Doxycycline and Minocycline act as Positive Allosteric Modulators of the PAC1 Receptor and Induce Plasminogen Activators in RT4 Schwann Cells

Featured Application: Targeting the PAC1-receptor has the potential to exert neuroprotective effects against a range of neurotoxic, neurodegenerative and inflammatory insults. The novel discovery of doxycycline and minocycline to act as positive allosteric modulators of the PAC1 receptor provides an exciting opportunity to therapeutically target this receptor. This study provides evidence for the ability of these tetracyclines to promote Schwann cells activities associated with axonal regeneration and neuroprotection through the induction of a PAC1-mediated proteolytic activity of tPA and uPA. Abstract: Regeneration of peripheral nerves depends on the ability of axons to navigate through an altered extracellular environment. It has been suggested that Schwann cells facilitate this process through their secretion of neuropeptides and proteases. Using the RT4-D6P2T Schwann cell line (RT4), we have previously shown that RT4 cultures endogenously express the neuropeptide PACAP, and respond to exogenous stimulation by inducing the expression of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) through PAC1 receptor activation. In this study, based on recent findings showing that doxycycline and minocycline act as positive allosteric modulators (PAMs) of the PAC1 receptor, we tested if treatment with these tetracyclines could induce the expression and activity of tPA and uPA in RT4 cells. Using ELISA and zymographic analyses, we demonstrate that doxycycline and minocycline reliably induce the secretion and activity of both tPA and uPA, which is paralleled by an increased expression levels, as shown by immunocytochemistry and Western blots. These actions were mediated, at least in part, by the PAC1 receptor, as PACAP6-38 mitigated tetracycline-induced expression and activity of tPA and uPA. We conclude that doxycycline and minocycline can act as PAMs of the PAC1 receptor to promote proteolytic activity in RT4 cells.


Introduction
Peripheral nerve injury can arise from systemic diseases or localised damage; however, unlike the central nervous system (CNS), the peripheral nervous system (PNS) displays a high regenerative capacity [1]. This is mainly driven by Schwann cells, the myelinating glia of the PNS that display high plasticity following peripheral nerve injury. Upon nerve injury, Schwann cells spontaneously reprogram to switch from a myelinating phenotype to a progenitor-like state that promotes nerve regeneration [2]. Part of this process includes the secretion of proteases by , like plasminogen activators, whose

ELISA tPA and uPA activity
The concentrations of uPA and tPA were determined by Enzyme Linked Immune Sorbent Assay (ELISA) commercial kits (Abcam, Cambridge, MA, USA; tPA [ab198510] and uPA [ab108917]) according to the manufacturer's instructions. RT4 cells were either left untreated (controls) or treated with increasing concentrations of either doxycycline or minocycline (10, 50, 100, 500 and 1000 ng/mL) and tPA and uPA levels assessed 24 hours later. Each condition was tested using five different batches of cells (n=5/group).

Cell viability assay
To assess cell viability, we used the cell proliferation kit I (Sigma-Aldrich, NSW, Australia). Cells were treated with increasing doses of either doxycycline or minocycline for 24 h as above. 10 μL of MTT labelling reagent was added to each well and incubated for 4 h. 100 μL of solubilisation solution was added to each well and incubated overnight at 37°C. Absorbance was measured at 565 nm in the TECAN infinite M1000-PRO ELISA reader (ThermoFisher Scientific, Victoria, Australia).

Zymography of tPA and uPA activity
The activity of tPA and uPA was determined by zymographic analysis using a procedure previously published [22,31]. Briefly, following indicated treatment, cells were collected with 1% Triton X-100 in PBS buffer. Equal amounts of protein were loaded onto a 12% polyacrylamide gel containing 2mg/ml casein in the presence of 5 μg/ml plasminogen. After electrophoresis, enzyme reaction was initiated by incubating the gel in 0.1 M glycine-NaOH (pH 8.3) at 37°C for 18 h, and lytic areas were identified after staining the gel with a solution of 30% methanol, 10% glacial acetic acid and 0.5% Coomassie blue G250. The gels were then destained and scanned with Bio-Rad Imaging system. Images were assessed semi quantitatively using ImageJ software. To exclude interference by matrix metalloproteinase, EDTA (2 mM) was included in the glycine-NaOH buffer during incubation. Gels that did not containing plasminogen were also run and failed to produce lytic areas corresponding to plasminogen activators (data not shown). Bands were identified based on their relative molecular weight.

Immunocytochemistry
To determine the expression of tPA in RT4 cells, immunocytochemistry was performed. Briefly, 1×10 4 cells were seeded in poly-L-lysine (Sigma-Aldrich, Castle Hill, NSW, Australia) pre-coated tissue culture coverslips (22mm Ø, Sarstedt, SA, Australia). Cells were fixed with 4% filtered paraformaldehyde (PFA: 4% in PBS pH 7.4) (Sigma-Aldrich, Castle Hill, NSW, Australia). Coverslips were then washed three times with ice cold PBS. Cell were then permeabilised for 10 minutes in PBS containing 0.25% Triton X-100 (Sigma-Aldrich, Castle Hill, NSW, Australia), followed 3× washes in PBS for 5 minutes. Thereafter, cells were soaked in 1.5% H2O2 / PBS solution for 15 min to quench endogenous peroxidases. Non-specific binding of antibodies was then prevented by incubating coverslips with 1% BSA in PBST for 30 minutes. Once completed, the cells were incubated in diluted rabbit anti-tPA primary antibody (Abcam, Cambridge, MA, USA [ab157469]; diluted 1:500 in PBST and 1% BSA) in a humidified chamber overnight at 4°C. The next day, the primary antibody was removed with 3× washes in PBS for 5 minutes. Cells were then incubated with secondary antibodies (goat anti-rabbit IgG H&L (HRP) [ab97051] at 1/5000 dilution) in 1% BSA in PBST for 2 h at room temperature with gentle oscillation. Secondary antibody solution was then removed, and cells were washed again three times with PBS for 5 minutes. A drop streptavidin peroxidase was then added to each well (containing the coverslip) and allowed to incubate for 30 min at room temperature, followed by 3 × 5 min washes in PBST on an orbital shaker. 3,3'-Diaminobenzidine (DAB) substrate (Sigma-Aldrich, Castle Hill, NSW, Australia) was added to each cell coverslip under a fume hood. Once the cells started turning brown, the reaction was stopped with 2× washes in PBS for 5 minutes each on a shaker. Counterstaining of nuclei was performed by dipping each coverslip into a staining dish of hematoxylin for 10 sec, followed by an acid bath (200 mL UltraPure water and 1-3 drops of acetic acid). Stained coverslips were then imaged using a Nikon Eclipse TS2 inverted microscope (magnification 20×). Scale bar is 60µm.

Western blot
Protein lysate were homogenised in RIPA buffer (Sigma-Aldrich, St. Louis, MO), which was supplemented with cOmplete™ULTRA protease inhibitor cocktail (Roche Life Science, North Ryde, NSW). Lysates were then sonicated twice at 50% power for 10 sec using an ultrasonic probe, followed by centrifugation prior to protein quantification. Protein concentrations were determined using the Bicinchoninic Acid (BCA) Assay Kit (Ther-moFisher Scientific, Waltham, MA).

Statistical analysis
Statistical analysis was performed using GraphPad Prism version 7.04 for Windows, GraphPad Software, San Diego California, USA, www.graphpad.com. All experimental data are reported as mean ± S.E.M. To assess statistical differences between three or more groups we used one-way analysis of variance (ANOVA) followed by Tukey post-hoc test, unless otherwise stated. P-values ≤ 0.05 were considered statistically significant.

Dose-response effects of doxycycline and minocycline on tPA and uPA secretion in RT4 cells
To investigate if doxycycline and minocycline were able to stimulate the secretion of tPA and uPA by RT4 Schwann-cell like cultures, we measured the amount of plasminogen activators released in culture media by enzyme-like immunosorbent assay (ELISA) after treatment with either antibiotics for 24 h. We utilized increasing concentrations of doxycycline and minocycline (0, 10, 50, 100, 500 and 1000ng/ml) to establish the optimal concentration to use in subsequent testing (Fig. 1). PACAP was used as a positive control in all experiments, which significantly increased tPA (***p<0.001 Vs Control) and uPA (*p<0.05 and **p<0.01) levels in culture media. We have already reported a dose-and time-dependent increase in plasminogen activators expression and activity by PACAP in RT4 cells in earlier studies [22].
Experimental data demonstrated that both doxycycline and minocycline dose-dependently increased the levels of secreted tPA and uPA in the supernatant of RT4 cells, although uPA induction was less remarkable than tPA (Fig. 1a-b and a'-b', respectively). Doxycycline reliably increased tPA secretion at 50ng/ml (*p<0.05 Vs Control, Fig. 1a), however, 100ng/ml doxycycline was required to significantly increase uPA levels as well (**p<0.01 Vs Control, Fig. 1a').
Minocycline treatment significantly increased tPA (**p<0.01 Vs Control, Fig. 1b) and uPA levels (*p<0.05 Vs Control, Fig. 1b') in the culture media at 50ng/ml; however, similarly to doxycycline, minocycline also peaked its activity at 100ng/ml (**p<0.01 Vs Control for both tPA and uPA). Accordingly, we identified 100ng/ml as the most effective concentration of both doxycycline and minocycline to use in subsequent experiments.

Effects of doxycycline or minocycline treatment on RT4 cell viability
To investigate if doxycycline or minocycline were toxic to cells, we treated RT4 cells with increasing concentrations of either doxycycline or minocycline and assessed viability using the MTT assay. At the concentrations tested, neither doxycycline nor minocycline were toxic to RT4 cells, as cells displayed normal growth rate up to 100ng/ml concentration (****p<0.0001 Vs Ctrl for both doxycline and minocycline), to then plateau at 500ng/ml (****p<0.0001 Vs Ctrl for both) and finally record a slight decline at the highest concentration tested (1000ng/ml, **p<0.01 Vs Ctrl for both, Fig. 2a and b). Figure 2. Cell viability of RT4 cells exposed to increasing concentrations of doxycycline or minocycline. Analysis of cell viability (MTT assay). RT4 cells were grown under normal conditions abd exposed to increasing concentrations (0, 50, 100, 500 or 1000ng/ml) of (A) doxycycline or (B) minocycline for 24 h. Values are reported as mean optical densities (OD) ± S.E.M. from two experiments, each using five batches of cells. **p < 0.01, ***p < 0.001 or ****p < 0.0001, as determined by ANOVA followed by Dunnett's post-hoc test.

Enzymatic activity of tPA and uPA in RT4 cells exposed to doxycycline or minocycline
Our next step was to establish if doxycycline or minocycline stimulated tPA and uPA enzymatic activity in RT4 cells using zymography. We used exogenous PACAP as our positive control [22]. RT4 cells were treated with either PACAP (100nM), doxycycline or minocycline (100ng/ml) for 24 h.
Quantification of both tPA and uPA lytic bands on gels (white bands corresponding to 70kDa [tPA] and 50kDa [uPA], Fig. 3a) confirmed that exogenous PACAP increases the activity of both plasminogen activators (**p<0.01 Vs Control). Both doxycycline and minocycline treatments significantly increased both tPA (**p<0.01 and ***p<0.001 Vs Control, respectively, Fig. 3b and c) and uPA activity (*p<0.05 Vs Control for both, Fig. 3b and c).

tPA-like immunoreactivity in RT4 cells exposed to PACAP, doxycycline or minocycline
We have previously demonstrated that treatment with PACAP or the PAC1 agonist maxidilan did not affect the distribution but only the expression of tPA in RT4 cells [22]. As such, we performed immunocytochemistry to determine if, similarly to PACAP, doxycycline or minocycline treatment increased the expression but not the cellular distribution of either plasminogen activators. Sections where the primary antibody was omitted were used as negative controls (Fig. 4a). Cells were either left untreated (Fig. 4b) or exposed to PACAP (Fig. 4c), doxycycline (Fig. 4d) or minocycline (Fig. 4e) for 24 h.
tPA-like immunoreactivity (IR) was moderate (about 25% of cell surface area) and mainly cytoplasmic in untreated cells (Fig. 4b). As expected, administration of PACAP significantly increased the expression of tPA-IR (***p<0.001 Vs Control, >40% cell surface area, Fig. 4c). Similarly to PACAP, both doxycycline and minocycline reliably increased the percentage of tPA + IR compared with controls (**p<0.01 Vs control, about 35% of cell surface area, Fig. 4d and e).

Pre-treatment with PACAP-38 blocks doxycycline-and minocycline-induced tPA and uPA protein expression levels
To evaluate whether PACAP6-38 induced reduction in plasminogen activators activities were also associated with a reduction in protein expression, we performed Western blot analyses in RT4 cells treated as per zymographic analyses.
Pre-treatment with PACAP6-38 prevented the induction of tPA expression by PA-CAP ($$p<0.01 Vs PACAP-treated) but also by doxycycline ($$$p<0.001 Vs doxycyclinetreated) and minocycline ($$p<0.01 Vs minocycline-treated) treated RT4 cells. Similarly, PACAP or tetracyclines'-induced uPA protein expression levels were prevented by PA-CAP6-38 pre-treatment. Specifically, PACAP6-38 completely rescued the induction of uPA protein levels caused by PACAP or doxycycline treatment ($$p<0.01 Vs corresponding drug treatment, Fig. 6b and b'). However, we observed only a partial reduction of uPA protein expression in RT4 cells that were pre-treated with PACAP6-38 and then exposed to minocycline ($p<0.05 Vs minocycline-treated, Fig. 6b and b'). ) in RT4 cells exposed to PACAP6-38 for 1h then treated with PACAP, doxycycline or minocycline for 24h. Total proteins were normalized to GAPDH, the loading control. Data represents means of n = 4 samples for each group. Results are expressed as mean ± S.E.M. *p < 0.05, **p < 0.01 or ***p < 0.001, as determined by ANOVA followed by Dunnett's post-hoc test.

Discussion
In the present study, we report for the first time that doxycycline and minocycline induce both the expression and activity of the 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 the novel finding that these tetracyclines may act as PAMs of the PAC1 receptor, first documented by Song and colleagues (2019) [23].
RT4 Schwann cells were utilized in this study. These immortalized Schwann cell lines share a similar transcriptional profile and biology with primary Schwann cells [32], making these cell lines 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 up-regulates 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 up-regulate PACAP expression and concurrently down-regulate 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 within Schwann cells following nerve injury. These findings align with other studies showing that these cell-types undergo important biochemical alterations after a nerve injury that allow cells to secrete factors that promote neuronal survival and axonal regeneration [33].
In the RT4 cell line, PACAP up-regulates 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 by two stages: (1) in which PACAP stimulates Schwann cells to secrete factors that promote debris clearance and axonal regrowth and (2) a second stage where PACAP promotes the accumulation of pro-myelinating factors by Schwann cells that will aid in remyelination 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 it plays a key role in both axon regeneration and the late 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 up-regulated and its expression was associated with nerve recovery. Additionally, when human induced pluripotent stem cell-derived sensory neurons were treated with PACAP, it enhanced axonal outgrowth in a dose dependent manner [36]. Together, these studies suggest a prominent role of PACAP induced PAC1 activity in mediating neuronal survival, axon regeneration and remyelination following peripheral nerve injury.
Targeting the PAC1 receptor has proven to be challenging [15]. Most studies have aimed to use the neuropeptide PACAP to stimulate PAC1 activity; nonetheless, as with most peptides, PACAP has limited bioavailability and is subject to rapid enzymatic degradation [37]. Despite being able to cross the blood brain barrier, albeit by passive diffusion or through the specific peptide transporter system 6 (PTS6), PACAP has an extremely short half-life [37]. In C57BL/6 mice, it was determined that the half-life of PACAP38 administered intravenously was less than two minutes [38]. Dipeptidyl peptidase IV (DPP IV) is the main enzyme involved in the degradation of PACAP, with mice lacking DPP IV showing significantly slower clearance of the peptide [38,39]. Despite these challenges, PACAP has a safe and wide therapeutic index and low doses of PACAP are still able to exert neuroprotection and other beneficial activities.
The recent discovery of doxycycline and minocycline as putative PAMs of the PAC1 receptor may provide an excellent opportunity to target the PAC1 receptor therapeutically. The two antibiotics have well-established neuroprotective and anti-inflammatory functions in the CNS [26,40] and some evidence suggests that, at least minocycline, may aid in nerve regeneration in bio-artificial nerve grafts that are free from Wallerian degeneration [41]. However, a few studies have shown that antibiotic treatment can inhibit Wallerian regeneration [42], an essential process of nerve rejuvenation, whereas others don't [43]. Therefore, it is not clear if the balance between beneficial and detrimental outcomes of tetracycline's treatment can ultimately result in an improved nerve recovery or not. Our data suggests that the up-regulation of plasminogen activators in RT4 Schwann cells implicates doxycycline and minocycline as agents that can improve extracellular matrix/debris clearance and consequently, promote axonal regrowth. However, additional work is required to confirm such beneficial activities using primary Schwann cells, neuronal/glial co-cultures and/or in vivo, as the repurposing of this class of antibiotics could indeed become a game changer in the treatment of nerve injury.

Conclusions
In conclusion, we have confirmed that pharmacological blockade of the PAC1 receptors (as well as VPAC receptor subtypes) by PACAP6-38 prevents tetracycline-induced activation of plasminogen activators by RT4 Schwann cells. Whilst the study was not aimed at identifying the existence of a PAC1 binding site for doxycycline or minocycline, the biochemical evidence provided supports their the novel function as PAMs of the PAC1-receptor in RT4 Schwann cells.