Neuron-specific Enolase 2 (Eno2) is an isozyme primarily distributed in the central and peripheral nervous systems and neuroendocrine cells. It promotes neuronal survival, differentiation, and axonal regeneration. Recent studies have shown that Eno2 localized on the cell membrane of motor neurons acts as a receptor for extracellular phosphoglycerate kinase 1 (ePgk1), which is secreted by muscle cells and promotes the neurite outgrowth of motor neurons (NOMN). However, interaction between Eno1, another isozyme of Enolase, and ePgk1 failed to return the same result. To account for the difference, we constructed seven point-mutations of Eno2, corresponding to those of Eno1, and verified their effects on NOMN. Among the seven Eno2 mutants, eno2-siRNA-knockdown NSC34 cells transfected with plasmid encoding the 419th aspartic acid mutated into serine (Eno2-[D419S]) or Eno2-[E420K] showed a significant reduction in neurite length. Moreover, the Eno2-ePgk1-interacted synergic effect on NOMN driven by Eno2-[D419S] was more profoundly reduced than that driven by Eno2-[E420K], suggesting that D419 was the more essential residue involved in NOMN mediated by Eno2-ePgk1 interaction. Eno2-ePgk1-mediated NOMN appeared to increase the level of p-Cofilin, a growth cone collapse marker, in NSC34 cells transfected with Eno2-[D419S] and incubated with ePgk1, thereby inhibiting NOMN. Furthermore, we conducted in vivo experiments using zebrafish transgenic line Tg(mnx1:GFP), in which GFP is tagged in motor neurons. In the presence of ePgk1, the retarded growth of axons in embryos injected with eno2-specific antisense morpholino oligonucleotides (MO) could be rescued by wobble-eno2-mRNA. However, despite the addition of ePgk1, the decreased defective axons and the increased branched neurons were not significantly improved in the eno2-[D419S]-mRNA-injected embryos. Collectively, these results lead us to suggest that the 419th aspartic acid of mouse Eno2 is likely a crucial site affecting motor neuron development mediated by Eno2-ePgk1 interaction, and, hence, mutations result in a significant reduction in the degree of NOMN in vitro and axonal growth in vivo.

Zebrafish is emerging as a model animal for phenotype-based drug screening. Drugs screened from the zebrafish platform have advanced into clinical trials, underscoring their translational potential. Amyotrophic lateral sclerosis is a progressive motor neurons (MN) degenerative disease with few approved drugs. Previously, supplementation with exogenous recombinant phosphoglycerate kinase 1 (Pgk1) was found to improve MN growth through its interaction with receptor Eno2. To bypass the high complexity and cost of full-length Pgk1 production, a short segment within Pgk1 (M08) was predicted as the key motif interacting with Eno2, and a zebrafish phenotypic screening platform was established to find the most neurotrophic compound(s) among M08 and its mutants. We first found that M08-injected zebrafish embryos significantly increased branched caudal primary MNs (CaPMNs). However, compared to M08 (59.20 ± 1.80%), M039, among 17 mutants further screened, showed even more improvement of branched CaPMNs, up to 74.54 ± 3.73%. Next, when we administered the M039 peptide to C9ORF72-knockdown ALS-like zebrafish embryos, it improved axonal growth and swimming ability. Then, we employed a cellular model as a secondary screen, and M039 exhibited improved neurite outgrowth of MN (NOMN) and reduced p-Cofilin in NSC34 neural cells grown in ALS-like condition. Therefore, by using a zebrafish MN phenotype as a primary screening platform, we identified a mutated short peptide M039 having the most pronounced positive effect on improving neurite growth among all 17 mutants in comparison to parental M08, demonstrating the feasibility of zebrafish screening as a cost-effective strategy for finding promising neuroprotective short peptides that serve as neurotherapeutic potentials.