The Asparaginyl Endopeptidase Legumain Is Essential for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

Abstract

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.

Secretome analysis of nerve repair mediating Schwann cells reveals Smad‐dependent trophism

“…In fish, ependymin is involved in memory formation (61) and was shown to promote neurite outgrowth of retinal ganglion cells (62). We further identified legumain, which was demonstrated to be essential for recovery after spinal cord injury in adult zebrafish (63). Because CNS regeneration is superior in fish as compared with mammals, legumain detection might shed new light into mammalian nerve regeneration and will have to be examined functionally in subsequent follow‐up studies.…”

Asparagine endopeptidase is an innovative therapeutic target for neurodegenerative diseases

“…The expression of AEP was increased in neurons of regenerative nuclei during the phase of axon regrowth/sprouting. Reducing the expression of AEP impaired axonal regeneration and locomotor recovery 24 .…”

Neuronal labeling patterns in the spinal cord of adult transgenic Zebrafish

“…We also stained some preparations for HuC/D (elav3) expressed in neurons, neurofilament (NF), synaptic vesicle antigen 2 (SV2), glial fibrillary acidic protein (GFAP), and choline acetyltransferase (ChAT). We examined spinal cord histology for each of these lines at the three regions considered for studies carried on adult zebrafish: the 8th (V8), the 15th (V15), and the 24th vertebrae (V24) (Becker et al, ; Bormann et al, ; Schweitzer et al, ; Reimer et al, ; Hui et al, ; Gabriel et al, ; Guo et al, ; Kyriakatos et al, ; Yu et al, ; Ausborn et al, ; Dias et al, ; Fang et al, ; Goldshmit et al, ; Kuscha et al,; Lin et al, ; Ma et al, ; Ogai et al, ; Ampatzis et al, ; Yu and Schachner, ; Liu et al, ; Vajn et al, ).…”