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

先前的一项研究表明，在体外实验中，发生在p150gled中的G59S替换部分损害了p150gled与微管的联系。然而，这种突变如何影响动力蛋白/动力蛋白复合体的功能并导致运动神经元退化尚不清楚。为了进一步研究这种运动神经元病的病理生物学机制，我们的具体目标是： 目的1：建立并鉴定p150胶合型G59S基因敲除小鼠。 目的：观察p150粘合G59S基因敲除小鼠脊髓运动神经元轴突运输的变化。 目的：进一步研究p150gled在动力蛋白介导的逆行转运中的作用。 为了研究dynactin p150gled中的G59S突变如何影响dynein/dynactin复合体的功能并促进运动神经元退变，我们建立了p150gled G59S敲入小鼠。我们发现G59S突变破坏了粘合的p150的稳定性，破坏了动力蛋白/动力蛋白复合体的功能，导致纯合子敲入小鼠的早期胚胎死亡。发育正常的杂合基因敲除小鼠在10个月后表现出类似MND的表型，包括神经肌肉接头处细胞骨架和突触小泡蛋白的过度积累，脊髓运动神经元的丢失，反应性星形胶质细胞增多，步态缩短，与野生型和年龄匹配的p150胶合杂合基因敲除小鼠相比。我们的研究结果表明，p150gled中的G59S突变破坏了p150gled的正常功能，并加速了运动神经元的退化。这项研究已由《神经科学杂志》(Lai等人，2007)发表，并在《神经科学杂志》(TWIJ)的《本周》杂志上予以重点介绍。 由于dynactin p150gled的缺失会导致早期胚胎死亡，我们决定建立dynactin p150gled条件性基因敲除(CKO)小鼠来研究dynactin p150gled是否在dynein介导的逆行运输中是必需的。为了获得dynactin p150胶合的CKO小鼠，我们通过在DCTN1基因的外显子2-4两侧加上一对loxP位点来修改DCTN1基因的基因座。为了从基因上抑制不同发育阶段神经元中dynactin p150的表达，我们将dynactin p150粘合的CKO小鼠与三个不同品系的Cre转基因小鼠杂交，其中Cre重组酶分别受巢蛋白、CaMKII和鸡肌动蛋白(CAG)启动子的控制。对于CAG-Cre转基因小鼠，转基因插入物含有Cre重组酶和突变形式的小鼠雌激素受体(ESR1)配体结合域的融合产物。突变的小鼠雌激素受体在生理浓度下不与天然配体结合，但会与合成配体4-羟基他莫昔芬结合。Cre/ESR1蛋白仅限于细胞质，在暴露于他莫昔芬后才能进入核间室。对这些突变小鼠的神经病理和行为表型的系统分析将使我们能够揭示dynactin p150在神经元分化、迁移、突触形成和存活中的作用。我们使用Cre/ESR1系统成功地从成年小鼠中删除了dynactin p150。令我们惊讶的是，抑制成年小鼠的dynactin p150不会引起任何明显的表型。似乎p135是dynactin p150gled的另一种翻译变体，可以补偿dynactin p150gled的丢失。Dynactin p150的缺失是否会影响神经元的发育还有待研究。 我们目前的研究首次证明，携带运动神经元疾病(MND)相关G59S替换的dynactin p150胶合亚单位的小鼠模型会出现许多与ALS和MND相关的症状，如运动神经元变性、反应性星形胶质细胞增生症和步态异常。由于Dctn1+/m小鼠含有一份野生型等位基因和一份G59S突变型等位基因，它在人类中忠实地复制了基因突变，可作为研究MND发病机制和测试潜在治疗方法的有用动物模型。我们提供的证据进一步证明p150胶合是细胞质动力蛋白细胞功能所必需的。Dctn1m/m和Dctn1-/-小鼠在8.5DPC之前死亡，与细胞质动力蛋白重链基因敲除小鼠相似。P150gled中的G59S突变不影响dynein/dynactin复合体的完整性，但在细胞系中过度表达时会诱导自身聚集。事实上，我们在Dctn1+/m小鼠大脑中观察到p150gled减少了约50%，没有从Dctn1m/m小鼠胚胎中检测到任何p150gled，这表明p150gled中的G59S突变导致突变的p150gled迅速降解。由于年龄匹配的Dctn1+/-小鼠没有出现Dctn1+/m小鼠中观察到的任何这些神经病理异常，我们的数据表明，仅减少50%的p150胶合不足以导致所检查的年龄的运动神经元退化。在Dctn1+/m小鼠中观察到的运动行为和神经病理缺陷也不能解释p135的增加，因为在Dctn1+/-小鼠中也发现了类似的p135增加。G59S突变可能通过进一步破坏动力蛋白/动力蛋白复合体的功能而对运动神经元施加额外的压力。一种有条件的p150粘着基因敲除小鼠模型，选择性地从成年运动神经元中去除p150粘着基因，将有助于研究dynein/dynactin介导的逆行转运是否对运动神经元的生存至关重要。此外，动态蛋白/动力蛋白介导的轴突逆行运输的实时成像将被用于研究p150粘合敲入和条件性KO小鼠潜在的轴突运输缺陷。

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.