br Results br Discussion In this

Results
Discussion In this study we provide evidence that the N-terminal end of CXCL12, a region containing CXCR4 binding and activation domains, is sufficient to induce both migration and proliferation of SVZ-born neuroblasts. Our experimental approach was to synthesize peptides that are analogous to CXCL12 N-terminal, introducing modifications such as replacement of the tripeptide C9-P10-C11 by a linker that separates the receptor binding and receptor activation domains or replacing C9 and C11 by A (PepA-A). As predicted, the synthetic peptides bearing only the binding domain (Pep3) or the activation domain (Pep1 and Pep2) showed little chemotactic activity, and were less active when compared to the complete CXCL12 molecule or to the peptide that had both domains (PepC-C). Similar results were obtained by Crump et al. (1997), using chimeras containing CXCL12 N-terminal end (receptor activation domain) and the REFFESH sequence (receptor binding domain) and showed that they were chemotactic for lymphocytes, demonstrating the chimeras contained the contact residues essential for functional CXCL12. Here, we not only corroborate those findings but in addition demonstrate that the alanine-modified peptide (PepA-A) was less chemotactic than CXCL12 and PepC-C and had no proliferative activity on CXCR4+ NSC or cerebellar NPC in vitro. Once we determined that PepC-C was as good chemotactic factor as CXCL12 in vitro we evaluated its ability to promote chemotaxis in vivo. It has been extensively described that after an injury to the AMG 925 or spinal cord the expression of CXCL12, among other chemokines and cytokines that are part of the inflammatory response, rises at the injury site. Following ischemic stroke, SCI or TBI, the blood–brain barrier becomes dysfunctional or disrupted, allowing circulating immune cells to infiltrate, interact with and activate resident immune cells. Activated glial cells (astrocytes, microglia and oligodendrocytes) start expressing chemokines such as CCL2, CCL3, CXCL1, CXCL2 and CXCL12, as part of the local inflammatory response (Kim et al., 1995; Takami et al., 1997; Hill et al., 2004; Pineau et al., 2010; Galindo et al., 2011). The current knowledge about the dynamics of chemokine expression in CNS injury and repair has been recently reviewed by Jaerve and Müller (2012). Using a TBI mouse model we show here that PepC-C, but not PepA-A, is able to promote migration of NSC from the SVZ towards the injury site. Twenty four hours after injury it was possible to observe that the number of BrdU prelabeled cells arriving at the traumatic injury and peptide administration site doubled when compared to animals subjected to injury but not treated or treated with vehicle or A-substituted peptide (PepA-A). We have not performed experiments to measure the peptide half-lives at the site of injury and injection, but there is data suggesting that the peptides would not last long. For example, a tripeptide analogous to the N-terminal of insulin-like growth factor I (IGF-I) lasted only 3h following intracerebroventricular administration in the presence of peptidase inhibitors (Baker et al., 2005). Based on that and on the fact that several proteases are secreted after injury to the CNS (Agrawal et al., 2008), we considered that the peptides would not last for 24h of in vivo experiment. To find a possible mechanism for the prolonged duration of chemotactic activity of PepC-C in vivo, we hypothesized that a gradient of CXCL12 could be formed if PepC-C stimulated the release of high amounts of CXCL12 at the injury site that would diffuse away from the source, reaching the SVZ cells. To verify that, we looked at Cxcl12 mRNA at the injury site 24h after injury and peptide administration and observed that PepC-C, but not PepA-A, doubled the local levels of Cxcl12 mRNA. There is no information regarding the modulation of CXCL12 expression by astrocytes after TBI, but based on data that shows upregulation of CXCL12 expression by astrocytes in response to ischemia (Hill et al., 2004; Miller et al., 2005), we investigated if those cells were the ones responsible for the increase in Cxcl12 mRNA we observed in vivo. To do that, we exposed astrocytes in culture to the peptides and measured CXCL12 protein by ELISA. Surprisingly, PepC-C alone was not able to induce the release of CXCL12 to the culture medium, but acted synergistically with CXCL12, increasing the concentration of CXCL12 in the culture medium after 24h. It is well documented that CXCL12 is internalized upon binding to CXCR4 (Amara et al., 1997; Signoret et al., 1997; Haribabu et al., 1997). When astrocytes were treated with CXCL12 alone the concentration of the chemokine in the culture medium after 24h was ~10ng/ml, that represents ~10% of the original input (100ng/ml), so the increase from ~10ng/ml to ~30ng/ml when cells were exposed to CXCL12+PepC-C could be explained by inhibition of CXCR4 internalization. To verify this we treated the astrocytes in the presence of a specific CXCR4 inhibitor (AMD3465) (Hatse et al., 2002) and did not observe any difference in the concentration of CXCL12 in the presence of peptides and/or CXCL12 and absence of AMD3465, indicating that PepC-C stimulated expression and secretion of CXCL12 rather than inhibited its internalization via CXCR4 activation. Two aspects are very interesting about these data. First, PepC-C alone cannot stimulate CXCL12 expression, it requires CXCL12 to be effective; and second, it does not involve CXCR4 signaling. Our suggestion is that this stimulatory effect could be elicited by the activation of CXCR7, a recently described G-protein coupled receptor for CXCL12 (Burns et al., 2006; Schönemeier et al., 2008; Odemis et al., 2010). The molecular mechanisms involved in PepC-C-induced CXCL12 expression/secretion by astrocytes were not the focus of the present work and deserve further investigation.