## 4 Discussion  
In the present study, we report for the first time that the antibiotics doxycycline and minocycline induce the expression and activity of two plasminogen activators, tPA and uPA, in RT4 Schwann cell-like cultures. Furthermore, we linked this induction to PAC1 receptor activity, as pre-treatment with the PAC1 receptor antagonist, PACAP6-38, mitigated this response. As such, this study provided additional evidence to support the recent finding suggesting that both tetracyclines may act as PAMs of the PAC1 receptor, which was first documented by [PERSON] and colleagues in 2019 [23]. Figure 6: Protein expression of tPA and uPA in treated RT4 cells. Western blots and densitometric analyses of tPA (**A**,**A**) and uPA (**B**,**B**) in RT4 cells exposed to PACAP6-38 for 1 h then treated with PACAP, doxycycline or minocycline for 24 h. Total proteins were normalized to GAPDH, the loading control. Data represent means of n = 4 samples for each group. Results are expressed as mean \(\pm\) S.E.M. * \(p<0.05\), ** \(p<0.01\) or *** \(p<0.001\) vs. Control; $ \(p<0.05\), $ \(p<0.01\) or $§§ \(p<0.001\) vs. corresponding drug-treatment groups, as determined by ANOVA followed by Sidak’s _post-hoc_ test. The entire study was carried out using RT4 Schwann cells. These immortalized Schwann cell lines share similar transcriptional profiles and biology with primary Schwann cells [32], hence they are considered as a viable alternative to study PNS myelin-producing glia. Our rationale to conduct this study was based on previous observations, in which we identified that both PACAP and the PAC1 receptors are endogenously expressed in RT4 Schwann cell lines [21]. Using these cells, we have shown that exogenous administration of PACAP upregulates the expression and activity of tPA via the PAC1 receptor and that conditions able to increase the endogenous PACAP/PAC1 signaling (i.e., serum starvation) can also increase the activity of plasminogen activators [22]. In addition, we have also determined that inflammatory factors to mimic the local microenvironment of nerve injury (lipopolysaccharide, aka LPS) can upregulate PACAP expression and concurrently downregulate a direct repressor of plasminogen activators expression (miR-340) in RT4 cells [5], corroborating the idea that the crosstalk between the PACAP neuropeptide system and the plasminogen activator system may be a critical step in the cascade of events that are initiated by Schwann cells following nerve injury. These findings align with other studies showing that these glial cells undergo important biochemical alterations that allow cells to secrete factors able to promote neuronal survival and axonal regeneration after nerve injury [33]. In the RT4 cell line, PACAP upregulates the expression of multiple myelin markers [34], an effect that suggests a broader involvement of the peptide in the nerve regeneration process after injury, summarized here in two distinct stages: (1) an early stage in which PACAP stimulates Schwann cells to secrete factors that promote debris clearance and axonal regrowth and (2) a late stage where PACAP promotes the accumulation of pro-myelinating factors by Schwann cells that aid in remyelination process once neurite regrowth is completed. This theory has been partly confirmed by recent evidence in vivo, where PACAP was found to promote myelin regeneration following sciatic nerve injury [6]. PACAP is significantly increased in peripheral neurons post-injury, and it is detectable at higher levels one month after nerve damage, suggesting that it may play a key role in both axonal regrowth and the consequent remyelination process [35]. Moreover, axon regeneration following injury is severely disrupted in PACAP knockout mice [20]. More recently, transcriptional profiling of the skin of patients undergoing surgery for carpal tunnel syndrome revealed that _ADCYAP1_ (the gene encoding for PACAP) was the most robustly upregulated and its expression was associated with nerve recovery.