【作者】 张菁；

【导师】 王兴龙；

【作者基本信息】 山东大学 ， 神经生物学， 2023， 博士

【摘要】 研究背景:Ras相关蛋白(Rab,Ras-related protein)是小GTP酶Ras超家族中目前已知最大成员分支,主要参与细胞内膜类结构的转运、加工过程,如囊泡的出芽、形成、输运、融合以及膜结构形态的维持等。Rab家族的蛋白结构具有较高的相似性,均包含4个与GDP/GTP结合,并发挥GTP酶活性的结构域(switch Ⅰ到switch Ⅳ)以及位于C端的P结构域。在switch Ⅱ结构域中存在一段较为保守的序列,其中的丝氨酸、苏氨酸可以被帕金森病(Parkinson’s disease,PD)相关蛋白激酶——富亮氨酸重复激酶2(Leucine-rich repeat kinase 2,LRRK2)磷酸化修饰,从而引发神经元内囊泡转运、自噬、溶酶体功能和纤毛形成等细胞过程的障碍,参与到疾病发病机制当中。在众多的Rab家族分子中,Rab10显得格外与众不同。首先,Rab蛋白一般具有较为固定的细胞内定位和相对单一的功能,但是Rab10几乎在细胞内所有的膜类细胞器上均有分布,功能十分复杂,可以参与调节内质网动力学、神经元轴突发育、纤毛形成及纤毛内转运、溶酶体管状化和胞外分泌及细胞自噬等多个过程;其次,Rab10在不同细胞、组织和发育阶段中发挥的作用不尽相同;此外,Rab10的磷酸化与多种神经退行性疾病密切相关。有研究报道,无论是在散发性PD还是家族性PD患者脑内,均可检测到LRRK2激酶活性及Rab10磷酸化水平的异常升高。而在阿尔茨海默病(Alzheimer’s disease,AD)患者脑内同样可以检测到Rab10磷酸化水平显著增高,并且这些异常磷酸化的Rab10与AD特征性神经病理改变——tau蛋白过度磷酸化导致的神经纤维缠结,具有高度的共定位。此外,Rab10还是Rab家族中与LRRK2亲和力最高的激酶底物。目前有已多项研究关注PD相关突变的LRRK2对Rab10异常磷酸化产生的效应。首先,LRRK2对Rab10的过度磷酸化会抑制细胞纤毛的发生,而促进纤毛的解聚。此外,LRRK2可以将Rab10、Rab8a和Rab35募集到发生应激的溶酶体上,并在此对它们进行磷酸化,从而促进溶酶体管状化和胞外分泌。当线粒体发生损伤时,Rab10可以在PINK1和PRKN的作用下,聚集到去极化的线粒体上,并招募自噬受体OPTN,促进线粒体自噬;而当LRRK2发生突变,激酶活性异常升高时,被磷酸化的Rab10无法再被募集到这些线粒体上,从而引发线粒体自噬障碍。但是,以上研究大多在LRRK2致病突变的体外细胞模型中展开,而Rab10磷酸化在体内,尤其是在脑内发挥的生理作用,仍亟待研究。研究目的:1.探讨Rab10 Thr73位点磷酸化对小鼠行为学表型的影响;2.探讨Rab10 Thr73位点磷酸化影响的主要脑区和细胞类型;3.探讨Rab10 Thr73位点磷酸化对脑内神经元形态、功能的影响及其分子机制。研究方法:1.构建模拟Rab10持续非磷酸化和持续磷酸化状态的小鼠模型利用CRISPR/Cas9技术,分别构建模拟Rab10持续非磷酸化状态和持续磷酸化状态的Rab10 T73V和T73D突变小鼠模型,并利用DNA测序、免疫蛋白印迹(Western Blot,WB)对Rab10的突变和表达水平进行了验证。2.检测Rab10突变小鼠的行为学表型对3月龄Rab10T73V/T73V小鼠和3月龄、5月龄、7月龄和9月龄Rab10T73D/+小鼠及同窝Rab10+/+小鼠进行一系列的行为学检测,包括:旷场试验、高架十字迷宫试验、明暗箱试验、Y迷宫试验、○迷宫试验、T迷宫试验、藏宝试验、筑巢试验、巴恩斯迷宫、加速旋转杆试验、悬尾试验、强迫游泳试验、条件性恐惧试验、代谢笼和新物体识别试验,用以检测小鼠自发活动度、焦虑样行为、重复性强迫样行为、学习记忆、抑郁样行为、运动协调能力和能量代谢等方面的表型。3.检测Rab10突变小鼠突触结构和功能的改变提取Rab10+/+及Rab10T73V/T73V小鼠纹状体、杏仁核、背侧海马、腹侧海马和前额叶皮层这5个与焦虑行为相关的脑区蛋白,WB检测VAMP1、VAMP2、Syntaxin 1、SHANK2、Synaptophysin、PSD95等突触蛋白的表达水平,确定Rab10 T73V突变影响的主要脑区。再对实验中确定的责任脑区——纹状体进行针对性检测:利用脑片膜片钳,记录中型棘突神经元(medium spiny neuron,MSN)微小抑制性突触后电流(miniature inhibitory postsynaptic current,mIPSC)和微小兴奋性突触后电流(miniature excitatory postsynaptic current,mEPSC),以检测突触功能;利用透射电镜观察突触密度、突触小泡的密度和直径、突触后致密斑的长度和宽度;对纹状体脑区进行抑制性突触前、后标志物GAD67和Gephyrin,兴奋性突触前、后标志物VGLUT1和PSD95的免疫荧光染色,分析抑制性突触和兴奋性突触的密度。4.检测Rab10突变小鼠神经元和胶质细胞的密度对3月龄实验小鼠进行尼氏染色,全景扫描小鼠皮层、背侧海马及纹状体脑区切片,计数这些脑区中神经元的密度。再对小鼠进行小胶质细胞标志物Iba1和星形胶质细胞标志物GFAP的免疫组化染色,统计纹状体脑区中小胶质细胞的密度和GFAP的表达情况。5.检测Rab10突变小鼠的神经元分支及树突棘发育情况对3月龄实验小鼠进行冠状位切片及高尔基染色,使用20倍镜沿z轴多层扫描小鼠纹状体、背侧海马、腹侧海马及杏仁核脑区的全景图像,计数、测量神经元树突分支数目及长度。再对小鼠进行树突标志物MAP2的实时荧光定量PCR和免疫荧光染色,分别检测MAP2 mRNA和蛋白表达水平。此外,体外培养胚胎16.5天小鼠原代神经元,培养3天后,进行神经元轴突标志物neurofilament和树突标志物MAP2的免疫荧光染色,测量、统计神经元轴突和小神经突的数目及长度。随后使用63倍油镜,沿z轴拍摄高尔基染色切片,统计树突棘密度及各类树突棘的占比情况。6.检测Rab10突变体的差异相互作用蛋白使用天然细胞裂解液提取3月龄Rab10+/+及Rab10T73V/T73V小鼠纹状体脑区总蛋白,利用Rab10抗体将组织中的Rab10蛋白免疫沉淀(Immunoprecipitation,IP)下来,应用串联质谱标签(tandem mass tag,TMT)定量蛋白质组学,分析野生型Rab10和Rab10 T73V突变体之间的差异相互作用蛋白,并对这些蛋白进行生物信息学分析,找到其中涉及的关键分子信号通路。研究结果:第一部分Rab10 T73V突变对小鼠的影响1.Rab10 T73V突变小鼠表现出特征性的焦虑样行为改变Rab10T73V/T73V小鼠较Rab10+/+小鼠,在旷场中央区活动的时间明显缩短。在高架十字迷宫和○迷宫中,Rab10T73V/T73V小鼠在开放臂或开放区域中活动的时间也呈现显著降低。在明暗箱试验中,Rab10T73V/T73V小鼠在明箱中活动的时间明显缩短,进入明箱的次数减少。此外,Rab10T73V/T73V小鼠在筑巢试验中撕碎的棉花相对较少,在藏宝试验中埋藏的玻璃珠数目也明显减少。而针对小鼠自发活动度、学习记忆、抑郁样行为、运动协调能力和能量代谢等方面的试验中,两组小鼠间未检测出显著差异。上述结果表明Rab10 T73V突变导致小鼠出现了焦虑样行为改变及重复性强迫样行为的损害。2.Rab10 T73V突变小鼠纹状体脑区突触蛋白表达谱发生改变,抑制性突触信号传递障碍在检测的5个与焦虑相关的脑区中,仅在纹状体脑区中检测到Rab10T73V/T73V小鼠突触蛋白表达的改变。其中,3个构成突触前SNARE复合物的蛋白VAMP1、VAMP2和Syntaxin 1及突触后膜蛋白SHANK2在Rab10T73V/T73V小鼠纹状体脑区中表达升高。进一步检测Rab10T73V/T73V小鼠纹状体脑区MSN突触电生理功能,发现mIPSC的频率显著降低,但波幅未出现明显改变;而mEPSC的频率和波幅均未表现出明显差异。此外,免疫荧光染色和透射电镜结果显示,抑制性突触和兴奋性突触的密度、突触后致密斑长度和宽度、突触前囊泡的密度及直径在两组小鼠间未见显著差别。这些结果提示Rab10 T73V突变损害了纹状体抑制性突触信号传导,但未对突触的结构产生显著影响。3.Rab10 T73V突变小鼠神经元密度、星形胶质细胞和小胶质细胞未出现明显改变尼氏染色并未检测到Rab10T73V/T73V小鼠与Rab1 0+/+小鼠在皮层、海马和纹状体脑区中神经元密度的差异。同时,Iba1标记的小胶质细胞和GFAP标记的星形胶质细胞在两组小鼠同样未见明显差别。因此,Rab10 T73V突变并不会对小鼠脑内神经元和胶质细胞的密度产生明显影响。4.Rab10 T73V突变小鼠纹状体脑区MSN树突分支增多高尔基染色显示,Rab10T73V/T73V小鼠纹状体MSN树突分支增多,总树突长度增长,但平均树突长度并不受影响。而背侧海马、腹侧海马及杏仁核中神经元形态均未见明显改变。进一步研究发现,与Rab1 0+/+小鼠相比,Rab10T73V/T73V小鼠纹状体脑区树突标志物MAP2的mRNA和蛋白表达水平均出现显著升高。因此,Rab10 T73V突变可以特异性地促进纹状体MSN树突的过度分支,引发神经元功能障碍。5.Rab10 T73V突变小鼠纹状体脑区MSN树突棘成熟度升高针对Rab1 0+/+和Rab10T73V/T73V小鼠不同脑区神经元树突棘进行比较、分析发现,T73V突变并不会影响脑内神经元树突棘的密度。进一步对这些树突棘进行分类比较发现,Rab10T73V/T73V小鼠纹状体MSN中成熟树突棘(短粗型、蘑菇型和分叉型)增多,占比更高;而未成熟的树突棘(丝状伪足型、细长型和细型),尤其是细型树突棘的比例显著降低。而背侧海马、腹侧海马和杏仁核神经元的树突棘成熟度未受明显影响。因而,Rab10 T73V突变可以特异性地促进纹状体MSN中树突棘过度成熟。6.T73V突变改变了 Rab10与细胞骨架组装相关的互作蛋白网与野生型Rab10相比,共有89个蛋白与Rab10 T73V突变体的结合发生改变,其中62个蛋白与Rab10 T73V突变体亲和力升高,另有27个蛋白与之亲和力降低。GO富集分析发现,这些差异结合蛋白大多定位于细胞骨架、突触、树突棘等细胞组分,与神经丝束的组装、肌动蛋白丝加帽和组装、突触囊泡的内吞、轴突发育和神经元投射的形成等生物过程密切相关。以上信息提示Rab10 T73V突变对神经元树突分支、树突棘和突触功能的影响,可能与神经元细胞骨架有关。第二部分Rab10 T73D突变对小鼠的影响1.Rab10 T73D纯合突变小鼠脑、肺和肾脏发育异常,出生时或出生后不久死亡大部分Rab10T73D/T73D小鼠在出生时或出生后1天内死亡,至断乳时,在获得的77只小鼠中仅发现1只Rab10T73D/T73D小鼠。此外,Rab10T73D/T73D新生鼠与Rab10T73D/+和Rab10+/+新生鼠相比,脑和肾脏重量明显降低,肺脏重量明显增加。HE染色发现,Rab10T73D/T73D新生鼠肺组织局部存在肺泡塌陷和肺不张,可能因此导致了新生鼠的死亡。2.Rab10 T73D杂合突变小鼠至9月龄未出现明显行为学改变3月龄、5月龄、7月龄和9月龄Rab10T73D/+小鼠在旷场试验、高架十字迷宫、Y迷宫、新物体识别试验和T迷宫中与Rab10+/+小鼠表现基本无异。因此至9月龄时,Rab10T73D/+小鼠尚未表现出焦虑和认知功能方面的行为学表型。3.Rab10 T73D突变抑制了脑内神经元树突分支的形成对仅存的一只3月龄Rab10T73D/T73D小鼠与同窝野生型对照小鼠进行高尔基染色分析发现:Rab10T73D/T73D小鼠背侧海马CA1区锥体神经元顶树突复杂度明显降低,分支减少,总树突长度缩短,但是平均树突长度并未发生显著改变;CA1区锥体神经元的基树突的分支数和总树突长度同样分别降低了 13.68%和16.68%,尽管并未达到统计学差异;Rab10 T73D突变的纹状体MSN的分支数较野生型明显减少,但平均树突长度和树突总长度并未发生改变。Rab10 T73D突变同样抑制了小鼠原代神经元轴突的发育,与Rab1 0+/+神经元相比,Rab10T73D/T73D神经元轴突数目减少,但轴突的总长度并未发生明显改变;Rab10T73D/T73D神经元小突起数目不变,而总长度增加。以上结果提示,Rab10 T73D突变会限制神经元树突分支发育,并且这种影响不具有明显的脑区特异性。4.Rab10 T73D突变降低了脑内神经元树突棘的密度与Rab1 0+/+小鼠相比,Rab10T73D/T73D小鼠背侧海马CA1区锥体神经元和纹状体MSN的树突棘密度均出现了显著降低。对这些树突棘进行分类统计发现,尽管Rab10 T73D突变神经元成熟和非成熟树突棘的密度均出现了降低,但各类树突棘的占比,并未发生显著改变。因此,Rab10 T73D突变主要降低了神经元树突棘的密度,但是对树突棘的成熟过程影响不大。结论及创新性:1.Rab10 Thr73位点磷酸化对纹状体参与的焦虑情绪相关神经环路至关重要,模拟Rab10持续非磷酸化状态会诱导小鼠出现焦虑样行为学改变。2.Rab10 Thr73位点磷酸化可能通过调节纹状体脑区SNARE复合物的功能,影响抑制性神经元神经递质的释放过程。3.Rab10 Thr73位点磷酸化对神经元树突分支和树突棘的发育起到重要调节作用,模拟Rab10持续磷酸化状态会阻碍神经元树突分支和树突棘的形成,而模拟Rab10持续非磷酸化状态则会特异性地诱导纹状体MSN树突分支增多、树突棘过度成熟。4.非磷酸化状态的Rab10是小鼠脑、肺和肾脏等重要组织器官发育所必须的,模拟Rab10持续磷酸化状态会导致小鼠出生时或出生后不久死亡。5.本研究利用模拟Rab10持续非磷酸化和持续磷酸化状态的小鼠模型,深入探究了Rab10 Thr73位点磷酸化在脑内的生理功能,揭示了 LRRK2对Rab10磷酸化修饰的重要生理意义,为基于LRRK2的各项治疗方案可能引发的副作用提供了实验证据支持。更多还原

【Abstract】 Background:Rab GTPase is by far the largest family of the Ras superfamily,they function as multifaceted coordinators in the regulation of intracellular membrane trafficking,which includes vesicle budding,uncoating,motility,fusion and the maintenance of membrane identity.Rab proteins possess four GDP/GTP binding and GTPase domains(switch Ⅰ throughⅣ),and a C-terminal motif for membrane binding.Most known phosphorylation sites in Rab proteins reside in the highly conserved switch Ⅱ motif,the Thr or Ser site of which could be phosphorylated by Parkinson’s disease(PD)associated LRRK2 kinase,and finally contributes to the pathogenesis of PD.Among the dozens of Rab GTPases,Rab 10 appears to be rather distinctive and attracts our great attention.Firstly,Rab 10 is ubiquitously distributed in intracellular membranes and is implicated in a myriad of membrane trafficking processes,such as endoplasmic reticulum dynamics,exocytic transport,axon development,ciliary transport and ciliogenesis,lysosome tubulation and exocytosis,and autophagy.Secondly,the function of Rab 10 varies with cell types and the stages of development involved.Most importantly,Rab 10 and its phosphorylation have been implicated in the pathogenesis of neurodegenerative diseases.Aberrantly enhanced LRRK2 kinase activity and concomitant elevated Rab 10 phosphorylation have been detected in the brains of sporadic and familial PD patients.There are also compelling evidences for the involvement of Rab 10 in the pathogenesis of Alzheimer’s disease(AD).Immunocytochemical analysis reveals that pRab 10-T73 is enriched in the hippocampus specimens of AD cases and highly co-localized with neurofibrillary tangles(NFTs),without any salient changes in total Rab10 level.Moreover,systematic proteomic and phosphoproteomic analysis identifies Rab10 as the substrate with the strongest affinity to LRRK2.Therefore,it is of great importance to uncover the physiological and pathological role of Rab10 and its phosphorylation.Recently,several studies have focused on the effect of Rab10 Thr73 phosphorylation by the PD related LRRK2 mutants.To be specific,studies revealed that Rab10 phosphorylation could strengthen its intrinsic ability to block ciliogenesis and promote deciliation.Furthermore,LRRK2 could be recruited to stressed lysosomes and phosphorylate Rab10,Rab8a and Rab35 therein,facilitating the tubulation and secretion lysosome.A recent study reveals that Rab10 and its phosphorylation have also been linked to mitophagy:Rab10 accumulates on depolarized mitochondria in a PINK1-and PRKN-dependent manner,recruits the autophagy receptor OPTN and facilitates mitophagy,which could be impaired by LRRK2 phosphorylation.However,these studies were mainly carried out by LRRK2 mutant cell models in vitro,and failed to decipher the role of Rab10 Thr73 phosphorylation in vivo under general physiological conditions,notably in the central nervous system,which is also of great importance and remains to be fully elucidated.Aims:1.To analyze the effect of Rab10 Thr73 phosphorylation on mouse behaviors.2.To find out the responsible brain region and cell type affected by Rab10 Thr73 phosphorylation.3.To investigate the effects of Rab10 Thr73 phosphorylation on the morphology and function of neurons in the brain and the underlying molecular mechanisms.Methods:1.Generation of non-phosphorylatable and phosphomimetic Rab10 mutant mice modelsNon-phosphorylatable Rab10 T73V and phosphomimetic Rab10 T73D mutant mice were generated using CRISPR/Cas9-mediated gene targeting.The mutation and the expression of Rab10 were confirmed by DNA sequencing and western blot(WB)analysis.2.Analysis of the behavioral phenotypes of Rab10 mutant mice3-month-old Rab10T73V/T73V mice,Rab10T73D/+mice at the age of 3,5,7,and 9 months,and age-matched littermates were subjected to behavior assessment batteries,including open field test(locomotor activity and anxiety-like behavior),elevated plus maze(anxiety-like behavior),light/dark box(anxiety-like behavior),Y maze(working memory),zero maze(anxiety-like behavior),T maze(working memory),marble-burying test(repetitive,compulsion-like behavior),nestlet-shredding test(repetitive,compulsion-like behavior),Barnes maze(long-term spatial memory),accelerating rotarod(motor coordination),tail suspension test(depression-like behavior),forced swimming test(depression-like behavior),fear conditioning(emotional memory),metabolic caging(energy metabolism),and novel object recognition test(recognition memory).3.Analysis of the changes in synaptic structure and function of Rab10 mutant miceTo identify the brain regions responsible for the behavioral deficits,we screened five anxiety-related brain areas of Rab10T73V/T73V mice,namely the striatum(STR),amygdala(AMY),dorsal hippocampus(DH),ventral hippocampus(VH),and prefrontal cortex(PFC),by immunoblotting to analyze the expression levels of VAMP1,VAMP2,Syntaxin 1,SHANK2,Synaptophysin,PSD95 and other synaptic proteins.Then we took the responsible brain region,STR,for further analysis.To determine the types and domains of the synapses affected,we recorded the miniature inhibitory postsynaptic current(mIPSC)and miniature excitatory postsynaptic current(mEPSC)by whole-cell patch-clamp recordings.The density of synapses,the density and diameter of synaptic vesicles,and the length and width of postsynaptic density(PSD)were observed by transmission electron microscopy.Immunofluorescent staining of inhibitory presynaptic and postsynaptic markers GAD67 and Gephyrin,and the excitatory presynaptic and postsynaptic markers VGLUT1 and PSD95,were performed to analyze the density of inhibitory and excitatory synapses in STR.4.Analysis of the density of neurons and glial cells in Rab10 mutant miceIn order to evaluate the density of neurons in cerebral cortex,DH and STR,Nissl staining was performed for 3-month-old mutant mice,and panoramic images were taken using VS 120 Virtual Slide Microscope.Immunohistochemical staining of Ibal and GFAP was performed to identify the density of microglia and astrocytes in STR respectively.5.Analysis of the development of dendritic branches and spines in Rab10 mutant miceCoronal sections from 3-month-old mutant mice were stained using the FD Rapid GolgiStain kit.Panoramic images of z-stacks of Golgi-stained dendrites were taken at 20 ×magnification by VS 120 Virtual Slide Microscope for STR,DH,VH and AMY.Then,the number of branches,average dendritic length and total dendritic length were counted and measured.The mRNA and protein levels of dendritic marker MAP2 were measured by real-time quantitative polymerase chain reaction(RT-qPCR)and immunofluorescent staining.In addition,E16.5 primary cortical neurons were collected.After 3 days of culture,immunofluorescent staining of neurofilament and MAP2 were performed to calculate the number and length of axons and minor neurites.Subsequently,Golgi stained sections were photographed along the z-axis with 63 × oil lens,the density and the types of dendritic spines were analyzed.6.Differential interacting proteins of Rab10 mutant were detectedTotal protein lysate was collected from 3-month-old Rab10+/+ and Rab10T73V/T73V mice using natural cell lysis buffer,and Rab10 protein was immunoprecipitation(IP)from the tissue by Rab10 antibody.Then the IP samples were analyzed by Tandem Mass Tag(TMT)-based quantitative proteomics to find out the proteins differentially interact with wild type Rab10 and Rab10 T73V mutant.Finally,those proteins were subjected to bioinformatics analysis,to identify the molecular signaling pathways involved.Results:Part Ⅰ:The effect of Rab10 T73V mutation on mice1.Rab10 T73V mutant mice exhibit anxiety-like behaviorCompared to Rab10+/+mice,Rab10T73V/T73V mice displayed significantly reduced activity in the center of the open field.Similarly,in the elevated plus maze and the zero maze,the Rab10T73V/T73V mice spent less time in the open arms or open sections.Rab10T73V/T73V mice also exhibited significantly decreased activity in the light chamber during the light/dark exploration task.Of note,the Rab10T73V/T73V mice displayed remarkable impairments in repetitive,compulsion-like social behaviors,because they shredded conspicuously fewer nestlets in the nestlet shredding test and buried significantly fewer marbles in the marble burying test compared to the wild-type group.In contrast,no discernible abnormalities were detected between Rab10T73V/T73V and Rab10+/+ mice,in terms of locomotor activity,cognitive function,depression-like behaviors,motor coordination and energy metabolism.Thus,deletion of the Thr73 phosphorylation in Rab10 gives rise to anxiety-like behavior.2.Rab10 T73V mutant mice show synaptic dysfunctionAmong the five anxiety-related brain areas screened,only STR showed intergroup differences in the expression of synaptic proteins.More specifically,in the STR of the Rab10 T73V mutant mice,a slight increase was observed in the postsynaptic protein SHANK2,and three presynaptic proteins,namely the SNARE proteins VAMP1,VAMP2 and Syntaxin 1 were elevated to a remarkable degree.Moreover,in recordings from the striatal MSNs,we found that the frequency,but not the amplitude,of mIPSC was selectively reduced by the Rab10 T73V mutation.In contrast,we observed no changes in the amplitudes or frequencies of the mEPSC.Transmission electron microscopy revealed that the synaptic densities,thelength and thickness of PSD,the density of synaptic vesicles,and the vesicle diameters were quite similar in the STR of the two groups.Immunostaining demonstrated that Rab10 T73V mutation did not induce any significant alteration in excitatory and inhibitory synapses.Collectively,these data suggest that the Rab10 T73V mutation disturbs the inhibitory synaptic transmission in STR,while the structure of synapses is significantly affected by the mutation.3.Rab10 T73V mutant mice do not show any changes in the density of neurons,microglia and astrocytesNo alteration in neuronal density was detected between Rab10T73V/T73V mice and Rab10+/+mice in cerebral cortex,hippocampus and STR.At the same time,there was no significant difference in Ibal-labeled microglia and GFAP-labeled astrocytes between the two groups.Therefore,Rab10 T73V mutation does not significantly affect the density of neurons and glial cells in the brain of mice.4.Rab10 T73V mice display altered neuronal morphology in STRSignificant increases in neurite number and total dendritic length were observed in striatal MSNs of Rab10 T73V mice,with no change in the average dendritic length.The effect of Rab10 phosphorylation on neuronal morphology appeared to be restricted to STR,as no arborization abnormality was observed in DH,VH and AMY.Consistently,there was an increase in the dendritic marker MAP2 expression in the T73V STR.Taken together,these results show that T73V mutation elicits neurite over-branching phenotypes in striatal MSNs,and induces neuronal dysfunction.5.Rab10 T73V mice display altered neuronal morphology in STRNo differences were observed in spine density between Rab10T73V/T73V and wild-type mice.Subsequent spine classification found that mature spines(stubby,mushroom,and branched)were relatively more prevalent in Rab10T73V/T73V mice,and immature spines(filopodia,long thin and thin),especially thin spines,were less prevalent.Likewise,the maturation processes of dendritic spines were largely unaffected in other brain regions.This suggests that the Rab10 T73V mutation accelerates the maturation of dendritic spines in striatal MSNs.6.The T73V mutation alters the Rab10 interacting protein network associated with cytoskeleton assemblyIn order to explore the effect of Rab10 T73V mutation on the function of Rab10 itself,Rab10 protein in the STR of Rab10+/+and Rab10T73V/T73V mice was extracted by IP,and analyzed by TMT-based quantitative proteomics.There were 89 proteins interacting differentially with Rab10,among which 62 proteins had increased affinity with Rab10 T73V mutant and 27 proteins had decreased affinity.Further GO enrichment analysis revealed that most of these proteins were located in cytoskeleton,synapses,and dendritic spines.In addition,they were closely associated with neurofilament bundle assembly,actin filament capping,synaptic vesicle endocytosis,axonogenesis,and neuron projection development.These results suggest that the effects of Rab10 T73V mutation on neuronal dendritic branches,dendritic spines and synaptic function may result from the regulation of phospho-Rab10 on neuronal cytoskeleton.Part Ⅱ:The effect of Rab10 T73D mutation on mice1.Homozygous Rab10 T73D mutant mice display abnormal brain,lung,and kidney development,and die at or shortly after birthMost Rab10T73D/T73D mice died at birth or a few hours after birth,and only one Rab10T73D/T73D mouse was found among the 77 mice obtained at the time of weaning.In addition,compared with Rab10T73D/+and Rab10+/+neonatal mouse,the weight of brain and kidney of Rab10T73D/T73D neonatal mice significaitly decreased,while the weight of lung was significantly increased.HE staining found the collapsed alveolar and atelectasis in the lung of Rab10T73D/T73D neonatal mice,which might result in the death of neonatal mice.2.Heterozygous Rab10 T73D mutant mice behave largely normal at 9 months of ageAt the age of 3,5,7,9 months,Rab10T73D/+mice behaved nearly the same as Rab10+/+mice in open field test,elevated plus maze,Y maze,zero maze,novel object recognition test,and T maze.Thus,Rab10T73D/+mice did not exhibit any phenotypes in anxiety-like behavior and cognitive function at least till 9 months of age.3.Rab10 T73D mutation inhibits the formation of dendritic branches in brainThe only remaining 3-month-old Rab10T73D/T73D mouse and it wild-type littermate were subjected to Golgi staining.It was found that DH CA1 pyramidal neurons of Rab10T73D/T73D mice possessed fewer branches and exhibited shorter total dendrite length,but the average dendrite length was not affected.The number of basal dendrites and the total dendrite length also decreased by 13.68%and 16.68%respectively,although the difference did not reach statistical significance.The number of branches in Rab10 T73D mutant striatal MSNs was also reduced compared with that of the wild type,but the average and total dendrite length did not change.Rab10 T73D mutation also inhibited the development of axons of primary neurons.Compared with Rab10+/+neurons,the number of axons of Rab10T73D/T73D neurons was reduced,but the total length of axons did not change significantly.The number of of Rab10T73D/T73D neurons remained unchanged,while the total minor neurite length increased.These results suggest that Rab10 T73D mutation could impair dendrite ramification,and this effect is universal and not restricted to specific brain regions.4.Rab10 T73D mutation reduces the density of dendritic spines in brainCompared with Rab10+/+ mice,the density of dendritic spines in DH CA1 pyramidal neurons and striatal MSNs of Rab10T73D/T73D mice decreased significantly.Further classification revealed that the proportion of different types of dendritic spines remained unchanged,although the density of mature and immature dendritic spines of Rab10 T73D mutant neurons both decreased.Therefore,Rab10 T73D mutation mainly impairs the density of dendritic spines,but had little effect on the maturation process of dendritic spines.Conclusions and innovation:1.Rab10 Thr73 phosphorylation probably plays an important role in the regulation of the striatal circuits for anxiety,and the non-phosphorylatable T73V mutation of Rab10 could induce anxiety-like behaviors in mice.2.Rab10 Thr73 phosphorylation may affect the release of inhibitory presynaptic neurotransmitter by regulating the function of SNARE complex in the striatum.3.Rab10 Thr73 phosphorylation plays an important role in dendritic arborization and the development of dendritic spines.The non-phosphorylatable T73V mutation of Rab10 specifically enhances neurite arborization and accelerates spine maturation in striatal MSNs.While phosphomimetic Rab10 T73D mutation inhibits the formation of dendritic branches and spines in the brain.4.Non-phosphorylated Rab10 is necessary for the development of brain,lung and kidney.The phosphomimetic T73D mutation of Rab10 causes perinatal death of mice.5.In this study,we used non-phosphorylatable and phosphomimetic Rab10 mutant mouse models and revealed the physiological significance of Rab10 Thr73 phosphorylation in the brain.This study could provide cues for understanding any side effects that may be observed during the development of LRRK2-based clinical treatments,as well as novel diagnostic and therapeutic targets for treating neurodegenerative diseases,such as AD and PD.更多还原