METHODS OF TREATMENT AND DIAGNOSTIC OF PATHOLOGICAL CONDITIONS ASSOCIATED WITH INTENSE STRESS
20230305023 · 2023-09-28
Inventors
- Jean-Michel Revest (Bordeaux, FR)
- Aline DESMEDT (Bordeaux, FR)
- Monique Vallee (Bordeaux, FR)
- Pier Vincenzo Piazza (Bordeaux, FR)
Cpc classification
A61K31/713
HUMAN NECESSITIES
C07K16/38
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a method for preventing or treating pathological conditions associated with intense stress such as Post-Traumatic Stress Disorder (PTSD) by targeting the endogenous PAI-1 (Type 1 Plasminogen Activator Inhibitor). In the present invention, inventors demonstrate that there is a shift in the balance between the expression of tPA and PAI-1 proteins in a hippocampal region of a preclinical model of Post-Traumatic Stress (PTSD), is responsible for the transition between moderate stress which increases memory and facilitates adaptation and intense stress intense stress which induce pathological memories. In conditions of moderate stress, glucocorticoid hormones (GC) increase the expression of the tPA protein in the hippocampal brain region which by triggering the Erk1/2.sup.MAPK cascade strengthens memory. When stress is particularly intense, very high levels of GC then increase the production of PAI-1 protein, which by blocking the activity of tPA induces PTSD-like memories. Accordingly, inhibition of PAI-1 activity represent a new therapeutic approach to this debilitating condition and PAI-1 body fluid level in patient after trauma could be a predictive biomarker of the subsequent appearance of PTSD.
Claims
1. A method of preventing or treating a pathological conditions associated with intense stress in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a PAI-1 antagonist, wherein said pathological conditions associated with intense stress is Post-Traumatic Stress Disorder (PTSD).
2. The method according to claim 1, wherein said PAI-1 antagonist directly binds to PAI-1 (protein or to a-nucleic sequence encoding PAI-1 and promotes tPA/plasmin activity to mediate the proteolytic processing of pro-BDNF to mature BDNF.
3. The method according to claim 1 wherein said PAI-1 antagonist is 1) an inhibitor of PAI-1 activity and/or 2) an inhibitor of PAI-1 gene expression.
4. The method according to claim 3 wherein said inhibitor of PAI-1 activity is selected from the group consisting of a small organic molecule, an anti-PAI-1 neutralizing antibody, a polypeptide and an aptamer.
5. The method according to claim 3 wherein the inhibitor of PAI-1 gene expression is selected from the group consisting of an antisense oligonucleotide, a nuclease, siRNA, shRNA and a ribozyme nucleic acid sequence.
6. A method for assessing a subject’s risk of having or developing pathological conditions associated with intense stress and treating the subject, said method comprising measuring the level of PAI-1 protein in a body fluid sample obtained from said subject, determining that the subject has a high level of PAI-1 protein compared to a control reference value, and treating the subject determined to have a high level of PAI-1 protein compared to a control reference value by administering a PAI-1 antagonist, wherein said pathological conditions associated with intense stress is Post-Traumatic Stress Disorder (PTSD).
7. The method according to claim 6 further comprising comparing said level of PAI-1 protein to a control reference value wherein: a higher level of PAI-1 than the control reference value is predictive of a high risk of having or developing a Post-Traumatic Stress Disorder (PTSD) and a lower level of PAI-1 than the control reference value is predictive of a low risk of having or developing a Post-Traumatic Stress Disorder (PTSD).
8. A method for monitoring the effect of a therapy for treating pathological conditions associated with intense stress in a subject and treating the subject comprising measuring the level of PAI-1 in a first body fluid sample obtained from said subject at t1, wherein when t1 is prior to therapy, measuring the level of PAI-1 in a second body fluid sample obtained from said subject at t2, wherein t2 is during or following therapy, and when t1 is during therapy, t2 is later during therapy or following therapy, determining that the level of PAI-1 in the sample measured at t2 is decreased as compared to the level of PAI-1 in the sample measured at t1, and treating the subject determined to have a high level of PAI-1 protein compared to a control reference value by administering the PAI-1 antagonist, wherein said pathological conditions associated with intense stress is Post-Traumatic Stress Disorder (PTSD).
9. The method according to claim 6, wherein the body fluid sample is blood sample and/or urine sample.
10. (canceled)
11. (canceled)
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13. (canceled)
Description
FIGURES:
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EXAMPLE 1
Material & Methods
[0196] Chemicals. In all the experiments we used a preformed water-soluble complex of corticosterone and 2-hydroxypropyl-β-cyclodextrin (#C174, Sigma, USA) (14-16). In mice, corticosterone (Cort; 2 mg/kg in a volume of 0.1 ml/10 g body weight) or vehicle (NaCl 0.9%) were administered i.p. immediately after the acquisition of fear conditioning in order to mimic the effect of intense trauma (5). Cort was used at 100 nM and 1000 nM on rat PC12 cells and at 10 nM and 1000 nM on rat hippocampal slices (14;16). Millipore (USA) provided the recombinant human BDNF (CAS Nb 218441-99-7, #GF029, 100 ng/side) and tPA inhibitor; stable recombinant mutant of human Type 1 Plasminogen Activator Inhibitor (PAI-1; CAS Nb: 140208-23-7, #528208, ranging from 30 to 240 ng/side) (16;18). The small-molecule inhibitor of PAI-1 activity Tiplaxtinin (PAI-039; CAS Nb: 393105-53-8, #1383, 5 ng/side) was provided by Axon MEDCHEM (The Netherlands).
[0197] Cell culture. PC12 cell line (ATCC CRL-1721) derived from a transplantable rat pheochromocytoma was used (14;16). PC12 cells were seeded on six well plates coated with poly-D-lysine at the appropriate concentration (10.sup.5 cells/well) in fresh, antibiotic-free medium (DMEM/F12 (#31330-038, Gibco, USA) + 10% Foetal Bovine Serum (FBS; #10270106, Fisher Scientific, USA)). Sixteen hours before Cort treatment, the medium was changed for a steroid-free culture medium (DMEM/F12 + 10% Charcoal/Dextran-treated FBS (#SH30068-03, Hyclone, Fisher Scientific, USA). PC12 cells (n=6/group) were treated with 100 nM and 1000 nM of corticosterone-HBC (Sigma, USA) then harvested after 3 h and the proteins and RNA extracted.
[0198] Hippocampal slice preparations and corticosterone treatment Hippocampal slice preparations have been described in detail previously (19). Briefly, adult male Sprague-Dawley rats (2-3 months old n=18, Charles River Laboratory, France) were used. Rats were then anesthetized with isoflurane and transcardially perfused with nearly frozen modified artificial cerebrospinal fluid (CSF) with 3 mM kynurenic acid. The modified CSF for perfusion contained: (in mM) 87 NaCl, 75 Sucrose, 25 Glucose, 5 KCl, 21 MgCl2, 0.5 CaCl.sub.2 and 1.25 NaH.sub.2PO.sub.4. After perfusion, the brains were quickly removed and sliced (300 .Math.m) in the coronal plane using a vibratome (Campden Instruments, UK). Immediately after cutting, slices were stored for 40 min at 32° C. in CSF ((in mM): 130 NaCl, 11 Glucose, 2.5 KCl, 2.4 MgCl.sub.2, 1.2 CaCl.sub.2, 23 NaHCO.sub.3, 1.2 NaH.sub.2PO.sub.4), equilibrated with 95% O.sub.2 / 5% CO.sub.2 then stored at room temperature for the rest of the experiment. Each brain slice was then treated for 1 h (60 min) and 3 h (180 min) with 10 nM and 1000 nM of Cort. One slice served as a control reference and did not undergo any treatment. Dorsal hippocampi were isolated, and proteins were extracted as previously described (14;16).
[0199] Protein extraction from brain tissues and immunoblotting analysis. A detailed description of protein extraction and immunoblotting analysis has been reported previously (14-16;20;21). Briefly, protein sample extracts from PC12 cells and mouse and rat hippocampi were performed in RIPA buffer containing protease and phosphatase inhibitors (#P8340 and #P0044, Sigma, USA) before being subjected to immunoblotting experiments. SDS-PAGE-separated proteins were then revealed with relevant antibodies. Rabbit polyclonal anti-PAI-1 antibodies were from Lifespan Biosciences (LSBio#C81062, 1/1000, WA, USA) and Epitomics (#3917-1, 1/3000, CA, USA), anti-tPA (#T5600-05G; 1/5000) was from US Biologicals (MA, USA), anti-Erk1/2.sup.MAPK (#06-182; 1/50000) was from Millipore (MA, USA), anti-Phospho-Erk1/2.sup.MAPK (#9101S; 1/1000) was from CST (MA, USA). Rabbit monoclonal antibodies anti-Phospho-Erk1/2.sup.MAPK (#4370; 1/5000) was from CST (MA, USA), anti-Phospho-TrkB (#2149-1; 1/5000) was from Epitomics (CA, USA), anti-TrkB (#610101; 1/2000) was from BD Biosciences (NJ, USA). Mouse monoclonal anti-Neuronal Class III β-Tubulin (TUJ1) (#MMS-435P; 1/20000) was from Eurogentec (Belgium), anti-α-tubulin (#N356, 1/50000) was from Amersham Life Sciences (Del, USA). CST provided secondary antibodies: anti-rabbit IgG, HRP-linked antibody (#7074, 1/5000) and anti-mouse IgG, HRP-linked antibody (#7076, 1/20000). In all experiments, βIII-tubulin or α-tubulin measures were used as a loading control. X-Ray films (Kodak, USA) were quantified by densitometry (optical density; OD) using a GS-800 scanner coupled with Quantity One software (Bio-Rad, CA, USA).
[0200] Quantitative PCR analysis. Samples of PC12 cells treated with Cort were homogenized in Tri-reagent (Euromedex, France) and RNA was isolated using a standard chloroform/isopropanol protocol (22). RNA was processed and analyzed using an adapted version of published methods (23). cDNA was synthesized from 2 .Math.g of total RNA using RevertAid Premium Reverse Transcriptase (Fermentas, Thermo Fisher Scientific, USA) and primed with oligo-dT primers (Fermentas, Thermo Fisher Scientific, USA) and random primers (Fermentas, Thermo Fisher Scientific, USA). qPCR was perfomed using a LightCycler® 480 Real-Time PCR System (Roche, Meylan, France). qPCR reactions were done in duplicate for each sample, using transcript-specific primers, cDNA (4 ng) and LightCycler 480 SYBR Green I Master (Roche) in a final volume of 10 .Math.l. The PCR data were exported and analyzed in a computer-based tool (Gene Expression Analysis Software Environment) developed at the Neurocentre Magendie (France). The Genorm method was used to determine the reference gene. Relative expression analysis was corrected for PCR efficiency and normalized against two reference genes. The ribosomal protein L13a (Rpl13a) and non-POU-domain-containing (Nono) genes were used as reference genes. The relative level of expression was calculated using the comparative (2.sup.-ΔΔCT) method (24). qPCR amplification used specific primers to specifically amplify Serpine1 gene encoding PAI-1 protein and Nono and Rpl13a as reference genes.
[0201] PAI-1 forward primer sequence (5′-3′): GGCACAATCCAACAGAGACAATC and reverse primer sequence (5′-3′): AGGCTTCTCATCCCACTCTCAA. (SEQ ID N°3 and SEQ ID N°4) Restraint Stress. Male C57BL/6J mice aged 2-3 months old (n=30) were obtained from Charles River Laboratory, France. Mice were placed into 50 ml conical centrifuge tubes fitted with a central puncture so as to allow ventilation. The tubes were placed in horizontal holders with strong light exposure, and the animals were held in this way for a continuous period of restraint. After 30 min, 1 h (60 min) and 3 h (180 min) of restraint, the mice and those from an unstressed control group were sacrified by decapitation, then the hippocampi and blood were collected and assayed for protein extraction (16;21).
[0202] Blood collection for corticosterone assay. Blood was rapidly collected in heparine-EDTA-coated tubes (Sarstedt, France) and centrifuged at 2,000 rpm (4° C., 20 min). Supernatant containing the blood plasma was stored at -20° C., and then processed for corticosterone assay. Plasma corticosterone (Corticosterone EIA kit #KO14-H1, Arbor Assays, Michigan, USA) levels were quantified by ELISA following the manufacturer’s instructions (16;21).
Behavioral Procedure.
[0203] Surgical procedure. Male C57BL/6J mice {n=20 in Experiment 1 (
[0204] Adaptive vs maladaptive (PTSD-like) fear memory. The behavioral model based on a general fear conditioning procedure has been fully described in a previous study (5). [0205] Pre-exposure - The day before fear conditioning, each mouse was placed individually in an opaque PVC chamber (30 × 24 × 22 cm) with an opaque PVC floor, for 2 min, in a brightness of 10 lux. This pre-exposure allowed the mice to acclimate and become familiar with the chamber used for the cue alone test (“safe context”). [0206] Induction of adaptive vs PTSD-like fear memory - Acquisition of fear conditioning was performed in a different context, consisting in a Plexiglas conditioning chamber (30 × 24 × 22 cm) with the floor connected to a shock generator, in a brightness of 110 lux, giving access to the different visual-spatial cues in the experimental room. Briefly, each animal placed in the conditioning chamber for 4 min received 2 footshocks (0.4 mA, 50 Hz, 1 s), which never co-occurred with 2 tone deliveries (70 dB, 1 kHz, 15 s). This tone-shock unpairing paradigm is known to make the contextual cues the primary stimuli that become associated with the footshock (5;26-28). Consequently, the phasic tone, although salient, is not predictive of the shock delivery, whereas the static contextual cues constitute the main predictor of the shock. Immediately after the acquisition of fear conditioning, mice received a systemic injection of either NaCl or Cort (see below for details). An adaptive fear memory will therefore be attested in control (NaCl-injected) mice by the expression of highly conditioned fear when re-exposed to the conditioning context and no conditioned fear when re-exposed to the irrelevant tone cue (in the safe context). In contrast, Cort-injected mice will display a maladaptive (PTSD-like) memory attested by an abnormally high fear response to the irrelevant tone (cue-based hypermnesia) together with a decreased conditioned fear to the conditioning context (contextual amnesia) (5). [0207] Memory tests - After fear conditioning, each animal was returned to its home cage and 24 h later, all mice were submitted to two memory tests during which freezing behavior, defined as a lack of any movement except for respiratory-related movements (29), was measured and used as an index of conditioned fear. During these two memory tests, animals were continuously recorded for off-line second-by-second scoring of freezing by an observer blind to the experimental groups. Mice were first re-exposed to the tone within the “safe” context during which three successive recording sessions of the behavioral responses were performed: one before (first 2 min), one during (next 2 min), and one after (last 2 min) tone presentation. Conditioned response to the tone is expressed by the percentage of freezing during the tone presentation compared to the levels of freezing expressed before and after tone presentation (repeated measures on 3 blocks of freezing). The strength and specificity of this conditioned fear is attested by a ratio that represents the increase in the percentage of freezing with the tone with respect to a baseline freezing level {i.e., pre- and post-tone periods mean: (tone - ((pre + post)/2)) / (tone + ((pre + post)/2))}. Then two hours later, mice were re-exposed to the conditioning context alone for 6 min (without the tone cue). Freezing to the context was calculated as the percentage of the total time spent freezing during the successive three blocks of 2-min periods of the test. While the first block is the critical block attesting difference between animals that are conditioned to the conditioning context and those that are not or less, the following two blocks are presented in order to assess a gradual extinction of the fear responses in the absence of shock. [0208] Molecular analysis - In the experiment (
[0209] Drug injections. Immediately after the acquisition of fear conditioning, mice were randomly divided into groups firstly according to first their systemic injection of Cort and secondly their specific intra-hippocampal infusion. Cort (2 mg/kg in a volume of 0.1 ml/10 g body weight) or vehicle (NaCl 0.9%) was administered i.p. (5) while PAI-1, mature BDNF and Tiplaxtinin were intra-hippocampally infused. [0210] Experiment 1 (
[0214] PAI-1 and BDNF were diluted in artificial CSF (aCSF) and Tiplaxtinin in 1.6 % dimethyl sulfoxide (DMSO) and then diluted in aCSF. Bilateral infusions of 0.3 .Math.l/side were administered into the dorsal hippocampus immediately after acquisition of fear conditioning at a constant rate (0.1 .Math.l/min).
[0215] Histology. A detailed description of the histological protocol was reported previously (14;15;30). Briefly, after completion of the behavioral study, animals were sacrificed in order to evaluate the cannulae placements.
[0216] Data analysis. All experiments involving mice and rats were performed according to the protocols approved by the Aquitaine-Poitou Charentes local ethical committee (authorization number APAF1S#7397-20161 02814453778 v2) in strict compliance with the French Ministry of Agriculture and Fisheries (authorization number D33-063-096) and European Communities Council Directive (2010/63/EU). All efforts were made to minimize animal suffering and to reduce the number of rodents used, while maintaining reliable statistics. All experiments were conducted with experimenters blind to drug treatment conditions; no randomization method for the constitution of the experimental groups was applied. The sample size was chosen to ensure adequate statistical power for all experiments. Statistical analyses were performed using analysis of variance (ANOVA) followed by either Newman Keuls or Fisher’s PLSD post-hoc test for pairwise comparisons. Student’s t-test was used for pairwise comparisons. A significance level of p<0.05 was used for all statistical analyses. Statistical significance was expressed as * = P<0.05; ** = P<0.01; *** or ### = P<0.005. All values were expressed as mean ± s.e.m.
Results
Corticosterone and Stress Stimulated the Expression of PAI-1 Protein
[0217] Our previous reports have revealed that the tPA/plasmin system induced by GC-activated GR is a core effector in the regulation of the pro-BDNF/BDNF balance allowing, through the activation of the TrkB/Erk1/2.sup.MAPK signaling cascade, the formation of normal fear memory (14-16). Interestingly, the major potential physiological inhibitor of this pro-memory cascade, the tPA inhibitor PAI-1 (Type 1 Plasminogen Activator Inhibitor), also displays Glucocorticoid Responsive Elements (GRE) in the regulatory sequences of its gene (31). Since the effects of GC and stress on PAI-1 are unknown, in a first experiment we treated PC12 cells, which expresses both endogenous GR and PAI-1 (14;32), with corticosterone (Cort) the major GC in rodents. After 3 h of treatment, Cort (100 and 1000 nM) strongly increased the expression of PAI-1 mRNA (
PAI-1 Protein is a Sufficient Condition to Induce PTSD-Like Memories
[0218] In a second series of experiments, we investigated whether changes in the expression of PAI-1 could determine the appearance of PTSD-like memories, which were evaluated using a previously described mouse model (5). Mice were submitted to a threatening situation - the delivery of an electric foot shock - when exposed to a specific context (conditioning cage). A discrete cue (a tone) was also repeatedly presented during conditioning but was never paired with shock delivery. In these conditions the context is the correct predictor of the threat (predicting context), whilst the cue although present with the threat does not predict it (non-predicting cue). Twenty-four hours after this conditioning procedure, animals were re-exposed first to the cue alone in a familiar and safe environment and then to the conditioning context without the cue (5). In control conditions Veh-injected mice showed a fear response (freezing) when exposed to the correct predictor of the threat, the predicting context, but not when exposed to the non-predicting cue (
PAI-1 Protein is a Necessary Condition to Induce PTSD-Like Memories
[0219] In the third series of experiments, we wanted to establish whether it was possible to block the development of PTSD-like memory. To address this issue, we first assessed the hypothesis whether PTSD-like memory induced by injection of PAI-1 (
[0220] Taken together the findings of these experiments indicated that an increase in PAI-1 levels triggered by high levels of GC is a sufficient and necessary condition to induce PTSD-like memories.
Discussion
[0221] Uncovering the molecular mechanisms of the shift from beneficial to harmful effects of stress and GC is a key question in understanding the pathophysiological mechanism through which life events can induce psychiatric disorders. Our results are of major importance because they provide the first molecular signaling model in which the beneficial and harmful effects of stress and GC on a cognitive process as memory can be dissociated. We previously showed that in moderate stress conditions, GC hormones induce the expression of the tPA protein which by increasing the production of mature BDNF triggers the activation of the TrkB/Erk1/2.sup.MAPK cascade which strengthens the memory trace of the stress-related event (14-16). Here we showed that the activity of the tPA/BDNF/TrkB/Erk1/2.sup.MAPK cascade is then inhibited by the delayed production of the tPA inhibitor PAI-1. However, in the case of particularly stressful conditions and very high levels of GC, the production of PAI-1 is triggered early on. PAI-1 then blocks the activity of tPA and inhibits the pro-mnesic BDNF/TrkB/Erk1/2.sup.MAPK signaling cascade, inducing PTSD-like memory. By lowering hippocampal PAI-1 activity, Tiplaxtinin (34-36) restored the formation of a hippocampal-dependent adaptive (“contextualized”) fear memory and thus normalizes traumatic memory.
[0222] Several lines of evidence support the involvement of impairment of BDNF processing mediated by PAI-1 in the pathophysiology of stress-related diseases. Firstly, impaired BDNF function has been associated with PTSD both in rodents and human (37). Secondly, PTSD patients have a higher risk of cardiovascular pathophysiologies and notably atherothrombosis (38), for which high levels of PAI-1 is a known risk factor (39). Thirdly, elevated PAI-1 levels and polymorphisms of the SERPINE1 gene encoding the PAI-1 protein have also been related to depression, another stress-induced condition (40;41).
[0223] An increase in PAI-1 could mediate the pathological effects of stress not only by decreasing the production of mature BDNF but also by promoting the accumulation of pro-BDNF that is no longer cleaved into mature BDNF by the tPA-activated plasmin. Indeed, although long considered to be inactive, pro-BDNF forms are able to form a ternary complex with the p75.sup.NTR and sortilin receptors, to induce neuronal cell death by apoptosis (42). In addition, pro-BDNF/p75.sup.NTR signaling has been shown to have the opposite effect on synaptic plasticity, inducing LTD whilst BDNF/TrkB signaling induces LTP (43). These results are consistent with brain imaging findings showing hippocampal atrophy reported in PTSD subjects (10).
[0224] Although the biphasic effects of activated GR have been described before in other contexts (44;45), the exact mechanism through which the dose-dependent effects of GC regulate the transcription of the PAI-1 encoding gene deserves further discussion. The PAI-1 promoter is known to have, in addition to GRE, response elements for the AP-1 transcription factor complex (Fos:Jun) (46). A plausible explanation of the observed dose-dependent effects is that when moderate levels of activated GR are produced, GR/AP1 heterodimers, which are known to promote reciprocal transcriptional interference, are mostly formed and prevent PAI-1 transcription through a protein-protein interaction mediated-sequestration process (47;48). In contrast, when high levels of activated GR are produced, for example after intense stress, GR/GR homodimers are now formed which are able to activate the transcription of the PAI-1 encoding gene.
[0225] In conclusion, our data show that the transition from adaptive to maladaptive stress-related memories is mediated by a shift in balance between tPA and PAI-1 proteins, with an adaptive increase in memory appearing when the ratio is in favor of tPA (16) and PTSD-like memory when it is in favor of PAI-1. As a consequence, PAI-1 levels after a traumatic event could be a predictive biomarker of the appearance of PTSD and pharmacological inhibition of PAI-1 activity a new therapeutic approach of this debilitating condition.
EXAMPLE 2 PAI BLOOD LEVEL IN RESPONSE TO RESTRAIN STRESS
Materials and Methods
[0226] Blood collection for PAI-1 assay. Blood was rapidly collected in heparine-EDTA-coated tubes (Sarstedt, France) and centrifuged at 2,000 rpm (4° C., 20 min). Supernatant containing the blood plasma was stored at -20° C., and then processed for PAI-1 assay. PAI-1 (Murine PAI-1 total antigen assay #MPAIKT-TOT, Molecular Innovation, Michigan, USA) expression levels were quantified by ELISA following the manufacturer’s instructions
[0227] Since PAI-1 circulates in the blood and was shown to be upregulated upon stress (
Table Section
[0228] TABLE-US-00001 Useful nucleotide and amino acid sequences for practicing the invention SEQ ID NO Nucleotide and amino acid sequences 1 (Human PAI-1 AA sequence) MQMSPALTCLVLGLALVFGEGSAVHHPPSYVAHLASDFGVR VFQQVAQASKDRNVVFSPYGVASVLAMLQLTTGGETQQQIQ AAMGFKIDDKGMAPALRHLYKELMGPWNKDEISTTDAIFVQ RDLKLVQGFMPHFFRLFRSTVKQVDFSEVERARFIINDWVKTH TKGMISNLLGKGAVDQLTRLVLVNALYFNGQWKTPFPDSSTH RRLFHKSDGSTVSVPMMAQTNKFNYTEFTTPDGHYYDILELP YHGDTLSMFIAAPYEKEVPLSALTNILSAQLISHWKGNMTRLP RLLVLPKFSLETEVDLRKPLENLGMTDMFRQFQADFTSLSDQE PLHVAQALQKVKIEVNESGTVASSSTAVIVSARMAPEEIIMDR PFLFVVRHNPTGTVLFMGQVMEP 2 (Human PAI-1 nucleic acid sequence) acagctgtgt ttggctgcag ggccaagagc gctgtcaaga agacccacac gcccccctcc agcagctgaa ttcctgcagc tcagcagccg ccgccagagc aggacgaacc gccaatcgca aggcacctct gagaacttca ggatgcagat gtctccagcc ctcacctgcc tagtcctggg cctggccctt gtctttggtg aagggtctgc tgtgcaccat cccccatcct acgtggccca cctggcctca gacttcgggg tgagggtgtt tcagcaggtg gcgcaggcct ccaaggaccg caacgtggtt ttctcaccct atggggtggc ctcggtgttg gccatgctcc agctgacaac aggaggagaa acccagcagc agattcaagc agctatggga ttcaagattg atgacaaggg catggccccc gccctccggc atctgtacaa ggagctcatg gggccatgga acaaggatga gatcagcacc acagacgcga tcttcgtcca gcgggatctg aagctggtcc agggcttcat gccccacttc ttcaggctgt tccggagcac ggtcaagcaa gtggactttt cagaggtgga gagagccaga ttcatcatca atgactgggt gaagacacac acaaaaggta tgatcagcaa cttgcttggg aaaggagccg tggaccagct gacacggctg gtgctggtga atgccctcta cttcaacggc cagtggaaga ctcccttccc cgactccagc acccaccgcc gcctcttcca caaatcagac ggcagcactg tctctgtgcc catgatggct cagaccaaca agttcaacta tactgagttc accacgcccg atggccatta ctacgacatc ctggaactgc cctaccacgg ggacaccctc agcatgttca ttgctgcccc ttatgaaaaa gaggtgcctc tctctgccct caccaacatt ctgagtgccc agctcatcag ccactggaaa ggcaacatga ccaggctgcc ccgcctcctg gttctgccca agttctccct ggagactgaa gtcgacctca ggaagcccct agagaacctg ggaatgaccg acatgttcag acagtttcag gctgacttca cgagtctttc agaccaagag cctctccacg tcgcgcaggc gctgcagaaa gtgaagatcg aggtgaacga gagtggcacg gtggcctcct catccacagc tgtcatagtc tcagcccgca tggcccccga ggagatcatc atggacagac ccttcctctt tgtggtccgg cacaacccca caggaacagt ccttttcatg ggccaagtga tggaaccctg accctgggga aagacgcctt catctgggac aaaactggag atgcatcggg aaagaagaaa ctccgaagaa aagaatttta gtgttaatga ctctttctga aggaagagaa gacatttgcc ttttgttaaa agatggtaaa ccagatctgt ctccaagacc ttggcctctc cttggaggac ctttaggtca aactccctag tctccacctg agaccctggg agagaagttt gaagcacaac tcccttaagg tctccaaacc agacggtgac gcctgcggga ccatctgggg cacctgcttc cacccgtctc tctgcccact cgggtctgca gacctggttc ccactgaggc cctttgcagg atggaactac ggggcttaca ggagcttttg tgtgcctggt agaaactatt tctgttccag tcacattgcc atcactcttg tactgcctgc caccgcggag gaggctggtg acaggccaaa ggccagtgga agaaacaccc tttcatctca gagtccactg tggcactggc cacccctccc cagtacaggg gtgctgcagg tggcagagtg aatgtccccc atcatgtggc ccaactctcc tggcctggcc atctccctcc ccagaaacag tgtgcatggg ttattttgga gtgtaggtga cttgtttact cattgaagca gatttctgct tccttttatt tttataggaa tagaggaaga aatgtcagat gcgtgcccag ctcttcaccc cccaatctct tggtggggag gggtgtacct aaatatttat catatccttg cccttgagtg cttgttagag agaaagagaa ctactaagga aaataatatt atttaaactc gctcctagtg tttctttgtg gtctgtgtca ccgtatctca ggaagtccag ccacttgact ggcacacacc cctccggaca tccagcgtga cggagcccac actgccacct tgtggccgcc tgagaccctc gcgccccccg cgcccctctt tttccccttg atggaaattg accatacaat ttcatcctcc ttcaggggat caaaaggacg gagtgggggg acagagactc agatgaggac agagtggttt ccaatgtgtt caatagattt aggagcagaa atgcaagggg ctgcatgacc taccaggaca gaactttccc caattacagg gtgactcaca gccgcattgg tgactcactt caatgtgtca tttccggctg ctgtgtgtga gcagtggaca cgtgaggggg gggtgggtga gagagacagg cagctcggat tcaactacct tagataatat ttctgaaaac ctaccagcca gagggtaggg cacaaagatg gatgtaatgc actttgggag gccaaggcgg gaggattgct tgagcccagg agttcaagac cagcctgggc aacataccaa gacccccgtc tctttaaaaa tatatatatt ttaaatatac ttaaatatat atttctaata tctttaaata tatatatata ttttaaagac caatttatgg gagaattgca cacagatgtg aaatgaatgt aatctaatag aagcctaatc agcccaccat gttctccact gaaaaatcct ctttctttgg ggtttttctt tctttctttt ttgattttgc actggacggt gacgtcagcc atgtacagga tccacagggg tggtgtcaaa tgctattgaa attgtgttga attgtatgct ttttcacttt tgataaataa acatgtaaaa atgtttcaaa aaaataataa aataaataaa 3) mouse PAI-1 forward primer sequence ggcacaatccaacagagacaatc 4) mouse PAI-1 reverse primer sequence aggcttctcatcccactctcaa
EXAMPLE 3: EVALUATING PAI-1 BLOOD EXPRESSION LEVEL IN HUMAN WITH PTSD
[0229] We recently showed that under high stress conditions the downregulation of the GMES signaling cascade, through Glucocorticoid-mediated PAI-1 increase in the hippocampus, underlies the transition from a normal to PTSD-like fear memory impairment as the one observed in PTSD patients (49).
[0230] Therefore our current hypothesis suggests that PAI-1 could be a promising target for the diagnosis and therapy of PTSD. To assess this hypothesis, we conducted a promising pilot study in collaboration with Dr. M. Trousselard (Chief Medical Officer of the NSCo Department, and coordinator of the IRBA) who have collected blood sample from French soldiers after a six month mission in Afghanistan. Some soldiers developed PTSD (i.e. PTSD+) afterwards while others who experienced the same violence during the fights did not develop PTSD (i.e. Control). We used Enzyme-Linked ImmunoSorbent Assay method (i.e. ELISA) to quantify PAI-1 blood level. Our study revealed significant higher levels of PAI-1 in the blood of soldiers with PTSD, compared to non PTSD soldiers (t = 2.333 df = 23,p < 0.0287), collected after their deployment to the war zone (
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