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

Abstract

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.

ECEL1 gene related contractural syndrome: Long-term follow-up and update on clinical and pathological aspects

“…Furthermore, in three patients (A1, A2 and B2), we noted the presence of fibres positive for nCAM suggesting a possible problem with innervation of some fibres ( Fig.4c and 4f). This finding supports the hypothesis, gathered from the Ecel1 mouse model, that the lack of peripheral motor axon arborisation contributes to the pathogenesis of DAD5 [19]. Analysis of muscle biopsies of further patients with ECEL1 gene mutations might help to clarify this point and to characterize further the histopathological spectrum of this condition, assessing if the condition is neurological rather than muscular in origin.…”

“…The jitter measurement was normal in three patients of our series examined by this means, effectively ruling out a neuromuscular junction defect. This is relevant in view of the suggestion, from the Ecel1 mouse model, of a contribution of the neuromuscular junction in the disease pathogenesis [19]. From a clinical diagnostic point of view, in view of the major clinical and pathological overlap with similar myopathic contractural syndromes, we strongly suggest adding ECEL1 gene into panel of genes responsible for congenital myopathies and / or distal arthrogryposis.…”

“…The ECEL1 protein is thought to play a role in neural and subsequently neuromuscular junction (NMJ) development during foetal life in mice and humans [14,16,17]. Indeed, mice deficient in the homologous murine gene, Ecel1, die of respiratory failure immediately after birth [17,18], and the rodent homologue knock-in animal model shows significant decrease of peripheral motor axons, arborization and failure of NMJs formation, suggesting this could be a key pathophysiological mechanism of ECEL1-related conditions [19].…”

Damage-induced neuronal endopeptidase (DINE) enhances axonal regeneration potential of retinal ganglion cells after optic nerve injury

“…22, 23 Subsequently, we have found that aberrant arborization of motor axons and failure of neuromuscular formation can be a primary cause of DA with the ECEL1 mutation. 24 In line with this, it is likely that DINE has an important role as a protease in the neurons where its expression is abundant.…”

Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina

“…13,14 Ecel1 knock-in mice with a pathogenic mutation causing type 5 DA have defects in the axonal arborization of the motor nerves in the limbs, suggesting that failure of the neuromuscular junctions could be the primary cause of DA in patients with Ecel1 mutation. 15 Ecel1 gene expression is synergistically regulated by axonal regeneration-associated transcriptional factors, such as ATF3, c-Jun, and STAT3, through the activation of Sp1. 16 This suggests that Ecel1 is associated with axonal regeneration.…”