Connexins in Nerve Regeneration and Inherited Neuropathy

描述（由申请人提供）： 这项授权的基本原则是连接蛋白32（Cx 32）是许旺细胞正常功能所必需的。虽然Cx 32的突变导致通道功能的改变，并明确与X连锁腓骨肌萎缩症（CMTX），这种疾病的发病机制仍有待阐明。Cx 32定位于髓鞘形成的许旺细胞的旁阳极和Schmidt-Lantermann切口，这导致了以下假设：Cx 32在非致密髓鞘内形成反射性间隙连接，并在髓鞘形成的许旺细胞的ab-和adaxonal细胞质之间提供“短路”途径。 然而，数据表明：1）缺乏Cx 32的小鼠显示出与髓鞘形成相关的再生能力降低; 2）Cx 32在原代雪旺细胞的培养物中表达和调节; 3）表达Cx 32的两种不同突变形式的雪旺细胞在异种移植模型中对再生具有显著不同的作用;以及4）从Cx 32敲除小鼠培养的雪旺细胞显示出死亡增加。这些发现导致了这样的假设，即Cx 32是非髓鞘相关的许旺细胞的正常功能所必需的，特别是那些响应于神经损伤而增殖并参与神经再生的许旺细胞。目的1将使用形态测量学，整个动物的电生理学和行为和生化评估，以检查的假设，Cx 32的损失是有害的正常再生能力。目的2将使用雪旺细胞培养和双膜片钳技术来检验以下假设：1）Cx 32在原代培养的增殖的成年雪旺细胞中功能性表达; 2）其表达水平受GGF调节（其被认为在沃勒变性期间诱导雪旺细胞增殖）; 3）Cx 32介导的细胞-细胞通道的丧失导致雪旺细胞死亡增加。目的3将使用原代培养的雪旺细胞和神经横断模型来检验Cx 32的表达是防止细胞凋亡和/或限制雪旺细胞增殖所必需的假设。本提案中概述的实验应该在理解Cx 32在雪旺细胞中的作用以及Cx 32突变如何导致遗传性周围神经病变方面发挥关键作用。

DESCRIPTION (provided by applicant): A fundamental principle underlying this grant is that connexin 32 (Cx32) is required for normal function of Schwann cells. Though mutations in Cx32 cause alterations in channel function and are clearly associated with the X-Linked Charcot-Marie-Tooth Disease (CMTX), the pathogenesis of this disorder remains to be elucidated. The localization of Cx32 to the paranodes and Schmidt-Lantermann incisures of the myelinating Schwann cell has lead to the hypothesis that Cx32 forms reflexive gap junctions within noncompact myelin and provides a "short circuit" pathway between the ab- and adaxonal cytoplasm of the myelinating Schwann cell. However, data suggest that: 1) mice lacking Cx32 show reduced capacity for regeneration associated myelination; 2) Cx32 is expressed and regulated in cultures of primary Schwann cells; 3) Schwann cells expressing two different mutant forms of Cx32 have strikingly different effects on regeneration in a xenograft model and 4) Schwann cells cultured from Cx32 knockout mice show increased death. These findings lead to the hypothesis that Cx32 is required for normal function of non-myelin-associated Schwann cells, especially those that are proliferating in response to nerve injury and participating in nerve regeneration. Aim 1 will use morphometry, whole animal electrophysiology and behavioral and biochemical assessments to examine the hypothesis that loss of Cx32 is detrimental to normal regenerative Capacity. Aim 2 will use Schwann cell culture and the dual patch clamp technique to examine the hypothesis that: 1) Cx32 is functionally expressed in proliferating adult Schwann cells in primary culture; 2) its expression levels are regulated by GGF (which is thought to induce Schwann cell proliferation during Wallerian degeneration); and 3) loss of Cx32 mediated cell-cell channels leads to increased Schwann cell death. Aim 3 will use primary Schwann cells in culture and the nerve transection model to examine the hypotheses that expression of Cx32 is required to prevent apoptotic cell death and/or limit proliferation of Schwann cells. The experiments outlined in this proposal should play a key role in understanding the role of Cx32 in the Schwann cell and how mutations in Cx32 lead to inherited peripheral neuropathy.