老年性痴呆相关基因Presenilins研究进展

新发现的与老年性痴呆（Ａｌｚｈｅｉｍｅｒ’ｓ ｄｉｓｅａｓｅ，ＡＤ）相关的两个基因 Ｐｒｅｓｅｎｉｌｉｎ和Ｐｒｅｓｅｎｉｌｉｎ２是目前国外分子生物学及老年医学的一个研究热点，本文就这两个基因的分子结构、基因突变、可能的功能和致病机理以及研究进展作一综述。

Sex effect on presenilins expression in post-natal rat brain

Presenilin 1 and presenilin 2 are widely expressed during brain development. Several mutations in these proteins have been associated with autosomal-dominant inherited forms of Alzheimer disease. Their expression is regulated by various cellular and extracellular factors, which change with age and sex. Both age and sex are key risk factors for Alzheimer’s disease, but the issue of whether the expression of presenilins is influenced by the sex during early postnatal development of the brain has been poorly investigated so far. In this study, we report that transcript levels of presenilins, and the subset of neurons expressing these proteins in various brain areas of the developing post-natal brain are different in male and female rats, suggesting that their function(s) may contribute to sexual dimorphism in the brain, both at morphological and functional levels.

Presenilins as endoplasmic reticulum calcium leak channels and Alzheimer's disease pathogenesis

Alzheimer disease(AD) is the most common neurodegenerative disorder worldwide and is at present,incurable.The accumulation of toxic amyloid-beta(Aβ) peptide aggregates in AD brain is thought to trigger the extensive synaptic loss and neurodegeneration linked to cognitive decline,an idea that underlies the'amyloid hypothesis'of AD etiology in both the familal(FAD) and sporadic forms of the disease.Genetic mutations causing FAD also result in the dysregulation of neuronal calcium(Ca2+) handling and may contribute to AD pathogenesis,an idea termed the'calcium hypothesis'of AD.Mutations in presenilin proteins account for majority of FAD cases.Presenilins function as catalytic subunit ofγ-secretase involved in generation of Aβ peptide Recently,we discovered that presenilns function as low-conductance,passive ER Ca2+ leak channels,independent of γ-secretase activity.We further discovered that many FAD mutations in presenilins result in loss of ER Ca2+ leak function activity and Ca2+ overload in the ER.These results provided potential explanation for abnormal Ca2+ signaling observed in FAD cells with mutations in presenilns.Our latest work on studies of ER Ca2+ leak channel function of presenilins and implications of these findings for understanding AD pathogenesis are discussed in this article.

Presenilins条件性双基因敲除小鼠海马与皮层超微结构的增龄性变化

为研究Presenilins条件性双基因敲除对于小鼠海马与皮层超微结构的影响,选用3、6及12月龄Presenilins 1/Presenilins 2双敲除小鼠(dKO)和同窝对照小鼠(CON),运用透射电子显微镜技术,分别观察海马与皮层突触、细胞核、线粒体、溶酶体超微结构的改变.结果发现:3月龄dKO小鼠海马与皮层溶酶体已向次级溶酶体转化.6月龄dKO小鼠海马与皮层的突触后致密物厚度显著降低;皮层细胞核膜内陷,核不规则;海马与皮层线粒体出现肿胀、嵴变形或消失;出现高电子密度的次级溶酶体,并伴有脂褐素小体出现.12月龄dKO小鼠海马与皮层突触后致密物厚度显著减小,突触间隙宽度显著增加;海马与皮层核膜内陷,染色质固缩沿核膜分布;线粒体严重受损,嵴大部分溶解;出现较多次级溶酶体和脂褐素小体.Presenilins条件性双基因敲除对小鼠海马与皮层有增龄性的病理影响,这些病理改变将为相关的药物评价和对阿尔茨海默病的深入研究提供形态学依据.

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.

Presenilin-1/Presenilin-2双基因敲除小鼠脑中单胺类神经递质变化的研究

条件性presenilins双基因敲除小鼠(dKO小鼠)表现出类似阿尔茨海默症(AD)的大部分神经退行性病症,如Tau蛋白磷酸化、神经元凋亡、皮层萎缩以及认知能力受损等.为探讨presenilins功能缺失、神经退行性症状与单胺类递质变化的相关性,利用毛细管电泳法检测6、9和12月龄dKO小鼠皮层、海马及其他前脑部位中各单胺类神经递质的含量.结果显示,与对照组相比,dKO小鼠皮层中单胺类神经递质在6月龄时显著降低,而随着年龄的增长,神经退行性病变加剧,递质浓度也均明显上升,在海马区,dKO小鼠单胺类递质则呈上升趋势,但仅6月龄时5-羟色胺和肾上腺素及12月龄时各递质的上升有统计学意义,前脑其他部位5-羟色胺和多巴胺递质在6、9月龄时与对照组相近,在12月龄时则显著降低,而去甲肾上腺素和肾上腺素在6月和12月龄时均呈降低趋势,且均有统计学差异(6月龄肾上腺素除外).实验表明,单胺类神经递质在presenilins双基因敲除的小鼠前脑各区域中的水平均发生了随龄化的变化,且在前脑皮层、海马与前脑其他区域的变化趋势各有不同,而单胺类递质的变化是presenilins双基因敲除导致的直接结果还是间接结果,单胺类递质在AD样神经退性行病变中的作用如何,还有待于进一步的研究.

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。

Alzheimer’s disease:epidemiology,genetics,and beyond

Alzheimer＇s disease （AD） is an increasing epidemic threatening public health. Both men and women are susceptible to the disease although women are at a slightly higher risk. The prevalence of AD rises exponentially in elderly people from 1% at age of 65 to approximately 40%-50% by the age of 95. While the cause of the disease has not been fully understood, genetics plays a role in the onset of the disease. Mutations in three genes （APP, PSENI, and PSEN2） have been found to cause AD and APOE4 allele increases the risk of the disease. As human genomic research progresses, more genes have been identified and linked with AD. Genetic screening tests for persons at high risk of AD are currently available and may help them as well as their families better prepare for a later life with AD.

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。

膜蛋白Presenilin 1的跨膜结构及催化机制

膜蛋白presenilin1(PS1)是γ分泌酶的催化组分,是催化产生β淀粉样蛋白(β-amyloid,Aβ)的关键蛋白酶,因此也是治疗阿尔茨海默病(Alzheimer’s disease,AD)的主要靶点.PS1属于膜内裂解蛋白酶家族,这是一类在膜脂双层内部催化肽键水解断裂的蛋白酶.PS1其独特的跨膜结构和催化机制虽然还未完全揭示,但近期相关的研究取得了重要成果:PS1有10个疏水区,跨膜9次,其N端位于胞内,C端位于胞膜外或者内质网腔内,亦或不同程度地插入膜内,2个起催化作用的天冬氨酸残基都位于疏水性的膜内,膜蛋白底物被催化水解时必须先结合到酶的疏水表面上来,然后再进入位于活性部位.虽然PS1的晶体从未获得,但2006年首次解析的膜内裂解蛋白酶GlpG的晶体结构和所提出的催化机理为PS1催化机理的揭示奠定了基础,也为设计和筛选PS1/γ分泌酶的特异性抑制剂提供了理论依据.

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。

果蝇Presenilin蛋白影响神经肌肉接点分布

为观察 Presenilin蛋白对突触可塑性的影响 ,本研究以果蝇神经肌肉接点分布为模型 ,发现 Presenilin突变 Psns3可导致神经肌肉接点数量的明显减少 ,而且当提高温度后 ,可造成接点数量的进一步减少。提示 ,Presenilin蛋白参与了突触的形成过程 ,进而可能会影响到学习记忆。结果还提示 。

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.

Converging links between adult-onset neurodegenerative Alzheimer’s disease and early life neurodegenerative neuronal ceroid lipofuscinosis?

Evidence from genetics and from analyzing cellular and animal models have converged to suggest links between neurodegenerative disorders of early and late life.Here,we summarize emerging links between the most common late life neurodegenerative disease,Alzheimer’s disease,and the most common early life neurodegenerative diseases,neuronal ceroid lipofuscinoses.Genetic studies reported an overlap of clinically diagnosed Alzheimer’s disease and mutations in genes known to cause neuronal ceroid lipofuscinoses.Accumulating data strongly suggest dysfunction of intracellular trafficking mechanisms and the autophagy-endolysosome system in both types of neurodegenerative disorders.This suggests shared cytopathological processes underlying these different types of neurodegenerative diseases.A better understanding of the common mechanisms underlying the different diseases is important as this might lead to the identification of novel targets for therapeutic concepts,the transfer of therapeutic strategies from one disease to the other and therapeutic approaches tailored to patients with specific mutations.Here,we review dysfunctions of the endolysosomal autophagy pathway in Alzheimer’s disease and neuronal ceroid lipofuscinoses and summarize emerging etiologic and genetic overlaps.

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.

Presenilin与AD的分子病理机制

Presenilin即早老基因,该基因及其表达蛋白在阿尔茨海默病的分子病理机制中占重要地位,本文综述了近年关于Presenilin蛋白在β淀粉样前体蛋白代谢和Notch受体信号传导中的作用,以及Presenilin蛋白与细胞凋亡关系。

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.

S3A-2 The Effect of PHA-543613 on Memory Disorders in Presenilin1 and Presenilin2 Conditional Double Knockout Mice

The mutation in the amyloid-beta precursor protein(APP)and presenilin genes(PSEN1 and PSEN2)cause autosomal dominant Alzheimer’s diease(ADAD)which is typically associated with early-onset familial Alzheimer’s disease(FAD),however,the mechanism by which presenilin mutations cause memory disorders and neurodegeneration remains poorly understood.In the present study,using Presenilin-1 and Presenilin-2 double knockout mice(cDKO mice),we observed that the impaired spatial reference memory,spatial working memory and contextual fear memory in cDKO mice.Consistently,deficits of basal synaptic transmission and LTP formation,as well as down-regulation of PI3K/Akt signaling pathway at hippocampus in cDKO mice.Furthermore,we found the expression levels ofα7-nicotinic ACh receptors(α7nAChRs),NMDAR and AMPAR composition subunits,which related to synaptic plasticity and memory,were decreased at hippocampus in cDKO mice.Importantly,all above deficits could be reversed byα7nAChR agonist PHA-543613.Taken together,our results indicate that knockout of PS1 and PS2 can disrupt the function ofα7nAChR,thereby down-regulate activation of PI3K/Akt signaling pathway,reduce the synaptic expression levels of NMDAR and AMPAR composition subunits at hippocampus,consequently cause neuronal apoptosis,disrupt basal synaptic transmission and LTP formation at hippocampus,fi nally impair hippocampal-dependent memory.