Pathophysiology of Conduction Block in HNPP.

描述（由申请人提供）：传导阻滞（CB），动作电位沿神经传播的失败，导致许多中枢和周围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发性神经病和多发性硬化症。然而，炭黑的分子基础尚不清楚。有趣的是，遗传性神经病伴压性麻痹（HNPP）患者（一种含有PMP22基因的染色体17p11.2拷贝缺失的遗传性疾病）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，通过NIH K08拨款的支持，PI的实验室使用HNPP的真实动物模型，pmp22杂合敲除小鼠（pmp22）研究了CB。我们发现，与野生型小鼠相比，pmp22小鼠可以更快地机械诱导CB。我们已经发现了频繁的局灶性轴突缩被副神经瘤包围（髓鞘过度折叠），这是HNPP的病理标志。我们假设，瘤突/轴突收缩易使PMP22缺陷神经发生机械诱导的脊髓灰质炎。此外，我们已经证明，在PMP22缺陷小鼠中去除p21激活的激酶1型（pak1）基因可以消除瘤/轴突收缩，这是一种新的信号机制。在本研究中，我们将进一步研究细胞和分子基础的发展和恢复的CB，形成的tomacula/轴突收缩，和PAK抑制剂的治疗潜力。针对这些目的，我们提出以下具体目标：目的1：验证在PMP22缺乏的情况下，瘤突/轴突收缩使神经容易机械诱导CB的假设。我们的初步结果显示pmp22小鼠在机械诱导下加速了脑皮层和轴突的收缩。在这个目的中，我们将首先使用另外一个有肿瘤/轴突狭窄的动物模型和一个没有这些病理的动物模型来确定脑脊炎易感性与肿瘤/轴突狭窄之间的关系。接下来，我们将研究这种倾向的潜在机制；这包括(1)由瘤突畸形引起的电生理效应和(2)可能从瘤突髓鞘泄漏电流，分流去极化电流，降低动作电位传播的安全系数。这些机制将研究使用共聚焦显微镜和三维电子显微镜描绘的细节几何特征的轴突畸形在tomacula。这些瘤突/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缺乏症中机械诱导CB倾向的机制提供见解。目的2：验证PAK1是番茄窝/轴突收缩形成所必需的假设。PAK1是一种丝氨酸-苏氨酸激酶，是PAK家族的一员（从PAK1到pak6），与cdc42和rac等小gtpase相互作用激活。PAK1 -/-小鼠缺乏PAK1不引起表型。然而，pak1-/-与pmp22小鼠杂交后，两种基因的双敲除消除了pmp22小鼠的番茄/轴突收缩。在本研究中，我们将测试去除番茄是否会逆转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缺乏症的瘤突/轴突收缩，将这一令人兴奋的发现转化为治疗。目的3：确定pmp22单倍不足延迟CB恢复的机制。我们的实验结果显示pmp22小鼠机械诱导的CB恢复延迟。在此目的下，将在pmp22小鼠中研究CB延迟恢复的细胞和分子机制。综上所述，这三个目标将确定使pmp22神经易发生机械诱导的CB的细胞和分子因素，并建立pmp22缺乏时形成突起/轴突收缩的分子信号通路。这些结果有望加深我们对CB分子基础的认识，从而为许多脱髓鞘疾病的发病机制提供见解。

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.