【作者】 王琪；

【导师】 杜蘅；

【作者基本信息】 山东大学 ， 神经病学， 2019， 博士

【摘要】 研究背景:阿尔茨海默病(Alzheimer’s disease,AD)是一种主要累及老年人的、以认知功能进行性下降为特征的神经退行性疾病。典型的AD病理表现包括脑内β-淀粉样蛋白(β-amloidy,A β)沉积形成的老年斑、Tau蛋白异常磷酸化导致的神经纤维缠结,以及突触损伤和神经元死亡。目前AD的发病机制尚不明确。淀粉样蛋白级联理论是目前医学界广为接受的一种假说。该假说认为β-淀粉样蛋白(β-amloidy,A β)生成与清除失衡导致的A β集聚是AD发生的起始事件,而A β42在其中发挥了关键作用。该假说进一步认为淀粉样前体蛋白(amyloid precursor protein,APP)基因、早老素 1(prescnilin 1,PS1)基因及早老素2(presenilin 2,PS2)基因突变是Aβ42产生的重要原因。但是研究发现散发性AD患者体内无APP或PS基因突变,因此该假说尚存在一定的局限性。近年来大量研究提示线粒体损伤是AD的重要病理改变之一,AD患者早期即可出现线粒体功能障碍,并且线粒体损伤随AD进展不断恶化最终影响突触及神经元功能。基于此,Swerdlow等人提出了适用于散发性AD的线粒体级联反应假说,该假说认为线粒体的功能障碍是导致AD发生的起始事件,Aβ沉积、tau磷酸化、神经元功能退化都是在线粒体功能障碍基础上发生的,而这些改变又进一步加重了线粒体的功能障碍。虽然线粒体级联反应假说也存在其固有的局限性,但其为AD的研究提供了新的思路。线粒体是神经元中重要的细胞器。除提供维持神经元功能所需的ATP外,线粒体通过提供能量、调节突触内钙稳态、介导突触/神经元核之间的对话以及调节氧自由基水平等功能,在维持突触可塑性和突触传递进而实现神经元功能中起到关键作用。值得注意的是线粒体的合成部位是神经元胞体,之后神经元将线粒体投送到突触部位。因此,正常的线粒体转运对维持突触功能至关重要;而线粒体转运障碍是导致神经元尤其是突触功能损伤的一个重要原因。近年来神经元线粒体转运障碍与AD状态下神经元损伤的关系不断得到关注。大量研究显示,线粒体运输障碍,尤其是线粒体顺轴向运输障碍,是导致突触功能障碍的原因之一。然而,神经元顺轴向运输障碍的具体分子机制目前尚未明了。KIF5A是kinesin-1家族的重要成员,特异性表达于各类神经元,参与神经元线粒体的顺轴向运输。既往研究表明,KIF5A表达下调可能是导致AD状态下神经元线粒体顺轴向运输障碍的原因。本文的目的是通过病理及分子生物学技术探究AD状态下,KIF5A导致神经元线粒体顺轴向运输障碍的具体分子机制。研究目的:1.明确AD状态下,参与神经元线粒体轴突运输相关蛋白的变化;2.明确AD状态下,KIF5A下降可能的机制;3.明确KIF5A下降对神经元线粒体顺轴向运输的影响;4.探究AD状态下,补充KIF5A对神经元线粒体顺轴向运输与神经元功能的影响。研究方法:1.人的样本:本研究人组织取自AD患者与认知功能健全者的颞叶与小脑组织,该部分组织来自于美国西南医学中心,并符合相关医学伦理要求。2.实验动物:本研究采用8月龄与12月龄的5xFAD小鼠用来模拟AD患者中晚期的病理改变。同时,采用出生24小时内的C57BL/6J小鼠用于原代神经元培养。3.原代神经元培养:使用出生24小时内的C57BL/6J小鼠,体外剥去脑膜,分离海马、大脑皮层,胰酶消化后种于含Neurobasal A、B27、L-谷氨酰胺、链霉素与青霉素的培养基中,置于含5%C02的37℃培养箱中。神经元种植密度为1×105/cm2(用于免疫蛋白印迹法检测)和0.1×105/cm2(线粒体运动分析)。4.免疫蛋白印迹法:利用Western Blot方法检测神经元线粒体相关蛋白表达水平的改变。5.免疫荧光染色:利用免疫荧光染色技术,分析8月龄与12月龄5xFAD小鼠脑内KIF5A表达水平的改变。6.慢病毒包被:目的质粒磷酸钙法转染HEK293T细胞,24小时后收集上清液,过滤、离心,所获沉淀即为慢病毒颗粒;7.KIF5A敲低模型构建:首先构建KIF5A靶向shRNA的慢病毒,并用其处理培养至第3天的原代神经元,持续作用7天后用于实验。8.KIF5A过表达模型构建:首先构建含有人KIF5A基因片段的慢病毒颗粒,并用其处理培养至第3天的原代神经元,作用7天后用于实验。9.A β 1-42寡聚体制备:用冰的HAM’S F-12溶解A β 1-42肽链,并用其处理神经元24小时,然后收集细胞用于实验。10.线粒体运动检测:使用尼康倒置荧光显微镜拍摄并分析线粒体运动相关指标;11.数据统计、分析:利用 Microsoft Excel(2016)与 Graphpad Prism5 进行数据的录入、统计与分析。两组间比较采用t检验,多组间比较采用单因素方差分析。P<0.0.5被认为有统计学意义。研究结果:1.KIF5A在AD患者颞叶组织内表达下调。利用免疫蛋白印迹法检测AD患者与认知功能健全者颞叶与小脑组织内线粒体转运相关蛋白KIF5A、KIF5B、KIF5C、Dynein、Syntabulin、Mirol以及线粒体标志物Tom40的表达水平,发现KIF5A在AD敏感脑区颞叶组织内下降明显,KIF5B在颞叶组织内有轻微下降,而小脑组织内没有观察到这种改变。进一步的相关性分析发现KIF5A表达水平的下降与年龄、死亡后取材时间无关。2.KIF5A下降与Aβ毒性密切相关。我们选用8月龄与12月龄5xFAD小鼠,利用免疫蛋白印迹与免疫荧光方法,发现KIF5A在小鼠皮层组织中的表达水平下降明显,KIF5B表达也有轻度下降,而KIF5C、Dynein、Syntabulin等蛋白表达水平没有明显改变。Mirol、Tom40则只有在12月龄小鼠皮层组织内出现表达下降。进一步用免疫蛋白印迹法检测Aβ处理的原代神经元,我们发现KIF5A表达水平仍然明显下降,而KIF5B、KIF5C、Dynein、Syntabulin等蛋白表达水平没有明显改变。Mirol表达水平轻度下调。3.KIF5A表达水平下调阻碍了神经元线粒体顺轴向运输。我们发现KIF5A表达下调降低了神经元线粒体顺轴向运输的数量与比率,同时线粒体顺轴向运输的速率明显低于对照组,而神经元线粒体逆轴向运输则不受影响。这种改变与Aβ介导的神经元线粒体运输障碍类似。4.KIF5A过表达有效挽救A β条件下神经元线粒体顺轴向运输障碍。A β处理KIF5A过表达与正常对照神经元,结果显示KIF5A过表达病毒处理过的神经元可以有效挽救A β引起的KIF5A表达下调,并提高神经元线粒体顺轴向运输的比例与速率。5.KIF5A过表达有效挽救A β引起的神经元损伤。KIF5A表达重建可以有效降低Aβ导致的神经元氧自由基升高,同时促进神经突触的形成。研究结论:1.KIF5A在AD患者颞叶组织中表达水平下降;2.KIF5A表达水平的下降与A β毒性密切相关;3.KIF5A表达下调是AD条件下神经元线粒体顺轴向运输障碍的原因之一;4.KIF5A表达重建可以有效挽救AD条件下神经元线粒体顺轴向运输障碍,并改善神经元与突触的功能。更多还原

【Abstract】 Background:Alzheimer’s disease(AD)is a common neurodegenerative disease characterized by cognitive decline,in particular progressive memory loss.Alzheimer’ s disease has been identified as a protein misfolding disease,caused by plaque accumulation of abnormally folded amyloid beta protein and tau protein in the brain,which cause synaptic dysfunction and neuron losses.The cause of Alzheimer’ s disease is poorly understood till now.The amyloid hypothesis,which is widely accepted,po_s tulated that extracellular amyloid beta,especially Aβ42,deposits are the fundamental cause of the disease.The hypothesis further suggests that the amyloid precursor protein(APP)gene,the presenilin 1(PS1)gene and the presenilin 2(PS2)gone mutation are important causes of Aβ 42 production.However,the study found that there is no APP or PS gene mutation in patients with sporadic AD,so the hypothesis still has certain limitations.In recent years,a large number of studies suggest that mitochondrial dysfunction is one of the important pathological changes of AD.Mitochondrial dysfunction can occur in AD patients early,and mitochondrial damage with AD progression is deteriorating and ultimately affects synaptic and neuronal function.Based on this,Swerdlow et al.proposed the mitochondrial cascade hypothesis for sporadic AD.The hypothesis is that mitochondrial dysfunction is the initial event leading to AD.Aβ deposition,tau phosphorylation,and neuronal degeneration are all based on mitochondrial dysfunction,which further aggravates mitochondrial dysfunction.Although the mitochondrial cascade hypothesis also has its inherent limitations,it provides a new idea for the study of AD.线Mitochondria are important organelles in neurons.In addition to providing the ATP required to maintain neuronal function,mitochondria maintain synaptic plasticity by providing energy,regulating synaptic calcium homeostasis,mediating dialogue between synapses/neuronal nuclei,and regulating oxygen free radical levels.And synaptic transmission plays a key role in achieving neuronal function.It is worth noting that the mitochondrial synthesis site is the neuronal cell body,after which the neuron delivers the mitochondria to the synaptic site.Therefore,normal mitochondrial transport is essential for maintaining synaptic function;mitochondrial transport disorders are an important cause of neuronal damage,especially synaptic function.In recent years,the relationship between neuronal mitochondrial transport disorders and neuronal damage in AD has been receiving attention.A large number of studies have shown that mitochondrial transport disorders,especially mitochondria,are one of the causes of synaptic dysfunction.However,the specific molecular mechanisms by which neurons are transported in the axial direction are not yet known.Kinesin superfamily proteins(KIFs)and cytoplasmic dynein are the major types of microtubule-dependent motor proteins,which are essential molecular machineries for intracellular transport.KIF5A is a key isoform of kinesin-1,which is a key molecular machinery in facilitating anterograde axonal mitochondrial transport.Previous studies have shown that down-regulation of KIF5A expression may be responsible for the disruption of anterograde axonal mitochondrial transport in AD.The purpose of this study is to explore the specific molecular mechanism of KIF5A leading to neuronal mitochondrial axial transport disorders in AD state by pathology and molecular biology techniques.Aims:1.To define changes of proteins revolved in mitochondrial axonal transport in AD;2.To find the possible mechanism of KIF5A decline in AD state;3.To clarify the effect of KIF5A downregulation on the anterograde axonal mitochondrial transport;4.To explore the effect of KIF5A restoration on axonal mitochondrial transport and neuronal function in AD state.Methods:1.Mice:8 and 12 months old nonTg and 5xFAD mice of mixed genders were used in the experiments.Day 0-1 pups from C57BL/6J strain mice were used for primary neuron culture.2 Human Samples:Frozen brain samples and paraffin-embedded brain slices were requested from UT Southwestern Medical Center ADC Neuropathology Core,supported by ADC grant(AG12300)under a protocol approved by The UT Southwestern Medical Center with informed consent from all subjects and the study adhered to the Declaration of Helsinki principles.3 Primary neurons culture:Cortices or hippocampi were dissected from Day 0 pups from C57BL/6J strain mice.Tissues were digested in trypsin and primary neurons were cultured in neuron culture medium(Neurobasal A with B27 supplement,L-glutamine,penicillin,and streptomycin).Mitochondria motility measurement was conducted on neurons with density of 0.1 × 105/cm2.The samples for Western Blot were collected from neurons with density of 1×105/cm2.4.Immunoblotting:Western Blot was used to detect changes in neuronal mitochondrial-related protein expression levels.5.Immunostaining:Brain tissues from 8 and 12 months old nonTg and 5× FAD mice were used to perform immunostaining,which to6.Generation of Lentivirus expressing mitochondria targeted DsRed:HEK293T cells(ATCC)were transiently cotransfected with lentivirus vector carrying mitochondria targeted DsRed by using calcium phosphate precipitation method.The lentivirus were collected from the culture medium and concentrated with Lenti-X Concentrator(Clontech).7.KIF5A knocking down in mouse primary neurons:Lentivirus-expressing shRNA targeted to mouse KIF5A was packaged with lentivirus shRNA construct.Mouse primary neurons were cultured for 3 days before infection with lentivirus.Neurons were treated and collected for experiments after a further 7 days in culture.8.KIF5A Overexpression in mouse primary neurons:Human KIF5A was inserted into lentivirus vector with human polyubiquitin promoter-C.Lentivirus containing KIF5A were packaged and applied on primary neurons similar as shRNA lentiviral vector.Mouse primary neurons were cultured for 3 days before infection with lentivirus at a m.o.i.of 5.Neurons were collected for experiments after 7 days infection.9.Oligomeric Aβ preparation:The peptide solution was resuspended in cold HAM’S F-12 to 100 u M and immediately vortexed for 30 s.The solution was then incubated at 4 0 C for 24 h.Oligomeric A β(1μM)was applied on neurons for 24 h before cell collection.10.Mitochondria motility measurement:Mouse primary neurons were infected by lentivirus expressing mtDsRed to visualized mitochondria.Time-lapse images were captured every 5 seconds for a total of 3 minutes under 40X oil objective on a Nikon inverted fluorescent microscope with an on-stage incubator(37℃,5%C02).The velocity of all mobile mitochondria was measured by using tracking system integrated in Nikon NIS Advanced Research software.11.Statistical analysisStatistical comparisons were performed using GraphPad Prism 5 software.One-way ANOVA followed by Bonferroni post hoc analysis,or unpaired two-way Student’ s t test were applied in data analysis.Numbers of replicates and P value are stated in each figure legend.All data were expressed as the mean±s.e.m.Significance was concluded when the P value was less than 0.05.Results:1.KIF5A is down-regulated in temporal lobe tissue of AD patients.Western blotting was used to detect the expression levels of mitochondrial transport-related proteins KIF5A,KIF5B,KIF5C,Dynein,Syntabulin,Mirol and mitochondrial marker Tom40 in the temporal and cerebellar tissues of patients with AD and non-AD subjects.KIF5A was found decreased significantly in temporal lobe,which is the AD-sensitive brain aera and KIF5B showed a slight decrease in the temporal lobe tissue,but no change was observed in the cerebellar tissue.Further correlation analysis found that the decrease in KIF5A expression levels was independent of age and time of death.2.The decrease in KIF5A is closely related to Aβ toxicity.We used 5xFAD mice at 8 months and 12 months of age.Using immunoblotting and immunofluorescence,we found that KIF5A decreased significantly in mouse cortex,KIF5B also decreased slightly,while KIF5C,Dynein,Syntabulin and other proteins did not change.Mirol and Tom40 only decreased in the cortex of 12-month-old mice.Further detection of Aβ-treated primary neurons by immunoblotting revealed that KIF5A expression levels were still significantly decreased,while KIF5B,KIF5C,Dynein,Syntabulin and other protein expression levels were not significantly changed.Mirol expression levels were slightly downregulated.3.Downregulation of KIF5A expression levels impeded the smooth transport of neuronal mitochondria.We found that down-regulation of KIF5A expression reduced the number and ratio of neuronal mitochondrial transport in the axial direction,while the rate of mitochondrial transport in the axial direction was significantly lower than that of the control group,while neuronal mitochondrial retrograde axonal mitochondrial transport was not affected.This change is similar to Aβ-mediated neuronal mitochondrial transport disorders.4.KIF5A overexpression effectively rescues neuronal mitochondrial axial transport disorders under Aβ conditions.A P treatment of KIF5A overexpression and normal control neurons,the results show that KIF5A overexpressing virus-treated neurons can effectively reduce the down-regulation of KIF5A expression induced by A p,and increase the proportion and rate of neuronal mitochondria transport in the axial direction.5.KIF5A overexpression effectively rescues neuronal damage caused by A p.Reconstruction of KIF5A expression level can effectively reduce the increase of oxygen free radicals in neurons induced by Aβ,and promote the formation of synapses.Conclusions:1.The expression level of KIF5A in temporal lobe tissue of AD patients decreased;2.The decrease in the expression level of KIF5A is closely related to the toxicity of Aβ;3.Down-regulation of KIF5A expression is one of the causes of neuronal mitochondrial axial transport disorder under AD conditions;4.Reconstruction of KIF5A expression level can effectively rescue the mitochondrial dysplasia of neurons under AD conditions and improve the function of neurons and synapses.更多还原