Motor Nerve Arborization Requires Proteolytic Domain of Damage-Induced Neuronal Endopeptidase (DINE) during Development.
Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease, which we originally identified as a nerve regeneration-associated molecule. Abundant expression of DINE is observed in regenerating neurons, as well as in developing spinal motor neurons. In line with this, DINE-deficient (DINE KO) embryos fail to arborize phrenic motor nerves in the diaphragm and to form proper neuromuscular junctions (NMJ), which lead to death shortly after birth. However, it is unclear whether protease activity of DINE is involved in motor nerve terminal arborization and how DINE participates in the process. To address these issues, we performed an in vivo rescue experiment in which three types of motor-neuron specific DINE transgenic mice were crossed with DINE KO mice. The DINE KO mice, which overexpressed wild-type DINE in motor neurons, succeeded in rescuing the aberrant nerve terminal arborization and lethality after birth, while those overexpressing two types of protease domain-mutated DINE failed. Further histochemical analysis showed abnormal behavior of immature Schwann cells along the DINE-deficient axons. Coculture experiments of motor neurons and Schwann cells ensured that the protease domain of neuronal DINE was required for proper alignment of immature Schwann cells along the axon. These findings suggest that protease activity of DINE is crucial for intramuscular innervation of motor nerves and subsequent NMJ formation, as well as proper control of interactions between axons and immature Schwann cells.



Damage-induced neuronal endopeptidase (DINE) is a membrane-bound metalloprotease; expression is abundant in developing spinal motor neurons, as well as in nerve-injured neurons. DINE-deficient (KO) embryos fail to arborize phrenic motor nerves in the diaphragm and to form a neuromuscular junction, leading to death immediately after birth. To address whether proteolytic activity of DINE is involved in this process, we performed in vivo rescue experiments with DINE KO mice. Transgenic rescue of DINE KO mice was accomplished by overexpression of wild-type DINE, but not by protease domain-mutated DINE. Immature Schwann cells were abnormally aligned along the DINE protease-deficient axons. Thus, the protease activity of DINE is crucial for motor axon arborization, as well as the interaction between axons and immature Schwann cells.
Department of Functional Anatomy and Neuroscience, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan.
Sakiko
S
Matsumoto
Department of Functional Anatomy and Neuroscience, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan skiryu@med.nagoya-u.ac.jp kiyama@med.nagoya-u.ac.jp.
Sumiko
0000-0001-6213-1258
S
Kiryu-Seo
Department of Functional Anatomy and Neuroscience, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan skiryu@med.nagoya-u.ac.jp kiyama@med.nagoya-u.ac.jp.
Hiroshi
0000-0001-5963-046X
H
Kiyama
D000818:Animals; D001369:Axons / Q000502:physiology*; D008666:Metalloendopeptidases / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D009046:Motor Neurons / Q000502:physiology* / Q000648:ultrastructure; D009416:Nerve Regeneration / Q000502:physiology; D009469:Neuromuscular Junction / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D010447:Peptide Hydrolases; D010791:Phrenic Nerve; D012583:Schwann Cells / Q000378:metabolism
D010447:Peptide Hydrolases; C120591:Ecel1 protein, mouse; D008666:Metalloendopeptidases; C407822:damage-induced neuronal endopeptidase
ECEL1; Schwann cell; branching; metalloprotease; motor neuron; neuromuscular junction