Pathophysiology of Conduction Block in HNPP.

描述(申请人提供)：传导阻滞(CB)是一种动作电位沿神经传播的失败，可导致许多中枢和外周神经系统脱髓鞘疾病的神经功能障碍，包括格林-巴利综合征、慢性炎症性脱髓鞘多神经病和多发性硬化症。然而，CB的分子基础还不是很清楚。有趣的是，遗传性压力性瘫痪(HNPP)是一种遗传性疾病，含有PMP22基因的染色体17p11.2的一个拷贝缺失，患者对周围神经的机械力异常敏感，并出现可逆性局灶性无力和感觉丧失，这可能是由于CB引起的。在过去的4年里，在NIH K08基金的支持下，PI的实验室使用了HNPP的真实动物模型-PMP22杂合基因敲除小鼠(PMP22)来研究CB。我们发现，在PMP22小鼠中，机械诱导CB比在野生型小鼠中更快。我们发现HNPP的病理特征之一--结旁绒毛(髓鞘过度折叠)导致局灶性轴索收缩。我们假设绒毛/轴突收缩使PMP22缺陷神经易于发生机械性CB。此外，我们已经证明，在PMP22缺陷小鼠中移除p21激活的激酶类型-1(PAK1)基因可以消除毛囊/轴突收缩，这是一种新的信号机制。在这个方案中，我们将进一步研究CB发生和恢复的细胞和分子基础，绒毛/轴突收缩的形成，以及PAK抑制剂的治疗潜力。为此，我们提出了以下具体目标：目的1：检验在PMP22缺乏时，绒毛/轴索收缩使神经易于发生机械性CB的假说。我们的初步结果显示，在PMP22小鼠的毛囊中，机械诱导的CB和轴突收缩加速。为了达到这个目的，我们将首先使用另外一个有绒毛/轴突收缩的动物模型和一个没有这些病理变化的动物模型来确定CB的易感性与毛斑/轴突收缩之间的关系。接下来，我们将研究这种易感性的潜在机制，其中包括(1)毛囊轴突畸形引起的电生理效应，以及(2)毛囊髓鞘可能发生的电流泄漏，分流去极化电流，从而降低动作电位传播的安全系数。这些机制将用共聚焦显微镜和三维EM来研究，以描绘毛斑区轴突畸形的详细几何特征。这些绒毛/轴突畸形的生理后果将通过阈值跟踪技术进行评估。这些结果将为PMP22缺乏症中机械诱发CB倾向的潜在机制提供洞察力。目的2：验证PAK1参与丘脑/轴索收缩形成的假说。PAK1是一种丝氨酸-苏氨酸激酶，是PAK家族(从PAK1到PAK6)的成员之一，它与CDC42和RAC等小分子GTP酶相互作用激活。在PAK1-/-小鼠中，PAK1的缺失导致没有表型。然而，在PAK1-/-与PMP22小鼠杂交后，这两个基因的双敲除消除了PMP22小鼠的绒毛/轴突收缩。为此，我们将测试去除毛囊是否能逆转PMP22缺陷小鼠对机械诱导的CB的易感性，并进一步探索这一新的信号通路。我们将尝试通过测试新合成的PAK抑制剂是否可以逆转PMP22缺乏症的绒毛/轴突收缩来将这一令人兴奋的发现转化为治疗。目的3：确定PMP22单倍性缺失延缓脐带血恢复的机制。我们的实验结果表明，在PMP22小鼠中，机械诱导的CB恢复延迟。为此，将在PMP22小鼠身上研究CB恢复延迟的细胞和分子机制。综上所述，这三个目标将确定PMP22神经易感于机械诱导CB的细胞和分子因素，并为PMP22缺乏症的丘脑/轴突收缩的形成建立分子信号通路。这些结果有望加深我们对CB的分子基础的了解，这可能为许多脱髓鞘疾病的发病机制提供深入的认识。 公共卫生相关性： 传导阻滞(CB)是一种电信号沿神经纤维传播的障碍，可导致多种神经功能障碍。易受压力性瘫痪的遗传性神经病患者经常出现局灶性无力和感觉丧失，这很可能是由CB引起的。本研究采用HNPP及其动物模型研究慢性阻塞性肺疾病的分子机制。

DESCRIPTION (provided by applicant): Conduction block (CB), a failure of action potential propagation along the nerve, causes neurological disabilities in a number of demyelinating diseases of the central and peripheral nervous systems, including Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, and multiple sclerosis. The molecular basis for CB, however, is not well understood. Interestingly, patients with hereditary neuropathy with liability to pressure palsies (HNPP), an inherited condition with a deletion of one copy of chromosome 17p11.2 containing the PMP22 gene, are abnormally sensitive to mechanical force on the peripheral nerve, and develop reversible focal weakness and sensory loss which are probably due to CB. In the past 4 years, through the support of an NIH K08 grant, the PI's laboratory has studied CB using an authentic animal model of HNPP, the pmp22 heterozygous knockout mouse (pmp22). We found that CB can be mechanically induced more rapidly in the pmp22 mice than that in wild-type mice. We have identified frequent focal axonal constrictions encased by paranodal tomacula (excessive myelin folding), a pathological hallmark of HNPP. We hypothesize that the tomacula/axonal constrictions predispose the PMP22 deficient nerves to develop mechanically induced CB. Moreover, we have shown that removal of the p21-activated kinase type-1 (pak1) gene in PMP22 deficient mice eliminates tomacula/axonal constrictions, a novel signaling mechanism. In this proposal we will further investigate the cellular and molecular basis for the development and recovery of CB, the formation of tomacula/axonal constrictions, and the therapeutic potential of PAK inhibitors. Toward these ends, we propose the following specific aims: Aim 1: Test the hypothesis that tomacula/axonal constrictions predispose nerves to mechanically induced CB in PMP22 deficiency. Our preliminary results have shown a hastened mechanically-induced CB and axonal constrictions in tomacula in pmp22 mice. In this aim, we will first determine the relationship between the predisposition of CB and tomacula/axonal constrictions using an additional animal model with tomacula/axonal constrictions and an animal model without these pathologies. We will next investigate potential mechanisms for this predisposition; these include (1) electrophysiological effects caused by axonal deformities in tomacula and (2) possible current leakage out of tomaculous myelin that shunts the depolarizing current to reduce the safety factor for action potential propagation. These mechanisms will be investigated using confocal microscopy and 3-dimentional EM to delineate detail geometric features of axonal deformities in tomacula. Physiological consequences of these tomacula/axon deformities will be evaluated by threshold tracking technique. These results will provide insights into the mechanisms underlying the propensity to mechanically-induce CB in PMP22 deficiency. Aim 2: Test the hypothesis that PAK1 is required for the formation of tomaculum/axonal constriction. PAK1, as a serine-threonine kinase and a member of the PAK family (from PAK1 to 6), interacts with small GTPases for its activation, such as cdc42 and rac. Deficiency of PAK1 in the pak1-/- mice causes no phenotype. After crossbreeding pak1-/- with pmp22 mice, however, double-knockout of both genes eliminates tomacula/axonal constrictions in pmp22 mice. In this aim, we will test whether removal of tomacula will reverse the susceptibility to mechanically-induced CB in PMP22 deficient mice, and further explore this novel signaling pathway. We will attempt to translate this exciting finding to therapy by testing whether newly synthesized PAK inhibitor can reverse tomacula/axonal constrictions in PMP22 deficiency. Aim 3: Identify the mechanisms by which haploinsufficiency of pmp22 delays the recovery of CB. Our experimental results have shown a delayed recovery of mechanically-induced CB in the pmp22 mice. In this aim, cellular and molecular mechanisms that underlie the delayed recovery of CB will be investigated in pmp22 mice. Taken together, these three aims will define cellular and molecular factors that predispose pmp22 nerves to mechanically induced CB, and establish molecular signaling pathway for the formation of tomaculum/axonal constriction in the PMP22 deficiency. Results are expected to deepen our understanding on the molecular basis of CB, which may render insights into the pathogenesis for many demyelinating diseases. PUBLIC HEALTH RELEVANCE: Conduction block (CB), a failure of propagation of electrical signal along nerve fibers, causes disabilities in a variety of neurological disorders. Patients with hereditary neuropathy with liability to pressure palsies present with frequent focal weakness and sensory loss, which are likely caused by CB. Our study investigates molecular mechanisms responsible for the CB using HNPP and its animal model.