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.
Highlights
In adult mammals, spinal cord injury (SCI) most often causes permanent disabilities due to failure to regenerate
Legumain expression is upregulated in the nucleus of the medial longitudinal fascicle after SCI To gain insights into the molecular mechanisms underlying successful spinal cord regeneration after SCI in adult zebrafish, we had performed microarray analysis to detect gene expression profile changes after SCI, when compared to sham-injured fish [2]
This analysis showed that legumain mRNA expression does not change during early phases after axotomy, i.e. 4 h (1.03860.067 (SCI) versus 1.00060.118 (CON), two-tailed t-test, P.0.05) and 12 h (0.95460.120 (SCI) versus 1.00060.089 (CON), two-tailed t-test, P.0.05) when compared to sham-injured fish, which were generally taken as controls
Summary
Spinal cord injury (SCI) most often causes permanent disabilities due to failure to regenerate. Zebrafish have developed into a powerful model to elucidate the molecular mechanisms underlying spinal cord regeneration, and regeneration of the adult CNS in general, raising the hope that the findings from zebrafish may lead to therapeutic approaches in mammals. To identify novel regeneration-conducive molecules, we have performed mRNA microarray expression profiling of the nucleus of the medial longitudinal fascicle (NMLF), a brainstem nucleus containing neurons capable of axonal regeneration after injury, hypothesizing that genes that are upregulated in expression after SCI contribute to successful recovery of locomotor functions. One of the molecules upregulated in neurons capable of axonal regeneration after SCI was legumain [2], the function of which in regeneration and in nervous system functions in general, is unknown
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