Plasminogen Activator Inhibitor-1 (PAI-1) deficiency predisposes to depression and resistance to treatments

Ten weeks old C57BL/129 PAI-1 knockout (PAI-1−/−) and C57BL/129 tPA knockout (tPA−/−) male mice and their corresponding wild-type littermates (PAI-1+/+; tPA+/+) were used in this study [6, 7]. The strain, number and age of mice used in each experiment are summarized in the Additional file 6: Table S1. All the experiments were approved by the French ministry of education and research (agreement numbers APAFIS#5115 and APAFIS#4359). The principal investigator (VA, personal license number 14–73) was accredited by the “Direction Départementale des Services Vétérinaires”. The animal investigations were performed under the current European directive (2010/63/EU) as incorporated in national legislation (Decree 87/848) and in authorized laboratories (GIP Cyceron; approval n° E14118001). Further details are described in the Additional file 1: Additional Materials and Methods.

We first set-up a depression screening system in mouse (n = 276; see Additional file 7: Table S2) to characterize the potential depressive-like phenotype of PAI-1 and tPA mice. To this aim, a range of behavioral tests were used for assaying each depression symptom: splash test, sucrose preference test, body weight, actimetry, rotarod, coat state, T-maze and forced swimming test. They are described in the Additional file 1: Additional Materials and Methods. Our methodological preclinical model thus consisted of exhaustively scoring primary (apathetic and anhedonic behaviors) and secondary (hypo- or hyperactivity, loss of motivation/effort, body weight, deficits of self-centered behaviors, cognitive deficits) depressive criteria (see Additional file 7: Table S2) by means of behavioral tests. One point was attributed when a significant difference was yielded between knockout and wild-type mice; the score was equal to 0 when there was no significant difference between groups. Thereafter, all the scores were added together forming a composite score. At the end, the depression-like phenotype was retained if and only if the composite score was at least equal to 4 points, including necessarily at least one of the two primary criteria.

Animals were deeply anesthetized with isoflurane 5%, and thereafter maintained with 2.5% isoflurane in a 70%/30% mixture of NO₂/O₂. A transcardial perfusion was performed with ice cold 0.9% NaCl heparinized. Further details are described in the Additional file 1: Additional Materials and Methods.

Tissues were dissociated in ice-cold TNT buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 0.5% Triton X-100; Sigma-Aldrich, Saint-Quentin Fallavier, France), centrifuged for 20 min (12,000 g at 4 °C), then protein concentration was quantified using the BCA method (Pierce, Rockford, IL, USA).

Proteins (20 μg) were electrophoresed in a 8% polyacrylamide gel under non reducing conditions. SDS was then exchanged with 2.5% Triton X-100 (Sigma-Aldrich). After washing off excess Triton X-100 with distilled water, the gel was carefully overlaid on a 1% agarose indicator support containing bovine fibrinogen, plasminogen and thrombin. Zymograms were then allowed to develop at 37 °C during several days and photographed at regular intervals using dark-ground illumination. Data were normalized to the mean value obtained for wild-type mice. Results shown are representative pictures of the zymograms and the corresponding quantifications made by calculating the area under the curve for each lysis band by using the ImageJ software (NIH).

Proteins (20 μg) were resolved on 4–20% SDS-PAGE gel (Bio-rad, Marnes-la-Coquette, France) under denaturing conditions and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with Tris-buffered saline (TBS: 10 mM Tris and 200 mM NaCl, pH 7.4) containing 0.05% Tween-20 and 1% BSA. Blots were incubated overnight at 4 °C in blocking buffer with the primary antibody: rabbit anti-mBDNF (1/5000; ab108319; Abcam, Paris, France), rabbit anti-proBDNF (1/100; ab72440; Abcam) or rabbit anti-actin (1/1000; A2066; Sigma–Aldrich). Membranes were then incubated with peroxidase-conjugated anti-rabbit secondary antibodies (1/50000; Sigma-Aldrich). Proteins were visualized with an enhanced chemiluminescence western blot detection reagent (ECL Prime Western Blotting System; GE Healthcare; RPN2232) using ImageQuant LAS 4000 Camera Camera (GE Healthcare, Chicago, IL, USA).

We developed and validated a novel UHPLC-MS/MS method for quantification of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) in mice brain structures. Briefly, the method consisted in crushing in formic acid aqueous solution in acetonitrile containing acetic acid. The samples were then centrifuged and injected in the UHPLC-MS/MS system and quantification was obtained by a 1/X2 weighted calibration curves using the internal standard method. Lower limits of quantification in the injected solutions were 0.1 ng/mL for DA and 5-HT, and 0.5 ng/mL for NA. For further detail, see Additional file 1: Additional Materials and Methods and Additional file 8: Table S3.

Escitalopram (ESC) and fluoxetine (FLX) were purchased from Carbosynth (Compton-Bershire, United Kingdom). Both drugs were dissolved in saline (NaCl 0.9%) and were administrated intraperitoneally (i.p.) at a concentration of 15 mg/kg (1 mg/ml) or 30 mg/kg (2 mg/ml) body weight. The drug dose was chosen based on previous preclinical studies [5, 11, 12, 17, 43]. Control groups received an equivalent volume of saline (vehicle: VEH). Mice were i.p. injected daily with ESC or FLX, or VEH, for 35 consecutive days. The behavioral tests were conducted from day 22 to day 35 of antidepressant treatment.

Statistical analyses were performed using STATISTICA PRO® v13.0 software (StatSoft, Inc.). The distribution of samples was studied with Shapiro-Wilk’s tests. When data were normally distributed, Student’s t-tests were used. When analysis of the data sets via parametric approaches turned out to be inappropriate due to violation of residual normality, non-parametric approaches where used (Mann-Whitney’s U-tests for independent samples or Wilcoxon’s signed-rank tests for matched samples). An alpha level of p < 0.05 was used for determination of significance in all statistical tests; all tests are two tailed.

After setting up a depression screening system in mouse (see Additional file 7: Table S2), we have characterized the behavioral phenotype of PAI-1−/− and PAI-1+/+ mice. In the splash test, PAI-1−/− mice exhibited decreased frequency of grooming behavior when compared to PAI-1+/+ control mice (Fig. 1a; U = 506; P = 0.042), thus pointing to apathetic behavior in PAI-1−/− mice (Table 1; score = 1). A significant alteration of the coat state of PAI-1−/− mice was found (Fig. 1b; PAI-1−/−: 2.58; PAI-1+/+: 1.27; U = 357.5, P = 0.00007) indicating a deficit of self-centered behaviors (Table 1; score = 1). PAI-1-deficient mice also decreased preference for sucrose containing water compared to PAI-1+/+ mice (Fig. 1c; t = 4.08, P = 0.00011), demonstrating an anhedonic behavior in PAI-1−/− mice (Table 1; score = 1). In addition, there was a significant difference in the body weight between the two genotypes (Fig. 1d; t = 2.08, P = 0.047; 24.12 g ± 1.60 for PAI−/− mice versus 25.54 g ± 2.11 for PAI-1+/+ mice) (Table 1; score = 1). In terms of actimetry, both groups of mice showed a similar locomotor activity (Fig. 1e; PAI-1−/−: number of movements = 746 ± 211; PAI-1+/+: number of movements = 694 ± 191; t = − 1.13, P = 0.261). In contrast, PAI-1−/− mice exhibited altered rotarod performance; indeed, latencies to fall were 57.35 s and 84.51 s for knockout and wild-type mice, respectively (Fig. 1f; t = 4.83, P = 0.00001). These results suggest impairment in motivation/effort in PAI-1 knockout mice (Table 1; score = 1). Finally, spatial cognitive abilities were examined by using a place recognition task in a T-maze which required the use of room cues. During the test phase, PAI-1+/+ mice discriminated correctly the newly opened arm from the familiar ones since they visited significantly more often the new arm than arms 1 and 2 (Fig. 1g; Arm 1 vs New arm: Z = 2.37, P = 0.018; Arm 2 vs New arm: Z = 2.10 P = 0.036), therefore indicating good spatial performance in control mice. PAI-1−/− mice entered more often in the new arm relative to familiar arm 1 (Fig. 1h; Arm 1 vs New arm: Z = 2.37, P = 0.018). However, the number of visits of the familiar arm 2 was not different from the newly open arm (Fig. 1h; Z = 1.68, P = 0.093). These observations reveal that the deficiency of PAI-1 alters the ability of mice to orientate properly (Table 1; score = 1). Overall, considering all behavioral procedures described above, PAI-1−/− mice obtained a composite score of 6 points, which is equivalent to six depressive-related symptoms of the screening system, including apathetic and anhedonic behaviors, two hallmarks of MDD in humans (Table 1, column 2; see Additional file 7: Table S2). Conversely, PAI+/+ mice showed any symptom. Altogether, these results demonstrate a depressive-like phenotype in PAI-1−/− mice. Interestingly, this deficit observed in PAI-1 −/− mice cannot be attributed to any brain weight or volume disparity (see Additional file 2: Figure S1; see Additional file 1: Additional Materials and Methods).

To determine whether the involvement of PAI-1 in depression was associated, or not, with its interaction with tPA, we also examined the behavioral phenotype of tPA−/− mice and that of their wild-type littermates with our depression screening system (see Additional file 7: Table S2; see Additional file 3: Figure S2). At the end of the behavioral tests, tPA knockout mice reached a composite score of 3 (Table 1, column 3). Importantly, they did not exhibit apathetic or anhedonic behavior. Thus, deficiency in tPA does not influence the depressive-like phenotype of mice compared to their wild-type littermates. This first set of behavioral tests suggests that the depressive-like phenotype observed in PAI-1−/− mice is tPA-independent.

The next step of our study examined cerebral tPA and BDNF levels in several brain structures related to depression (prefrontal cortex, hippocampus, hypothalamus) in PAI-1−/− and PAI-1+/+ mice. tPA activity was similar in both groups of mice whatever the brain areas (Fig. 2a: t = − 0.12, P = 0.907; Fig. 2c: t = − 0.52, P = 0.618; Fig. 2e: t = − 1.51, P = 0.170). These data argue for a role of PAI-1 in depression-like behaviors by a mechanism independent of its role of tPA inhibitor. To ascertain this claim, we measured the levels of mBDNF in the same areas of PAI-1−/− and PAI-1+/+ mice. Our results revealed that the neurotrophin levels did not differ between groups (Fig. 2b: t = − 2.04, P = 0.076; Fig. 2d: t = − 0.91, P = 0.387; Fig. 2f: t = 1.65, P = 0.138). These data thus confirm the role of PAI-1 in depression, by a mechanism independent of the tPA–plasminogen–BDNF axis.

Given the monoaminergic hypothesis of MDD, we measured the levels of 5HT, NA and DA in brain structures (prefrontal cortex, hippocampus, hypothalamus, dorsal raphe) in PAI-1−/− and PAI-1+/+ mice. Our data demonstrate a significant decrease of 5-HT levels in prefrontal cortex of PAI-1 knockout mice when compared with those of PAI-1+/+ mice (Fig. 3a; U = 0, P = 0.008). In hippocampus and dorsal raphe, the levels of DA were also significantly reduced in PAI-1−/− mice when compared to control mice (Fig. 3b, d; Hippocampus: U = 0, P = 0.008; Raphe: U = 0, P = 0.008). The levels of NA were not affected by PAI-1 deficiency whatever the brain area (Fig. 3a: U = 6, P = 0.222; Fig. 3b: U = 8, P = 0.421; Fig. 3c: U = 10, P = 0.690; Fig. 3d: U = 4; P = 0.095). Altogether, these data reveal a strong relationship between the amounts of 5-HT and DA present in specific cerebral structures and the expression of PAI-1.

Because SSRIs are the most frequently prescribed antidepressants [41], we tested the effects of treatment with escitalopram, or fluoxetine, in PAI-1−/− mice. For this pharmacological study, we replaced the cognitive task of our screening behavioral tests by the forced swimming test (FST) as it is the gold standard assay for screening of antidepressant drugs. We first applied an escitalopram chronic treatment (35 days) at 15 mg/kg. The data showed that none of the depressive-like symptoms was counterbalanced by the escitalopram treatment in PAI-1−/− mice (Fig. 4a: U = 114.5, P = 0.207; Fig. 4b: t = − 0.99, P = 0.328; Fig. 4c: U = 105, P = 0.118; Fig. 4d: U = 100, P = 0.083; Fig. 4e: t = − 1.43, P = 0.161; Fig. 4f: t = − 1.36, P = 0.186). This was also true for the FST since the duration of immobility was similar for the two groups (Fig. 4g: U = 112, P = 0.274). We then examined the effect of a higher dose of the same antidepressant, i.e. 30 mg/kg i.p. This second experiment confirmed our previous observation that chronic treatment with escitalopram did not exert antidepressant-like effect in PAI-1−/− mice, whatever the dose administered or the assessed symptom (see Additional file 4: Figure S3a: U = 65, P = 0.712; see Additional file 4: Figure S3b: t = 1.11, P = 0.281; see Additional file 4: Figure S3c: U = 56, P = 0.378; see Additional file 4: Figure S3d: t = − 0.86, P = 0.397; see Additional file 4: Figure S3e: U = 58, P = 0.443; see Additional file 4: Figure S3f: t = − 0.92, P = 0.369; see Additional file 4: Figure S3 g: t = 0.96, P = 0.348). To further understand whether this lack of efficacy was specific to escitalopram or generalized to other SSRIs, we subjected PAI-1−/− mice to treatment with fluoxetine, another SSRI largely used in clinic. Our data once again demonstrated the ineffectiveness of the antidepressant to reverse depressive symptoms of PAI-1−/− mice (see Additional file 5: Figure S4a: U = 68.5, P = 0.611; see Additional file 5: Figure S4b: U = 77.5, P = 0.979; see Additional file 5: Figure S4c: U = 53, P = 0.303; see Additional file 5: Figure S4d: t = − 1.28, P = 0.214; see Additional file 5: Figure S4e: t = − 1.16, P = 0.259; see Additional file 5: Figure S4f: t = 1.86, P = 0.078; see Additional file 5: Figure S4 g: U = 47.5; P = 0.160).