Partial cleavage of CXCL at the N

Partial cleavage of CXCL12 at the N-terminus by distinct peptidases results in loss of chemotactic activity and impairment in CXCR4 receptor affinity (Cho et al., 2010; Delgado et al., 2001; Levesque et al., 2003). The proteolytic cleavage of CXCL12 by matrix metalloproteinase 2 and 9 (MMP-2 and 9) removes selectively the first four N-terminus Epigallocatechin manufacturer from the full length molecule, generating the truncated form CXCL12(5-67) (McQuibban et al., 2001). Previous work (Denoyer et al., 2012; Van Raemdonck et al., 2014; Vergote et al., 2006; Zhu et al., 2009) showed that CXCR3, natural receptor of CXCL9, 10 and 11, acts as signalling receptor for CXCL12(5-67). These authors also demonstrated in those independent studies that CXCL12(5-67) affects the viability of differentiated cell types in the CNS, but its effect on neural stem cells remains poorly studied.
Results
Discussion In the healthy brain, MMP-2/9 modulate neural progenitor cell migration from the SVZ to the olfactory bulb along the rostral migratory stream (Bovetti et al., 2007), whereas following brain lesions, MMP-2/9 participate on migration of neural progenitors into an injured area. Besides migration, MMPs play regulatory activity in different tissues, processing active molecules as surface receptors, growth factors and chemokines (Ben-Hur et al., 2006; Kang et al., 2008; Lee et al., 2006). CXCL12 is one of the many substrates of MMP-2/9, and cleavage in a specific site at N-terminal portion of CXCL12 generates CXCL12(5-67) (McQuibban et al., 2001). In addition, enhanced MMP-2/9 expression is involved in the pathophysiologic mechanisms of brain injury and neurodegenerative diseases (Hoehna et al., 2012; Kaplan et al., 2014; Stomrud et al., 2010; Uckermann et al., 2011). Here we showed that CXCL12(5-67) impairs NSCs migration. These results corroborated with the reports of Peng et al. (2012) which demonstrated that the proteolytic processing of CXCL12 by MMP-2/9 reduces foetal neural progenitor cell migration. We also observed no difference on migration when we increased CXCL12(5-67) concentration, suggesting a migration abolishment rather than receptor affinity reduction. These findings are in accordance with many other studies showing that most of CXCL12 receptor activation sequence disrupts migration mediated by CXCR4 receptor. When we tried to recover the full-length molecule chemotactic capacity by adding CXCL12 N-terminal peptide (KPVSLSYR-NH2, pep NH2) together with CXCL12(5-67), still no effect on migration was observed. This observation indicates that the spatial arrangement of these sequences in the full-length molecule is important for binding and activation of CXCR4 receptor. Chemokines have been reported to act in an optimal concentration, with no improvement in the cell migration when concentration increases (Ottoson et al., 2001; Poznansky et al., 2000). Furthermore, it was reported that an increase in CXCL12 concentration above the optimal range blocks cell migration by ligand dimer formation, downregulation of receptor expression or its desensitization (Pelletier et al., 2000). Corroborating these observations, here we saw no difference in migration when cells were treated with increasing concentrations of CXCL12. Surprisingly, the N-terminal peptide enhanced CXCL12 chemotaxis at 100ng/mL, but caused no effect at 200ng/mL. Since the peptide per se does not induce chemotaxis, the effect observed may be due to an interaction between the peptide and the full-length molecule, preventing CXCL12 dimer formation, thus improving the chemotactic activity. Additionally, high concentrations of CXCL12 can induce dimerization of CXCR4 receptors, affecting signal transduction, which also can help to explain why CXCL12 chemotactic effect is not concentration dependent (Zlatopolskiy and Laurence, 2001). As migration is one of multiple steps of neurogenesis, disruption of this process can ultimately affect newborn neurons survival. But here we aimed to assess the direct effect of CXCL12(5-67) on NSCs survival. Even though CXCL12(5-67) has been implicated in neurodegeneration by HIV and retinal degeneration (Denoyer et al., 2012; Vergote et al., 2006; Zhang et al., 2003), but its role in adult NSCs remained unclear. Here we showed, for the first time, that CXCL12(5-67) induces the intrinsic apoptosis pathway in adult NSCs. Treatment with CXCL12(5-67) induced activation of caspases 3 and 7 early as after 4h of treatment in vitro, and apoptosis was also confirmed using the annexin V binding assay.