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

Abstract

Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease that we identified as a nerve regeneration-associated molecule. The expression of DINE is upregulated in response to nerve injury in both the peripheral and central nervous systems, while its transcription is regulated by the activating transcription factor 3 (ATF3), a potent hub-transcription factor for nerve regeneration. Despite its unique hallmark of injury-induced upregulation, the physiological relevance of DINE in injured neurons has been unclear. In this study, we have demonstrated that the expression of DINE is upregulated in injured retinal ganglion cells (RGCs) in a coordinated manner with that of ATF3 after optic nerve injury, whereas DINE and ATF3 are not observed in any normal retinal cells. Recently, we have generated a mature DINE-deficient (KOTg) mouse, in which exogenous DINE is overexpressed specifically in embryonic motor neurons to avoid aberrant arborization of motor nerves and lethality after birth that occurs in the conventional DINE KO mouse. The DINE KOTg mice did not show any difference in retinal structure and the projection to brain from that of wild–type (wild type) mice under normal conditions. However, injured RGCs of DINE KOTg mice failed to regenerate even after the zymosan treatment, which is a well-known regeneration-promoting reagent. Furthermore, a DINE KOTg mouse crossed with a Atf3:BAC Tg mouse, in which green fluorescent protein (GFP) is visualized specifically in injured RGCs and optic nerves, has verified that DINE deficiency leads to regeneration failure. These findings suggest that injury-induced DINE is a crucial endopeptidase for injured RGCs to promote axonal regeneration after optic nerve injury. Thus, a DINE-mediated proteolytic mechanism would provide us with a new therapeutic strategy for nerve regeneration.

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

“…However, previous work suggested that Ecel1 deletion has tended to reduce RGCs but did not significantly affect the survival of injured RGCs in Ecel1 KO Tg mice. 19 One possible explanation is that the permanent deletion of Ecel1 in Ecel1 KO Tg mice might change the expression of other genes or signaling pathways to compensate for the loss of Ecel1. In the retina of Ecel1 KO Tg mice, no significant difference was seen in the number of RGCs or the axonal projections of the optic nerve to the superior colliculus and lateral geniculate nucleus.…”

“…In the retina of Ecel1 KO Tg mice, no significant difference was seen in the number of RGCs or the axonal projections of the optic nerve to the superior colliculus and lateral geniculate nucleus. 19 However, Ecel1 is expressed in the embryonic neural retina. 9 Thus, the retinal network surrounding the RGCs might somehow be changed.…”

“…In particular, brain-derived neurotrophic factor is known to have a powerful neuroprotective effect against RGC damage, such as occurs in optic nerve crush and ischemia/reperfusion models. Kaneko et al 19 clearly showed that Ecel1 has the potential to induce axonal regeneration of the RGCs after optic nerve crush. However, we could not find any protective effect against oxidative stress or ER stress in vitro ( Supplementary Fig.…”

“…ATF3 is also known as a core transcriptional factor that initiates nerve regeneration and is colocalized with Ecel1. Furthermore, Ecel1 promotes axonal regeneration in mice after optic nerve crush, 19 suggesting that injury-induced Ecel1 expression is promoted by NGF deprivation to ameliorate the effects of axonal damage and ceramideinduced apoptosis in RGCs.…”

“…17,18 In the field of retinal research, a recent study of mature Ecel1deficient mice (KO Tg ) showed that Ecel1 had a role in promoting axonal regeneration after optic nerve injury. 19 In addition, a protease-mediated mechanism might contribute to the activation of antioxidant enzymes and the prevention of neuronal death. 5,17 However, the neuroprotective effect of Ecel1 in vivo is still unclear.…”