Assembly and Axonal Transport of Neurofilaments

描述（由申请人提供）：神经丝是填充空间的细胞骨架聚合物，其功能是增加轴突的横截面积。这些聚合物沿着轴突以快速的速度运输，但总体速度很慢，因为运动被长时间的停顿所打断。神经丝在轴突口径生长期间在轴突中积聚，并且它们也在广泛的神经退行性疾病中异常和过度地积聚，最显著的是肌萎缩性侧索硬化症。破坏神经丝组装的突变也会导致一种形式的腓骨肌萎缩症。我们研究的长期目标是了解轴突中神经丝的运输和组装动力学以及导致神经丝在发育和疾病中积累的机制。我们提出三个具体目标。目的1研究神经丝在轴突中的组装动力学。轴突神经丝在体内可以长达100 5 m或更长，它们也可以与可溶性池交换亚基，但对这些过程如何发生知之甚少。我们提出，神经丝可以延长端到端退火的预先形成的细丝，他们可以交换亚基的添加和损失的亚基沿着的长度的细丝，一个过程，我们术语插入亚基交换。我们将使用细胞融合以及光漂白和光活化策略来测试这些假设在培养的神经元。我们还将把我们的研究扩展到其他类型的中间丝蛋白，以确定我们的发现是否更普遍适用。目的二是研究磷酸化在神经丝转运中的作用。我们提出，磷酸化的神经丝的CDK5和ERK 1/2激酶调节其运输的添加剂和剂量依赖性的方式增加的时间比例的神经丝花费暂停。我们将在培养的神经元中通过使用定点诱变来模拟特定位点的磷酸化或非磷酸化状态来测试这些假设。我们还将直接使用药理学抑制剂和组成型活性或显性负性激酶构建体来操纵激酶活性。目的3探讨神经丝停顿的功能意义。我们认为，神经丝停顿是轴突神经丝含量的一个关键决定因素。我们将检验神经丝磷酸化通过增加它们在轴突中的停留时间而引起轴突神经丝积累的假设。我们还将使用长期的髓鞘培养来验证这一假设，即髓鞘细胞通过增加暂停时间的比例来局部减缓神经丝的运输。使用活细胞成像来研究培养中的髓鞘轴突中的神经丝转运是本目标的一个特别创新的方面。调节神经丝暂停的机制可能是导致轴突中神经丝过度积累的疾病过程的靶点。 公共卫生相关性：神经丝是临床上感兴趣的，因为它们在广泛的衰弱性神经障碍中的神经细胞中过度积累，所述衰弱性神经障碍包括运动神经元疾病、巨大轴突神经病、腓骨肌萎缩症、阿尔茨海默病、帕金森病、糖尿病性神经病和脊髓性肌萎缩症。这些积聚通过破坏正常功能所必需的其他轴突成分的运动而加剧疾病进展。我们对轴突中神经丝组装和运动的研究将阐明可能导致神经丝在轴突中积累的基本机制，并可能最终导致治疗策略的发展，以减轻这些细胞骨架堵塞，从而延缓这些悲惨疾病的进展。

DESCRIPTION (provided by applicant): Neurofilaments are space-filling cytoskeletal polymers that function to increase the cross-sectional area of axons. These polymers are transported along axons at fast rates, but the overall rate is slow because the movements are interrupted by prolonged pauses. Neurofilaments accumulate in axons during the growth of axonal caliber and they also accumulate abnormally and excessively in a wide range of neurodegenerative diseases, most notably amyotrophic lateral sclerosis. Mutations that disrupt neurofilament assembly also cause one form of Charcot-Marie-Tooth disease. The long-term goal of our research is to understand the transport and assembly dynamics of neurofilaments in axons and the mechanisms that cause neurofilaments to accumulate in development and disease. We propose three specific aims. Aim 1 is to investigate the assembly dynamics of neurofilaments in axons. Axonal neurofilaments can be 100 5m or more in length in vivo and they can also exchange subunits with a soluble pool, but little is known about how these processes occur. We propose that neurofilaments can lengthen by end-to-end annealing of pre-formed filaments and that they can exchange subunits by addition and loss of subunits along the length of the filaments, a process that we term intercalary subunit exchange. We will use cell fusion as well as photobleaching and photoactivation strategies to test these hypotheses in cultured neurons. We will also extend our studies to other types of intermediate filament proteins to establish whether our findings are more generally applicable. Aim 2 is to investigate the role of phosphorylation in neurofilament transport. We propose that phosphorylation of neurofilaments by CDK5 and ERK1/2 kinases regulates their transport in an additive and dose-dependent manner by increasing the proportion of the time the neurofilaments spend pausing. We will test these hypotheses in cultured neurons by using site-directed mutagenesis to mimic phosphorylated or non- phosphorylated states at specific sites. We will also manipulate kinase activities directly using pharmacological inhibitors and constitutively active or dominant negative kinase constructs. Aim 3 is to investigate the functional significance of neurofilament pausing. We propose that neurofilament pausing is a critical determinant of axonal neurofilament content. We will test the hypothesis that neurofilament phosphorylation causes axonal neurofilaments to accumulate by increasing their residence time in axons. We will also use long-term myelinating cultures to test the hypothesis that myelinating cells locally slow neurofilament transport by increasing the proportion of the time that they spend pausing. The use of live-cell imaging to study neurofilament transport in myelinating axons in culture is a particularly innovative aspect of this Aim. The mechanisms that regulate neurofilament pausing are likely targets for disease processes that lead to excessive accumulation of neurofilaments in axons. PUBLIC HEALTH RELEVANCE: Neurofilaments are of clinical interest because they accumulate excessively in nerve cells in a wide range of debilitating neurological disorders including motor neuron disease, giant axonal neuropathy, Charcot-Marie- Tooth disease, Alzheimer's disease, Parkinson's disease, diabetic neuropathy and spinal muscular atrophy. These accumulations exacerbate the disease progression by disrupting the movement of other axonal components which are essential for normal function. Our studies on neurofilament assembly and movement in axons will illuminate the basic mechanisms that can cause neurofilaments to accumulate in axons and may ultimately lead to the development of therapeutic strategies to alleviate these cytoskeletal log-jams, thereby delaying the progression of these tragic diseases.