reduce the production of the huntingtin protein in subjects with Huntington's disease,was prematurely interrupted because the drug failed to show higher efficacy than placebo and,at highest doses,led to worsened outcomes.A 28-week trial of tofersen,an antisense oligonucleotide for superoxide dismutase 1 in patients with amyotrophic lateral sclerosis with superoxide dismutase 1 gene mutations failed to show significant beneficial effects but the 1-year open label extension of this study indicated better clinical and functional outcomes in the group with early tofersen therapy.A trial of venglustat,a potent and brain-penetrant glucosylceramide synthase inhibitor,in Parkinson's disease subjects with heterozygous glucocerebrosidase gene mutations revealed worsened clinical and cognitive performance of patients on the enzyme inhibitor compared to placebo.We concluded that clinical trials in neurodegenerative diseases with a genetic basis should test monoclonal antibodies,antisense oligonucleotides or gene editing directed against the mutated enzyme or the mutated substrate without dramatically affecting physiological wild-type variants.

野生型与突变型PS1真核表达载体的构建及其在SH-SY5Y细胞中的表达

目的为了对我们发现的中国人家族性阿尔茨海默病早老素-1(presenilin1,PS1)基因新的点突变进行蛋白功能研究,分别构建野生型和突变型PS1(G289T)与绿色荧光蛋白(EGFP)共表达载体,并检测其在SHSY5Y细胞内的表达。方法利用含人全长PS1cDNA的pcDNA3·1(zeo+),采用定点突变技术,构建PS1(G289T)-pcDNA3·1(zeo+)载体。采用基因重组技术构建野生型和突变型PS1与绿色荧光蛋白共表达载体。应用脂质体将携带野生型和突变型PS1的质粒转染至SH-SY5Y细胞,检测报告基因表达,RT-PCR检测PS1mRNA表达。结果经限制性内切酶酶切图谱分析及DNA测序证实融合蛋白表达载体构建成功;RT-PCR产物经测序显示突变型PS1mRNA在SH-SY5Y中有表达。结论成功构建了人野生型和突变型PS1与EGFP共表达载体并成功转染至SH-SY5Y细胞,为进一步的研究工作奠定了基础。

鼠PS1-GFP融合蛋白表达载体的构建研究

Presenilin1(PS1)的突变是早发家族性老年痴呆发生的重要因素之一。目前对于PS1结构和功能的研究表明,PS1作为一种多次跨膜并具有γ-secretase活性的蛋白酶,很可能参与众多的细胞信号传导通路,影响细胞分化和调亡、组织及个体的发育等等。但对于PS1的精细结构和功能目前了解甚少,为探讨PS1的精细结构和功能,本研究构建了鼠PS1-GFP融合蛋白表达载体。方法:我们设计了扩增PS1的cDNA全长的引物,以C57BL/6小鼠9天胚的RNA为模板,利用RT-PCR的方法从C57BL/6小鼠9天胚中获得PS1的cDNA,经测序确认后,将其克隆到pMD18-TVector中。设计引物:上游5’-GCCGAATTCTATGACAGAGATACCTGCACC-3’;下游5’-GAAGGATCCGATATAAAACTGATGGAATGC-3’,上游含有EcoRI酶切位点,下游含有BamHI酶切位点。利用高保真DNA聚合酶通过PCR方法将pMD18-T-PS1质粒中的PS1基因亚克隆到pEGFP-C1载体中,获得pEGFP-C1-PS1融合蛋白表达载体,然后利用EcoRI和BamHI从pEGFP-C1-PS1融合蛋白中切下PS1基因,从pMD18-T-PS1质粒中切下载体部分,经过连接,获得中间载体,利用该重组载体中的EcoRI和SalI酶切位点,酶切连接入pEGFP-N2载体中,获得pEGFP-N2-PS1融合蛋白表达载体,经测序鉴定后备用。

PS1基因突变敲入小鼠B免疫记忆细胞功能变化

探讨PS1基因突变敲入小鼠B免疫记忆细胞功能变化。OVA免疫PS1基因突变敲入小鼠,通过ELISA抗体检测观察B细胞免疫记忆功能;分离小鼠脾淋巴细胞,检测脾淋巴细胞增殖实验、B细胞表面抗原表达情况。痘苗病毒免疫正常和基因突变敲入小鼠两次,在免疫后的一年进行痘病毒腹腔攻毒实验,观察PS1基因突变敲入小鼠存活情况。通过分离中枢和外周淋巴细胞进行体外功能检测表明,PS1基因突变敲入小鼠细胞增殖功能明显降低(P<0.0001);B记忆细胞表面蛋白表达有差异(P=0.045);痘苗攻毒实验结果表明,PS1基因突变敲入小鼠死亡率明显增高(P<0.0001)。研究结果表明,PS1基因在调控B记忆细胞功能方面发挥重要作用,但是通过调控T细胞功能而发挥间接还是直接针对B细胞发挥调控作用,还需要进一步研究结果确认。

MicroRNA and mRNA profiling of cerebral cortex in a transgenic mouse model of Alzheimer’s disease by RNA sequencing

In a previous study,we found that long non-coding genes in Alzheimer’s disease(AD)are a result of endogenous gene disorders caused by the recruitment of microRNA(miRNA)and mRNA,and that miR-200a-3p and other representative miRNAs can mediate cognitive impairment and thus serve as new biomarkers for AD.In this study,we investigated the abnormal expression of miRNA and mRNA and the pathogenesis of AD at the epigenetic level.To this aim,we performed RNA sequencing and an integrative analysis of the cerebral cortex of the widely used amyloid precursor protein and presenilin-1 double transgenic mouse model of AD.Overall,129 mRNAs and 68 miRNAs were aberrantly expressed.Among these,eight down-regulated miRNAs and seven up-regulated miRNAs appeared as promising noninvasive biomarkers and therapeutic targets.The main enriched signaling pathways involved mitogen-activated kinase protein,phosphatidylinositol 3-kinase-protein kinase B,mechanistic target of rapamycin kinase,forkhead box O,and autophagy.An miRNA-mRNA network between dysregulated miRNAs and corresponding target genes connected with AD progression was also constructed.These miRNAs and mRNAs are potential biomarkers and therapeutic targets for new treatment strategies,early diagnosis,and prevention of AD.The present results provide a novel perspective on the role of miRNAs and mRNAs in AD.This study was approved by the Experimental Animal Care and Use Committee of Institute of Medicinal Biotechnology of Beijing,China(approval No.IMB-201909-D6)on September 6,2019.

Presenilin mutations and their impact on neuronal differentiation in Alzheimer’s disease

The presenilin genes(PSEN1 and PSEN2)are mainly responsible for causing early-onset familial Alzheimer’s disease,harboring~300 causative mutations,and representing~90%of all mutations associated with a very aggressive disease form.Presenilin 1 is the catalytic core of theγ-secretase complex that conducts the intramembranous proteolytic excision of multiple transmembrane proteins like the amyloid precursor protein,Notch-1,N-and E-cadherin,LRP,Syndecan,Delta,Jagged,CD44,ErbB4,and Nectin1a.Presenilin 1 plays an essential role in neural progenitor maintenance,neurogenesis,neurite outgrowth,synaptic function,neuronal function,myelination,and plasticity.Therefore,an imbalance caused by mutations in presenilin 1/γ-secretase might cause aberrant signaling,synaptic dysfunction,memory impairment,and increased Aβ42/Aβ40 ratio,contributing to neurodegeneration during the initial stages of Alzheimer’s disease pathogenesis.This review focuses on the neuronal differentiation dysregulation mediated by PSEN1 mutations in Alzheimer’s disease.Furthermore,we emphasize the importance of Alzheimer’s disease-induced pluripotent stem cells models in analyzing PSEN1 mutations implication over the early stages of the Alzheimer’s disease pathogenesis throughout neuronal differentiation impairment.

Subcellular localization of PS1 based on PS1/GFP fusing protein

Mutations in presenilin 1(PS1)gene are closely associated with the early onset of familial Alzheimer’s disease(EOFAD).The fusion genes,GFP-PS1(recombinant plasmid pEGFP-C1-PS1)and PS1-GFP(recombinant plasmid pEGFP-N2-PS1)were constructed to study the subcellular localization of PS1 holoprotein.Recombinant plasmids were transiently transfected into two cell lines,HEK293 and CHO,respectively,using the green fluorescence from GFP(green fluorescence protein)as the PS1 localization signal.Then,we observed green fluorescence with a SPOT II(Olympus,BH2)and CONFOCAL microscope(Olympus,FV300)under 488 nm.The results show that PS1 located on the nuclear envelope.A few can be found on the cellular membrane and in the cytosol in a non-homogeneous distribution.

Human neuroblastoma cells transfected with two Chinese presenilin 1 mutations are sensitized to trophic factor withdrawal and protected by insulin-like growth factor-1

Background Two novel presenilin 1 （PS1） mutations, V97L and A136G, were recently found to be involved in the early-onset of Alzheimer＇s disease in two Chinese families. This research aimed to verity their pathological effects. Methods The human neuroblastoma SH-SY5Y cells stably transfected with these two Chinese presenilin 1 mutations were established to explore whether they are sensitive to, or influenced by, serum deprivation and protected by insulin-like growth factor-1 （IGF-1）. Apoptosis rate, glucose uptake of the cells and the expression of glucose transport protein 1 （GLUT1） on cell membranes were examined. Results The V97L or A136G mutants significantly decreased the cells viability and increased the apoptosis rate when compare to PSlwt and mock transfected cells. IGF-1 was found to improve the viability of these two kinds of mutant cells significantly, and to show a protective effect for the mutants when they were treated with trophic deprivation. The glucose uptake of each transfected cell line increased to about 25% after IGF-1 treatment, GLUT1 expression on the cell membrane increased modestly by about 15%-20%. Conclusions Enhanced sensitivity to trophic withdrawal in the cells transfected with the two Chinese PS1 mutations may contribute to the neuron apoptosis, IGF-1 provided a protective effect to cells, possibly through an enhanced glucose transport and mitochondrial activities.

Gene polymorphism in apolipoprotein E and presenilin-1 in patients with late-onset Alzheimer's disease

Objective To evaluate the association of apolipoprotein E (apoE) and presenilin 1 (PS 1) gene polymorphism with late onset Alzheimer's disease (AD) Methods A case control study was undertaken to detect the polymorphism of apoE and PS 1 by polymerase chain reaction and digestion with the endonucleases of BspL Ⅰ, Hha Ⅰ and BamH Ⅰ Results The frequencies of apoE ε3/4 genotype and ε4 allele in late onset AD (n=42) were significantly higher than those of age matched controls ( P <0 05) The frequencies of the apoE intron 1 enhancer (IE1) G/G genotype and G allele in late onset AD were also significantly higher than those in controls ( P <0 05) The frequencies of the PS 1 1/1 genotype but not the 1 allele in AD were significantly higher than those in controls ( P <0 05) The apoE ε4 allele was associated with a tripling of risk for late onset AD compared with that with no ε4 allele (odds ratio: 2 932) Homozygosity of the G allele in IE1 and 1/1 genotype in PS 1 was associated with a doubling of risk for late onset AD, and odds ratios were 2 223 and 2 066, respectively When the apoE ε4 was controlled, the association between the IE1 G/G genotype AD was no longer statistically significant ( P >0 05) We sequenced the exon 4 of apoE in patients with late onset AD, and found no other genetic polymorphism or mutation except for apoE ε4 and IE1 G alleles associated with AD Conclusion apoE ε4 gene appears to be the strongest gene risk factor for late onset AD and its apparent association between the IE1 G/G genotype and late onset AD is a consequence of the association between the ε4 and IE1 G/G genotype The PS 1/1 genotype is weakly associated with late onset AD