The Function of dynactin p150glued in Axonal Transport and Motor Neuron Diseases

dynactin p150glued 在轴突运输和运动神经元疾病中的作用

• 批准号: 7964106
• 负责人: Huaibin Cai
• 金额: $ 15.91万
• 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/7964106
• 关键词: 4-Hydroxy-Tamoxifen; Accounting; Actins; Address; Adult; Affect; Age; Age-Months; Alleles; Animal Model; Axonal Transport; Behavioral; Binding; Brain; Cell Line; Cell physiology; Classification; Coiled-Coil Domain; Complex; Cytoplasm; Cytoskeleton; Data; Defect; Development; Dynein ATPase; Embryo; Estrogen Receptors; Exons; Exposure to; Functional disorder; Gait; Gait abnormality; Gene Mutation; Genes; Glycine; Human; Image; Journals; Kinesin; Knock-in Mouse; Knockout Mice; Ligand Binding Domain; Ligands; Link; Lower Motor Neuron Disease; Macromolecular Complexes; Mediating; Microtubules; Missense Mutation; Molecular; Motor; Motor Neuron Disease; Motor Neurons; Mus; Mutant Strains Mice; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Junction; Neurons; Neurosciences; Nuclear; Organelles; Phenotype; Physiological; Proteins; Publishing; Research Personnel; Research Project Grants; Role; Site; Spinal; Staging; Stress; Symptoms; Synaptic Vesicles; Syndrome; System; Tamoxifen; Testing; Therapeutic; Time; Transgenes; Transgenic Mice; Transport Vesicles; Variant; Vesicle; Work; astrogliosis; base; calmodulin-dependent protein kinase II; dynactin; dynactin 1; in vitro Assay; migration; motor neuron degeneration; mouse model; mutant; nestin protein; neuron development; neuronal cell body; promoter; protein complex; recombinase; retrograde transport; synaptogenesis; trafficking

A previous study suggests that the G59S substitution occurring in p150glued partially compromises the association of p150glued with microtubules in an in vitro assay. However, how this mutation affects the function of the dynein/dynactin complex and contributes to motor neuron degeneration is unclear. To further study the pathobiological mechanism of this type of motor neuron disease, our specific aims are: Aim 1: To generate and characterize p150glued G59S knock-in mice. Aim 2: To examine any alterations in axonal transport of spinal motor neurons derived from p150glued G59S knock-in mice. Aim 3: To further investigate the role of p150glued in dynein-mediated retrograde transport. To study how the G59S mutation in dynactin p150glued affects the function of the dynein/dynactin complex and contributes to motor neuron degeneration, we generated p150glued G59S knock-in mice. We found that the G59S mutation destabilizes p150glued and disrupts the function of dynein/dynactin complex, resulting in early embryonic lethality of homozygous knock-in mice. Heterozygous knock-in mice, which developed normally, displayed MND-like phenotypes after 10 months of age, including excessive accumulation of cytoskeletal and synaptic vesicle proteins at neuromuscular junctions, loss of spinal motor neurons, increase of reactive astrogliosis, and shortening of gait compared with wild-type littermates and age-matched p150glued heterozygous knockout mice. Our findings indicate that the G59S mutation in p150glued abrogates the normal function of p150glued and accelerates motor neuron degeneration. This work has been published by the Journal of Neuroscience (Lai et al., 2007), and highlighted in the This Week in the Journal (TWIJ) by the Journal of Neuroscience. Because the loss of dynactin p150glued leads to early embryonic lethality, we decided to generate dynactin p150glued conditional knockout (CKO) mice to investigator whether dynactin p150glued is required in dynein-mediated retrograde transport. To generated dynactin p150glued CKO mice, we modified the DCTN1 gene locus by flanking the exons 2-4 of DCTN1 gene with a pair of Loxp sites. To genetically inhibit the expression of dynactin p150glued at different developmental stages of neurons, we then crossed the dynactin p150glued CKO mice with three different lines of Cre transgenic mice in which the Cre recombinase is under the control of nestin, CaMKII, and chick β-actin (CAG) promoters, respectively. For the CAG-Cre transgenic mice, the transgene insert contains a fusion product involving Cre recombinase and a mutant form of the mouse estrogen receptor (Esr1) ligand binding domain. The mutant mouse estrogen receptor does not bind natural ligand at physiological concentrations but will bind the synthetic ligand, 4-hydroxytamoxifen. Restricted to the cytoplasm, the Cre/Esr1 protein can only gain access to the nuclear compartment after exposure to tamoxifen. A systematic analysis of neuropathological and behavioral phenotypes of these mutant mice will allow us to reveal the role of dynactin p150glued in neuron differentiation, migration, synaptogenesis, and survival. We have successfully deleted dynactin p150glued from adult mice using the Cre/Esr1 system. To our surprise, inhibition of dynactin p150glued in adult mice does not cause any overt phenotypes. It appears that p135, an alternative translational variant of dynactin p150glued may compensate for the loss of dynactin p150glued. Whether the loss of dynactin p150glued affects the neuron development remains to be investigated. Our present study demonstrates for the first time that a mouse model carrying a motor neuron disease (MND)-linked G59S substitution in the dynactin p150glued subunit develops many symptoms related to ALS and MND, such as motor neuron degeneration, reactive astrogliosis, and abnormal gait. Because Dctn1+/m mice contain one copy of wild-type allele and one copy of G59S mutant allele, it faithfully replicates the genetic mutation in humans and may serve as a useful animal model for studying the pathogenic mechanism of MND and testing potential therapeutics. We provided evidence to further demonstrate that p150glued is required for the cellular functions of cytoplasmic dynein. Dctn1m/m and Dctn1-/- mice died before 8.5 dpc, similar to cytoplasmic dynein heavy chain knockout mice. The G59S mutation in p150glued does not affect the integrity of the dynein/dynactin complex, except for inducing self-aggregation when over-expressed in cell lines. In fact, we observed around a 50% reduction of p150glued in Dctn1+/m mouse brains and failed to detect any p150glued from Dctn1m/m mouse embryos, suggesting that the G59S mutation in p150glued leads to a rapid degradation of mutant p150glued. Because age-matched Dctn1+/- mice did not develop any of these neuropathological abnormalities as observed in Dctn1+/m mice, our data indicate that a 50% reduction of p150glued alone is not sufficient to cause motor neuron degeneration at the age having been examined. Neither does the increase of p135 account for the motor behavioral and neuropathological deficits observed in Dctn1+/m mice, because a comparable increase of p135 was also found in Dctn1+/- mice. The G59S mutation may exert additional stress to motor neurons through further disruption of the function of dynein/dynactin complex. A conditional p150glued knockout mouse model that selectively removes p150glued from adult motor neurons will be useful to address whether dynein/dynactin-mediated retrograde transport is essential for the survival of motor neurons. In addition, real time imaging of dynein/dynactin-mediated axonal retrograde transport will be employed to investigate potential axonal trafficking defects in both p150glued knock-in and conditional KO mice.

先前的一项研究表明，在P150GLOUD中发生的G59S取代部分损害了体外测定中P150GLES与微管的缔合。然而，这种突变如何影响动力蛋白/dynactin复合物的功能并有助于运动神经元变性。为了进一步研究这种运动神经元疾病的病理生物学机制，我们的具体目的是： AIM 1：生成和表征P150GLOUD G59的敲入小鼠。 目标2：检查源自P150G59S敲入小鼠的脊柱运动神经元的轴突转运的任何变化。 AIM 3：进一步研究P150GLEL在动力蛋白介导的逆行转运中的作用。 为了研究Dynactin P150GLUED中的G59S突变如何影响Dynein/Dynactin复合物的功能并有助于运动神经元变性，我们产生了P150GLEAD G59S敲入小鼠的功能。我们发现，G59S突变破坏了P150GLID并破坏Dynein/Dynactin复合物的功能，从而导致纯合敲击小鼠的早期胚胎致死性。正常发育的杂合敲击小鼠在10个月大后显示出MND样表型，包括在神经肌肉连接处的细胞骨架和突触囊泡蛋白的过度积累，脊柱运动神经元的丧失，反应性星形胶质症的增加以及缩短了反应性星形胶质病，并增加了步态。搭配野生型窝窝和年龄匹配的p150glued杂合淘汰小鼠。我们的发现表明，P150GLOUD中的G59S突变消除了P150GLED的正常功能，并加速了运动神经元变性。这项工作已由《神经科学杂志》（Lai等，2007）发表，并在本周的《神经科学杂志》（TWIJ）中强调。 由于Dynactin P150glued的损失导致早期的胚胎致死性，因此我们决定生成Dynactin P150glued有条件敲除（CKO）小鼠到研究者到研究者是否需要在Dynein介导的逆行运输中Dynactin P150glued。为了生成Dynactin P150GLOUD CKO小鼠，我们通过将DCTN1基因的外显子2-4用一对LoxP位点侧翼，修改了DCTN1基因座。为了在遗传上抑制在神经元不同发育阶段的Dynactin P150GLLE的表达，然后我们用三种不同的CRE转基因小鼠越过Dynactin P150GLOUD CKO小鼠，其中CRE重物组合酶在NESTIN，CAMKII，CAMKII，CAMKII，CAMKI-ATCATIN，以及CREβ-肌动蛋白的控制下（CAG）启动子。对于CAG-CRE转基因小鼠，转基因插入物包含涉及CRE重组酶的融合产物和小鼠雌激素受体（ESR1）配体结合结构域的突变形式。突变小鼠雌激素受体不以生理浓度结合天然配体，而是结合合成配体4-羟基莫昔芬。 CRE/ESR1蛋白仅限于细胞质，只能在暴露于他莫昔芬后进入核室。对这些突变小鼠的神经病理学和行为表型的系统分析将使我们能够揭示Dynactin P150Gll在神经元分化，迁移，突触发生和生存中的作用。使用CRE/ESR1系统，我们成功地从成年小鼠中删除了Dynactin P150。令我们惊讶的是，成年小鼠中对Dynactin P150GL的抑制不会引起任何明显的表型。看来，p135是Dynactin P150GLUED的另一种翻译变体可以弥补Dynactin P150GLUED的损失。 Dynactin P150GlueD的损失是否会影响神经元发育仍有待研究。 我们目前的研究首次表明，携带运动神经元疾病（MND）连接的G59s替代的小鼠模型在Dynactin P150GLUED亚基中会出现许多与ALS和MND相关的症状，例如运动神经元变性，反应性星形胶质细胞和异常步态， 。由于DCTN1+/M小鼠包含一个野生型等位基因的副本和一份G59S突变等位基因的副本，因此它忠实地复制了人类的遗传突变，并且可以作为研究MND的致病机制和测试潜在疗法的有用动物模型。我们提供了证据，以进一步证明细胞质动力蛋白的细胞功能需要P150GLUED。 DCTN1M/M和DCTN1 - / - 小鼠在8.5 dpc之前死亡，类似于细胞质动力蛋白重链敲除小鼠。 p150glued中的G59S突变不会影响动力蛋白/dynactin复合物的完整性，除非在细胞系中过表达时诱导自我聚集。实际上，我们观察到DCTN1+/M小鼠大脑中的P150GLLES降低了约50％，未能检测到从DCTN1M/M小鼠胚胎中检测到的任何P150GLID，这表明P150GLEADS中的G59S突变使P150GLUED突变的铅迅速去分解。由于年龄匹配的DCTN1 +/-小鼠没有在DCTN1+/M小鼠中观察到的这些神经病理学异常中的任何一种，因此我们的数据表明，单独使用P150GLED的50％降低不足以引起该年龄的运动神经元。 p135的增加也没有说明在DCTN1+/M小鼠中观察到的运动行为和神经病理学缺陷，因为在DCTN1 +/-小鼠中也发现了p135的可比增加。 G59S突变可能会通过进一步破坏动力蛋白/dynactin复合物的功能对运动神经元施加额外的压力。有条件的P150敲除小鼠模型，有选择地去除成人运动神经元中的P150GLEL，对于解决Dynein/dynactin介导的逆行转运对于运动神经元的存活至关重要。此外，将采用Dynein/dynactin介导的轴突逆行转运的实时成像来研究P150GLUED敲入和条件KO小鼠的潜在轴突运输缺陷。