Connexins in Nerve Regeneration and Inherited Neuropathy

描述(由申请人提供)： 这项授权背后的一个基本原理是，雪旺细胞的正常功能需要连接蛋白32(Cx32)。虽然Cx32基因突变导致通道功能改变，并与X连锁Charcot-Marie-Tooth病(CMTX)明显相关，但该疾病的发病机制仍有待阐明。Cx32定位于髓鞘雪旺细胞的副阳极和Schmidt-Lantermann切迹，导致假设Cx32在非致密的髓鞘内形成反射性缝隙连接，并在髓鞘雪旺细胞的ab和轴突细胞质之间提供了一条“短路”途径。然而，数据表明：1)缺乏Cx32的小鼠再生相关髓鞘形成能力降低；2)Cx32在原代雪旺细胞培养中表达和调控；3)表达两种不同突变形式的Cx32的雪旺细胞在异种移植模型中对再生有显著不同的影响；4)从Cx32基因敲除小鼠培养的雪旺细胞显示死亡增加。这些发现导致了一种假设，即Cx32是非髓鞘相关雪旺细胞正常功能所必需的，特别是那些因神经损伤而增殖并参与神经再生的细胞。目的1将使用形态计量学、全动物电生理学以及行为和生化评估来检验Cx32缺失对正常再生能力有害的假说。目的2将利用雪旺细胞培养和双膜片钳技术来验证以下假设：1)Cx32在原代培养的增殖期成人雪旺细胞中有功能表达；2)其表达水平受GGF(被认为在沃勒变性过程中诱导雪旺细胞增殖)的调节；3)Cx32介导的细胞-细胞通道的丢失导致雪旺细胞死亡增加。目的3将利用原代培养雪旺细胞和神经横断模型来验证Cx32表达是防止雪旺细胞凋亡和/或限制雪旺细胞增殖的假说。这项提案中概述的实验应该在理解Cx32在雪旺细胞中的作用以及Cx32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.