Assembly and Axonal Transport of Neurofilaments

描述(申请人提供)：神经丝是一种充满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快速运输，但总体速度很慢，因为运动会被长时间的停顿打断。神经丝在轴突口径的生长过程中积累在轴突中，它们也在广泛的神经退行性疾病中异常和过度积累，最明显的是肌萎缩侧索硬化症。破坏神经丝组装的突变也会导致一种形式的Charcot-Marie-Tooth病。我们研究的长期目标是了解神经丝在轴突中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。我们提出了三个具体目标。目的1研究神经细丝在轴突中的组装动力学。在活体中，轴突神经丝可以长达100 5米或更长，它们也可以与可溶池交换亚单位，但这些过程是如何发生的，人们知之甚少。我们认为神经丝可以通过预先形成的细丝的端到端的退火来延长，并且它们可以通过沿细丝长度的亚基的添加和丢失来交换亚基，这一过程我们称之为插入亚基交换。我们将使用细胞融合以及光漂白和光激活策略在培养的神经元中测试这些假设。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适用于一般情况。目的2研究磷酸化在神经丝运输中的作用。我们认为，CDK5和ERK1/2激酶对神经丝的磷酸化通过增加神经丝停顿时间的比例，以一种相加的和剂量依赖的方式调节它们的运输。我们将在培养的神经元中通过使用定点突变来模拟特定位置的磷酸化或非磷酸化状态来测试这些假说。我们还将直接使用药理抑制剂和结构性活性或显性负性激酶构建物来操纵激酶活性。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关键决定因素。我们将检验神经丝磷酸化导致轴突神经丝通过增加它们在轴突中的停留时间而积聚的假设。我们还将使用长期髓鞘培养来测试这一假设，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髓鞘轴突中的神经丝运输是这一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神经丝在轴突中过度积累的疾病过程的靶标。与公共卫生相关：神经丝具有临床意义，因为它们在一系列衰弱的神经系统疾病中过度积累在神经细胞中，包括运动神经元病、巨大轴索神经病、夏科-玛丽-图思病、阿尔茨海默病、帕金森病、糖尿病神经病变和脊髓性肌萎缩。这些堆积扰乱了对正常功能至关重要的其他轴突成分的运动，从而加剧了疾病的进展。我们对神经丝在轴突中的组装和运动的研究将阐明导致神经丝在轴突中积累的基本机制，并最终可能导致开发治疗策略来缓解这些细胞骨架的堵塞，从而延缓这些悲剧性疾病的进展。

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.