Pathophysiology of Conduction Block in HNPP.

描述（由申请人提供）：传导阻滞（CB）是一种动作电位沿神经沿着传播的失败，在许多中枢和外周神经系统脱髓鞘疾病中导致神经功能障碍，包括格林-巴利综合征、慢性炎症性脱髓鞘性多发性神经病和多发性硬化症。然而，CB的分子基础还不清楚。有趣的是，患有遗传性神经病易患压力麻痹（HNPP）的患者，一种含有PMP 22基因的染色体17p11.2的一个拷贝缺失的遗传性疾病，对周围神经上的机械力异常敏感，并发展可逆的局灶性无力和感觉丧失，这可能是由于CB。在过去的4年中，通过NIH K 08基金的支持，PI的实验室使用HNPP的真实动物模型，pmp 22杂合基因敲除小鼠（pmp 22）研究了CB。我们发现，CB可以更迅速地机械诱导pmp 22小鼠比野生型小鼠。我们已经确定了频繁的局灶性轴突收缩包裹的结旁tomacula（过度的髓鞘折叠），一个病理标志的HNPP。我们推测，tomacula/轴突收缩倾向于PMP 22缺陷的神经发展机械诱导CB。此外，我们已经表明，去除PMP 22缺陷小鼠中的p21激活激酶1型（pak 1）基因消除了tomacula/轴突收缩，这是一种新的信号传导机制。在这个建议中，我们将进一步研究CB的发展和恢复的细胞和分子基础，tomacula/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：测试的假设，tomacula/轴突收缩倾向于机械诱导的CB在PMP 22缺陷的神经。我们的初步研究结果表明，加速机械诱导的CB和轴突收缩tomacula在pmp 22小鼠。在这个目标中，我们将首先确定CB的易感性和tomacula/轴突收缩之间的关系，使用一个额外的动物模型与tomacula/轴突收缩和动物模型没有这些病理。接下来我们将研究这种倾向的潜在机制;这些机制包括（1）由tomacula中的轴突畸形引起的电生理学效应和（2）可能的电流泄漏出tomacula髓鞘，分流去极化电流以降低动作电位传播的安全系数。这些机制将使用共聚焦显微镜和三维电镜描绘详细的几何特征的轴索畸形在tomacula的调查。这些tomacula/轴突畸形的生理后果将通过阈值跟踪技术进行评价。这些结果将提供深入了解的机制的倾向，机械诱导CB的PMP 22缺陷。目的2：检验PAK 1是形成tomaculum/轴突收缩所必需的假设。PAK 1是一种丝氨酸-苏氨酸激酶，属于PAK家族（PAK 1 - 6），与小的GTP酶（如cdc 42和rac）相互作用而激活。pak 1-/-小鼠中PAK 1的缺乏不引起表型。然而，在pak 1-/-与pmp 22小鼠杂交后，两个基因的双重敲除消除了pmp 22小鼠的tomacula/轴突收缩。在这个目标中，我们将测试去除tomacula是否会逆转PMP 22缺陷小鼠对机械诱导CB的易感性，并进一步探索这种新的信号通路。我们将尝试通过测试新合成的PAK抑制剂是否可以逆转PMP 22缺乏时的tomacula/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pmp 22单倍不足延迟CB恢复的机制。我们的实验结果表明，机械诱导的CB在pmp 22小鼠的延迟恢复。在这个目标中，CB延迟恢复的细胞和分子机制将在pmp 22小鼠中进行研究。 总之，这三个目标将确定细胞和分子因素，使pmp 22神经易受机械诱导CB，并建立分子信号通路的形成tomaculum/轴突收缩的PMP 22缺陷。这些结果有望加深我们对CB的分子基础的理解，这可能有助于了解许多脱髓鞘疾病的发病机制。

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.