ECEL1 mutation implicates impaired axonal arborization of motor nerves in the pathogenesis of distal arthrogryposis

The membrane-bound metalloprotease endothelin-converting enzyme-like 1 (ECEL1) has been newly identified as a causal gene of a specific type of distal arthrogryposis (DA). In contrast to most causal genes of DA, ECEL1 is predominantly expressed in neuronal cells, suggesting a unique neurogenic pathogenesis in a subset of DA patients with ECEL1 mutation. The present study analyzed developmental motor innervation and neuromuscular junction formation in limbs of the rodent homologue damage-induced neuronal endopeptidase (DINE)-deficient mouse. Whole-mount immunostaining was performed in DINE-deficient limbs expressing motoneuron-specific GFP to visualize motor innervation throughout the limb. Although DINE-deficient motor nerves displayed normal trajectory patterns from the spinal cord to skeletal muscles, they indicated impaired axonal arborization in skeletal muscles in the forelimbs and hindlimbs. Systematic examination of motor innervation in over 10 different hindlimb muscles provided evidence that DINE gene disruption leads to insufficient arborization of motor nerves after arriving at the skeletal muscle. Interestingly, the axonal arborization defect in foot muscles appeared more severe than in other hindlimb muscles, which was partially consistent with the proximal-distal phenotypic discordance observed in DA patients. Additionally, the number of innervated neuromuscular junction was significantly reduced in the severely affected DINE-deficient muscle. Furthermore, we generated a DINE knock-in (KI) mouse model with a pathogenic mutation, which was recently identified in DA patients. Axonal arborization defects were clearly detected in motor nerves of the DINE KI limb, which was identical to the DINE-deficient limb. Given that the encoded sequences, as well as ECEL1 and DINE expression profiles, are highly conserved between mouse and human, abnormal arborization of motor axons and subsequent failure of NMJ formation could be a primary cause of DA with ECEL1 mutation.

膜结合金属蛋白酶内皮素转化酶样1（ECEL1）新近被确定为一种特定类型的远端关节挛缩症（DA）的致病基因。与大多数DA致病基因不同，ECEL1主要在神经元细胞中表达，这表明在具有ECEL1突变的一部分DA患者中存在独特的神经源性发病机制。本研究分析了啮齿动物同源物损伤诱导的神经元内肽酶（DINE）缺陷小鼠肢体的发育性运动神经支配和神经肌肉接头形成。在表达运动神经元特异性绿色荧光蛋白（GFP）的DINE缺陷肢体中进行整体免疫染色，以观察整个肢体的运动神经支配情况。尽管DINE缺陷的运动神经从脊髓到骨骼肌显示出正常的轨迹模式，但它们在前肢和后肢的骨骼肌中表现出轴突分支受损。对10多种不同后肢肌肉的运动神经支配进行系统检查，证明DINE基因破坏导致运动神经到达骨骼肌后轴突分支不足。有趣的是，足部肌肉的轴突分支缺陷似乎比其他后肢肌肉更严重，这与在DA患者中观察到的近端 - 远端表型不一致部分相符。此外，在受严重影响的DINE缺陷肌肉中，受神经支配的神经肌肉接头数量显著减少。此外，我们构建了一种带有致病性突变的DINE敲入（KI）小鼠模型，该突变最近在DA患者中被发现。在DINE KI肢体的运动神经中明显检测到轴突分支缺陷，这与DINE缺陷肢体相同。鉴于编码序列以及ECEL1和DINE的表达谱在小鼠和人类之间高度保守，运动轴突的异常分支以及随后神经肌肉接头形成失败可能是具有ECEL1突变的DA的主要病因。