Pathogenic Mechanisms of Dynactin p150glued in ALS and Parkinson's Disease

Dynactin p150 与 ALS 和帕金森病的致病机制

• 批准号: 8736660
• 负责人: Huaibin Cai
• 金额: $ 23.94万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8736660
• 关键词: 129S4/SvJaeSor Mouse; Affect; Age-Months; Alternative Splicing; Amino Acid Substitution; Animals; Axon; Axonal Transport; Base Pairing; Behavioral; Binding; Binding Proteins; Brain; Breeding; Cell Nucleus; Cells; Cessation of life; Chimera organism; Chromosomes, Human, Pair 6; Complex; Crossbreeding; Cytoskeleton; Cytoskeleton Alteration; DCTN2 gene; DNA; Defect; Deposition; Dynein ATPase; Embryo; Endosomes; Exons; Female; Fibroblasts; Functional disorder; Future; Generations; Genes; Genetic; Genomics; Glycine; Golgi Apparatus; Human; Huntington Disease; Hypercapnic respiratory failure; Imaging Techniques; Internal Ribosome Entry Site; Intracellular Space; Intracellular Transport; Kinesin; Knock-in Mouse; Lead; Life; Link; Lower Motor Neuron Disease; Lysosomes; Mediating; Mental Depression; Microtubules; Midbrain structure; Minus End of the Microtubule; Missense Mutation; Mitosis; Mitotic spindle; Modeling; Modification; Molecular; Motor; Motor Neuron Disease; Motor Neurons; Movement; Mus; Mutant Strains Mice; Mutation; Neomycin; Neomycin resistance gene; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Organelles; Parkinson Disease; Pattern; Phenotype; Physiological; Positioning Attribute; Property; Protein Family; Proteins; Reporting; Resistance; Role; Sedimentation process; Sensory; Serine; Site; Skin; Southern Blotting; Spinal; Staining method; Stains; Sucrose; Symptoms; Syndrome; TNFRSF5 gene; Tamoxifen; Tandem Repeat Sequences; Testing; Transgenic Mice; Vesicle; Western Blotting; antibiotic G 418; base; blastocyst; brain tissue; dimer; dopaminergic neuron; dynactin; early embryonic stage; embryonic stem cell; gamma Tubulin; in vitro Assay; in vivo; interest; male; mature animal; migration; motor neuron degeneration; nervous system disorder; protein complex; protein expression; recombinase; research study; retrograde transport; selective expression; vector

1. Generation of dynactin p150glued conditional KO mice Since p150glued homozygous KO mice are early embryonic lethal, we decided to generate p150glued conditional KO mice in order to study the function of p150glued in postmitotic neurons. Mouse p150glued protein is encoded by the Dctn1 gene at chromosome 6. A 9.3 kb genomic DNA fragment carrying exons 28 of Dctn1 gene was isolated for genetic modifications. Briefly, a 1.4 kb genomic DNA fragment containing Dctn1 exons 2 to 4 was inserted between two tandem repeat loxP sites in order to build the conditional KO targeting vector. The targeted ES cell selection marker, neomycin (neo) resistance gene flanked with two FRT sites, was then inserted between the first loxP site and exon 2. The targeting vector was linearized and transfected into 129/SvJ ES cells, which were later subjected to G418 selection. The G418 resistant ES clones were picked and screened by Southern blot analysis for the correctly targeted clones. Two positive ES clones were expanded and injected into blastocysts, and the resulting male chimera mice were bred with WT C57BL/6J female mice to obtain Dctn1+/loxP-FRT-neo-FRT-ex2&4-loxP mice. These Dctn1+/loxP-FRT-neo-FRT-ex2&4-loxP mice were then crossed with FLP Tg mice 129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J to remove the FRT-neo-FRT cassette. The resulting Dctn1+/loxP-ex2&4-loxP (Dctn1+/loxP) animals were intercrossed to generate homozygous Dctn1loxP/loxP animals. When Dctn1loxP/loxP mice were crossed with a line of inducible Cre Tg mice B6.Cg-Tg (CAG-cre/Esr1)5Amc/J, we turned off the expression of p150glued in the one month old creEsr1/Dctn1loxP/loxP mice by administrating the animals tamoxifen, which triggered the activation of Cre recombinase and deleted the exons 2-4 of Dctn1 gene. Western blot analyses demonstrated a nearly complete elimination of p150glued protein expression in brain tissues. Concomitantly, the expression level of alternative spliced dynactin p135 protein was up-regulated in p150glued conditional KO mouse brains. Interestingly, the p150glued conditional KO mice appeared normal and did not displayed any obvious behavioral phenotypes up to 18 months of age. These results indicate that p150glued may not perform any critical physiological functions in adult animals. 2. The loss of dynactin p150glued did not affect the complex formation of other dynactin components The vertebrate dynactin complex has been reported to migrate as a 19S protein heteromultimer resolved by 520% sucrose gradient sedimentation. To test the integrity of the dynactin complex in p150glued conditional KO mice, we performed sedimentation analysis on a 520% sucrose gradient using brain extracts from WT and KO mice. We found that the migration patterns of dynactin p135, p50, and Arp1 as well as DIC from p150glued conditional KO brains were the same as those from WT controls, indicating that the deletion of p150glued does not impair the formation of remaining dynactin complex with DIC. 3. The loss of dynactin p150glued altered the subcellular distribution of dynactin complex in primary cultured fibroblasts Since dynactin p135 mainly exists in neurons, we wondered whether the loss of p150glued may cause severe disruption of remaining dynactin protein complex in non-neuronal cells such as skin fibroblasts (FB). We derived primary cultured FBs from CreEsr1-positive or negative p150glued conditional mice and treated these cells with tamoxifen for four days. The presence of p150glued was almost completely abolished in tamoxifen-treated CreEsr1-positive p150glued conditional KO FBs. However, sucrose gradient sedimentation experiments revealed the same migration patterns of dynactin p50 and Arp1 as well as DIC from p150glued conditional KO FBs as cells from CreEsr1-negative controls. These observations are consistent with our earlier findings in brain tissues. We then examined the subcellular distribution of dynactin proteins in p150glued conditional KO FBs. In control CreEsr1-negative FBs, p50 and Arp1 staining typically presented as large patches adjacent to the nucleus By contrast, p50 and Arp1 staining in CreEsr1-positive FBs was evenly distributed in the entire intracellular space as small clusters. These results indicate that p150glued may serve as anchor points for perinuclear localization of the dynactin protein complex. 4. Generation of tetO-dynactin p150glued Tg mice We previously generated G59S p150glued knock-in mice to model an autosomal familial form of lower motor neuron disease. Interestingly, three more missense mutations of G71A, G71R, and Q74P have been recently identified in the same CAP-Gly domain of p150glued that cause Perry syndrome. To study the pathogenic mechanism of p150glued Perry mutations in the degeneration of nigrostriatal DA neurons, we decided to generate G71R p150glued inducible Tg mice that selectively express human G71R p150glued in the midbrain DA neurons. We have generated multiple founders of tetO-G71R p150glued Tg mice. As controls, tetO-WT p150glued and tetO-G59S p150glued Tg founder mice were also generated. At present, we are crossbreeding these tetO-p150glued mice with PITX3-IRES-tTA mice to drive the expression of WT, G71R or G59S p150glued in midbrain DA neurons. The redistribution of dynactin family proteins in p150glued-deficent FBs may reflect a disengagement of dynactin complex with dynein-mediated centripetal transport or a loss of p150glued as the anchor points for the attachment of dynactin proteins to the minus end of microtubules. Future studies will be planned to test these two possibilities. We will examine the dynein/dynactin-dependent retrograde transport of lysosomes and endosomes in p150glued conditional KO FBs by live imaging techniques, and also check the targeting of p150glued-lacking dynactin complex to the minus end of microtubule assemblies by co-immunostaining with gamma-tubulin and other microtubule minus end binding proteins. More importantly, we are going to evaluate how the loss of p150glued impacts the function of neurons. We speculate that p150glued may affect the ER-to-Golgi transport in neurons, and will test this hypothesis in primary cultured neurons prepared from p150glued conditional KO mice.

1。dynactin P150glued有条件的KO小鼠的一代 由于P150GLOUD纯合子KO小鼠是早期的胚胎致死性，因此我们决定生成P150GLOUD的有条件的KO小鼠，以研究有丝分裂后神经元中P150GLEL的功能。小鼠p150glued蛋白在6染色体上由DCTN1基因编码。分离出9.3 kb的基因组DNA片段携带DCTN1基因28的外显子28分离出来进行遗传修饰。简而言之，在两个串联重复的LOXP位点之间插入了一个1.4 kb的基因组DNA片段，其中包含DCTN1外显子2至4，以构建条件的KO靶向载体。然后将靶向ES细胞选择标记物，新霉素（NEO）抗性基因侧翼，两侧是两个FRT位点，然后在第一个LOXP位点和外显子2之间插入。将靶向载体线性化并转染到129/SVJ ES细胞中，后来接受G418选择。挑选了G418抗ES克隆，并通过Southern印迹分析筛选正确靶向的克隆。将两个阳性ES克隆扩展并注入胚泡，并用WT C57BL/6J雌性小鼠繁殖所得的雄性嵌合体小鼠，以获得DCTN1+/LOXP-FRT-FRT-FRT-NEO-FRT-FRT-EX2和4-LOXP小鼠。然后将这些DCTN1+/LOXP-FRT-NEO-FRT-EX2和4-LOXP小鼠与FLP TG小鼠129S4/SVJAESOR-GT（ROSA）26Sortm1（FLP1）DYM/J交叉以删除FRT-NEO-NEO-FRT CASSETTE。将所得的DCTN1+/LOXP-EX2和4-loxP（DCTN1+/LOXP）动物间隔以产生纯合DCTN1LOXP/LOXP动物。 When Dctn1loxP/loxP mice were crossed with a line of inducible Cre Tg mice B6.Cg-Tg (CAG-cre/Esr1)5Amc/J, we turned off the expression of p150glued in the one month old creEsr1/Dctn1loxP/loxP mice by administrating the animals tamoxifen, which triggered the activation of Cre recombinase and deleted the DCTN1基因的外显子2-4。 Western印迹分析表明，在脑组织中几乎完全消除了P150粘液蛋白的表达。同时，在有条件的KO小鼠大脑中，替代剪接dynactin P135蛋白的表达水平上调。有趣的是，P150 g的条件KO小鼠看起来正常，并且没有显示出长达18个月大的行为表型。这些结果表明，P150GLUED可能在成年动物中没有执行任何关键的生理功能。 2。dynactin p150glued的损失不会影响其他dynactin成分的复杂形成 据报道，脊椎动物驱体复合物作为19S蛋白异杀剂迁移，该蛋白异杀剂由520％的蔗糖梯度沉降解决。为了测试在P150GLOUD条件KO小鼠中的Dynactin复合物的完整性，我们使用WT和KO小鼠的脑提取物对520％的蔗糖梯度进行了沉降分析。我们发现，Dynactin P135，P50和ARP1的迁移模式以及P150GLUED条件性KO大脑的DIC与WT对照组的迁移模式相同，表明P150GLUED的删除并不会损害DIC剩余的Dynactin Complecter的形成。 3。dynactin p150glued的损失改变了原代培养成纤维细胞中Dynactin复合物的亚细胞分布 由于Dynactin P135主要存在于神经元中，因此我们想知道P150GLOUD的丧失是否会导致非神经元细胞（例如皮肤成纤维细胞（FB））中剩余的Dynactin蛋白复合物的严重破坏。我们从CREESR1阳性或阴性P150GLOUD条件小鼠中得出了原代培养的FB，并用他莫昔芬治疗了这些细胞四天。在他莫昔芬处理的CREESR1阳性P150GLUED条件KO FBS中，P150Glued的存在几乎完全被完全废除。然而，蔗糖梯度沉积实验揭示了Dynactin P50和ARP1的相同迁移模式以及来自p150gl的条件KO FBS的DIC作为CREESR1阴性控制的细胞。这些观察结果与我们早期在脑组织中的发现一致。然后，我们检查了p150glued条件KO FBS中测能素蛋白的亚细胞分布。在对照CREESR1阴性FBS中，P50和ARP1染色通常以相反的核对与小簇相邻的creesr1阳性FBS中的P50和ARP1染色相邻，在整个细胞内空间中均匀分布。这些结果表明，P150GLUED可以用作Dynactin蛋白复合物核核定位的锚点。 4。二氧蛋白p150glued TG小鼠的产生 我们先前生成了G59S P150GLOUD敲入小鼠，以建模下运动神经元疾病的常染色体家族形式。有趣的是，最近在引起佩里综合征的P150gly的同一帽盖域中发现了G71A，G71R和Q74P的三个错义突变。为了研究Nigrostriatal DA神经元退化中P150GLOUD PERRY突变的致病机制，我们决定生成G71R P150GLUED诱导型TG小鼠，该诱导型TG小鼠在中脑DA Neurrons中选择性地表达人G71R P150GLED。我们已经产生了多个TETO-G71R P150GLUED TG小鼠的创始人。作为对照，还生成了TETO-WT P150GLUED和TETO-G59S P150GLUED TG创始人小鼠。目前，我们正在将这些TETO-P150GLIED小鼠与PITX3-IRES-TTA小鼠杂交，以驱动中脑DA神经元中WT，G71R或G59S P150GLEL的表达。 p150glued缺乏的FBS中dynactin家族蛋白的重新分布可能反映了dynactin复合物与动力蛋白介导的中心转运的脱离接触，或者是p150gll的损失，作为连接到微管蛋白的附着点的锚点。未来的研究将计划测试这两种可能性。我们将通过实时成像技术检查p150gloup的条件KO FBS中溶酶体和内体的逆行逆行转运，并通过共同抑制胶结蛋白和其他微型蛋白和其他原型光谱的微调和其他原型原蛋白和其他Micrub蛋白和其他Micrub蛋白和其他Micrub蛋白和其他Micrub蛋白和其他Micrub蛋白和其他Micrub蛋白和其他Micrub蛋白和其他Micrub蛋白和其他Micrub蛋白来检查P150glueld-lack-lack-lacking Dynactin复合物的靶向。更重要的是，我们将评估P150GLAID的损失如何影响神经元的功能。我们推测，P150GlueD可能会影响神经元中的ER到高尔基体的转运，并将检验由P150GLUED条件KO小鼠制备的原代培养神经元中的假设。