Investigating a novel form of Charcot-Marie-Tooth disease

描述（由申请人提供）：我们已经确定了一种新形式的腓骨肌萎缩症（CMT）-第一个在NEFL基因中具有隐性突变（E210 X）的家族，该基因编码神经丝光（NFL）。这种突变的纯合子儿童发展为进行性的、严重的感觉和运动神经病变，传导缓慢，进入传统的“脱髓鞘范围”。我们发现突变蛋白在培养的细胞中不形成中间丝。在一个病人的腓肠神经活检的有髓轴突的密度显着减少，和剩余的轴突小，缺乏中间丝的电子显微镜（EM）。这些发现表明E210 X突变可能是无效等位基因，因此NEFL的纯合无效等位基因导致人类严重的进行性神经病变。后一个结论是完全出乎意料的，因为研究了Nef 1缺失小鼠和鹌鹑（其在Nef 1中具有无义突变），发现其没有神经病，尽管发现有髓鞘的PNS轴突缺乏神经丝（NF）并且在发育期间未能正常扩大。这种差异促使我们用EM重新检查一些“老的”（至少一岁）Nefl缺失小鼠，其显示不仅在外周神经中，而且在视神经和脊髓中的有髓鞘轴突退化，这表明NFL是维持PNS和CNS中的有髓鞘轴突所必需的。然而，这些小鼠一直保持近交系，因此不能排除其他基因导致这些病理学发现的可能性。为了明确地检验这一假设，我们提出将小鼠与它们以前的背景（C57/B16）反向杂交，并通过在3、6、12、18和24月龄时通过光学显微镜、免疫细胞化学和EM检查L4和L5腹根、坐骨神经、股神经和视神经以及颈脊髓和腰脊髓来比较年龄匹配的Nelf -/-、Nelf和Nelf同窝出生仔。具体目的如下：1）确定Nef 1缺失小鼠的PNS中轴突变性的时间和模式。2)研究Nefl基因敲除小鼠PNS中传导减慢的细胞基础。3)确定Nefl缺失小鼠CNS中轴突变性的时间和模式。这些数据将确定神经纤维是否对维持PNS和CNS中的有髓轴突至关重要;这种作用在先前的研究中尚未得到证实。如果发现轴突变性的证据，该模型可用于进一步研究，以确定为什么轴突完整性需要神经丝，并了解这些患者以及其他类型CMT患者的轴突变性机制。 公共卫生相关性：拟议的研究将确定神经纤维是否是必不可少的维持有髓轴突在PNS和中枢神经系统，这样的作用还没有被证明在以前的研究。如果发现轴突变性的证据，该模型可用于进一步研究，以确定为什么轴突完整性需要神经丝，并了解这些患者以及其他类型CMT患者的轴突变性机制。

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.