Investigating a novel form of Charcot-Marie-Tooth disease

描述(申请人提供)：我们已经发现了一种新型的夏科-玛丽-图思病(CMT)--第一个编码神经丝状纤维光(NFL)的NEFL基因发生隐性突变(E210X)的家系。这种突变的纯合子儿童发展成进行性的、严重的感觉和运动神经病，传导缓慢，完全进入传统的“脱髓鞘范围”。我们发现突变蛋白在培养细胞中不形成中间丝。1例患者的腓肠神经活检电子显微镜下见髓鞘轴突密度明显降低，残存轴突较小，缺少中间丝。这些发现表明，E210X突变很可能是零等位基因，因此NEFL的纯合子零等位基因会导致人类严重的进行性神经病。后一个结论是完全出乎意料的，因为对NEFL缺失的小鼠和箭鸟(在NEFL中含有无义突变)进行了调查，发现它们没有神经病变，尽管发现有髓的PNS轴突缺乏神经细丝(NFS)，并且在发育过程中无法正常扩大。这种差异促使我们重新检查了一些患有EM的NEFL基因缺失的“老年”(至少一岁)小鼠，结果显示，不仅在外周神经，而且在视神经和脊髓中，NFL都是退化的有髓轴突，这表明NFL对于维持三叉神经节和中枢神经系统的有髓轴突是必需的。然而，这些小鼠一直保持着近交系，因此不能排除其他基因导致这些病理结果的可能性。为了明确验证这一假设，我们建议将小鼠回交到它们以前的背景(C57/BL6)，并通过在3、6、12、18和24个月龄时对L4和L5腹根、坐骨神经、股神经和视神经以及颈脊髓和腰髓进行光学显微镜、免疫细胞化学和EM检查来比较年龄匹配的NELF-/-、NELF-/-、NELF-/-和NELF-/-小鼠，其具体目的如下：1)确定NEFL缺失小鼠PNS中轴突变性的时间和模式。2)探讨NEFL基因缺失小鼠三叉神经核传导减慢的细胞基础。3)确定NEFL基因敲除小鼠中枢神经系统轴突变性的时间和模式。这些数据将确定在三叉神经节和中枢神经系统中，神经纤维是否是维持有髓轴突所必需的；这种作用在以前的研究中还没有得到证实。如果发现轴突变性的证据，该模型可以用于进一步的研究，以确定为什么轴突完整需要神经丝，并了解这些患者以及其他类型CMT患者的轴突退化机制。 与公共卫生相关：拟议的研究将确定神经纤维是否对维持三叉神经节和中枢神经系统的有髓轴突是必不可少的；这种作用在以前的研究中尚未得到证实。如果发现轴突变性的证据，该模型可以用于进一步的研究，以确定为什么轴突完整需要神经丝，并了解这些患者以及其他类型CMT患者的轴突退化机制。

DESCRIPTION (provided by applicant): We have identified a novel form of Charcot-Marie-Tooth disease (CMT) - the first family with a recessive mutation (E210X) in the NEFL gene, which encodes neurofilament light (NFL). Children homozygous for this mutation develop a progressive, severe, sensory and motor neuropathy with slow conduction well into the traditional "demyelinating range". We found that the mutant protein did not form intermediate filaments in cultured cells. The density of myelinated axons was markedly reduced, and the remaining axons were small and lacked intermediate filaments in a sural nerve biopsy from one patient by electron microscopy (EM). These findings indicate that the E210X mutation is likely a null allele, so that homozygous null alleles of NEFL cause a severe progressive neuropathy in humans. The latter conclusion was completely unexpected as the Nefl-null mice and quiver quail (which harbor a nonsense mutation in Nefl) were investigated and found not to have a neuropathy, in spite of the finding that myelinated PNS axons were devoid of neurofilaments (NFs) and failed to enlarge normally during development. This discrepancy prompted us to re-examine some "old" (at least one- year-old) Nefl-null mice with EM, which showed degenerating myelinated axons not only in peripheral nerves, but also in optic nerve and spinal cord, suggesting that NFL is required for the maintenance of myelinated axons in both PNS and CNS. However, these mice had been maintained an inbred line, so the possibility that other genes contributed to these pathological findings could not be excluded. To definitely examine this hypothesis, we propose to back crossing the mice to their former background (C57/Bl6) and compare age- matched Nelf -/-, Nelf , and Nelf littermates by examining the L4 and L5 ventral roots, sciatic, femoral and optic nerves, and cervical and lumbar spinal cord by light microscopy, immunocytochemistry and EM at 3, 6, 12, 18 and 24 months of age, with the following Specific Aims: 1) Determine the timing and pattern of axonal degeneration in the PNS of Nefl-null mice. 2) Investigate the cellular basis of slowed conduction in the PNS of Nefl-null mice. 3) Determine the timing and pattern of axonal degeneration in the CNS of Nefl-null mice. These data will establish whether NFs are essential for the maintenance of myelinated axons in both PNS and CNS; such a role has not been demonstrated in prior studies. If evidence of axonal degeneration is found, this model can be used for further investigation to determine why neurofilaments are required for axonal integrity and to understand the mechanism of axonal degeneration in these patients and perhaps patients with other kinds of CMT. PUBLIC HEALTH RELEVANCE: The proposed study will establish whether NFs are essential for the maintenance of myelinated axons in both PNS and CNS; such a role has not been demonstrated in prior studies. If evidence of axonal degeneration is found, this model can be used for further investigation to determine why neurofilaments are required for axonal integrity and to understand the mechanism of axonal degeneration in these patients and perhaps patients with other kinds of CMT.