Abstract:
Post-traumatic stress disorder (PTSD) is a severe stress-related psychiatric condition triggered by traumatic life-threatening events, characterized notably by an altered memory profile. Although clinically well-documented, no specific biomarker exists. This translational study identifies plasminogen activator inhibitor-1 (PAI-1) as a brain risk factor for PTSD, thereby supporting its potential as a blood-derived biomarker. Mice with genetically ablated PAI-1 were protected from developing a PTSD-like memory profile. Conversely, mice exhibiting PTSD-like cognitive impairment showed increased blood PAI-1 levels, correlating with their profile severity. In the brain, PAI-1 levels were specifically increased in the dorsal hippocampus, a key region for cognitive functions and in the etiology of PTSD. Finally, a longitudinal study of soldiers revealed that those developing PTSD symptoms exhibit rising blood PAI-1 levels over a 12-month period. Its significant association with various indicators of PTSD-related psychological distress attests to PAI-1's potential as a blood biomarker and brain therapeutic target for PTSD.

Abstract:
Cannabis use disorder (CUD) is widespread, and there is no pharmacotherapy to facilitate its treatment. AEF0117, the first of a new pharmacological class, is a signaling-specific inhibitor of the cannabinoid receptor 1 (CB(1)-SSi). AEF0117 selectively inhibits a subset of intracellular effects resulting from Delta(9)-tetrahydrocannabinol (THC) binding without modifying behavior per se. In mice and non-human primates, AEF0117 decreased cannabinoid self-administration and THC-related behavioral impairment without producing significant adverse effects. In single-ascending-dose (0.2 mg, 0.6 mg, 2 mg and 6 mg; n = 40) and multiple-ascending-dose (0.6 mg, 2 mg and 6 mg; n = 24) phase 1 trials, healthy volunteers were randomized to ascending-dose cohorts (n = 8 per cohort; 6:2 AEF0117 to placebo randomization). In both studies, AEF0117 was safe and well tolerated (primary outcome measurements). In a double-blind, placebo-controlled, crossover phase 2a trial, volunteers with CUD were randomized to two ascending-dose cohorts (0.06 mg, n = 14; 1 mg, n = 15). AEF0117 significantly reduced cannabis' positive subjective effects (primary outcome measurement, assessed by visual analog scales) by 19% (0.06 mg) and 38% (1 mg) compared to placebo (P < 0.04). AEF0117 (1 mg) also reduced cannabis self-administration (P < 0.05). In volunteers with CUD, AEF0117 was well tolerated and did not precipitate cannabis withdrawal. These data suggest that AEF0117 is a safe and potentially efficacious treatment for CUD.ClinicalTrials.gov identifiers: NCT03325595 , NCT03443895 and NCT03717272 .

Abstract:
Stressful events trigger a set of complex biological responses which follow a bell-shaped pattern. Low-stress conditions have been shown to elicit beneficial effects, notably on synaptic plasticity together with an increase in cognitive processes. In contrast, overly intense stress can have deleterious behavioral effects leading to several stress-related pathologies such as anxiety, depression, substance use, obsessive-compulsive and stressor- and trauma-related disorders (e.g., post-traumatic stress disorder or PTSD in the case of traumatic events). Over a number of years, we have demonstrated that in response to stress, glucocorticoid hormones (GCs) in the hippocampus mediate a molecular shift in the balance between the expression of the tissue plasminogen activator (tPA) and its own inhibitor plasminogen activator inhibitor-1 (PAI-1) proteins. Interestingly, a shift in favor of PAI-1 was responsible for PTSD-like memory induction. In this review, after describing the biological system involving GCs, we highlight the key role of tPA/PAI-1 imbalance observed in preclinical and clinical studies associated with the emergence of stress-related pathological conditions. Thus, tPA/PAI-1 protein levels could be predictive biomarkers of the subsequent onset of stress-related disorders, and pharmacological modulation of their activity could be a potential new therapeutic approach for these debilitating conditions.

Abstract:
The central serotonin(2B) receptor (5-HT(2B)R) modulates 5-HT and dopamine (DA) neuronal function in the mammalian brain and has been suggested as a potential target for the treatment of neuropsychiatric disorders involving derangements of these monoamine systems, such as schizophrenia, cocaine abuse and dependence and major depressive disorder. Studies in rats and mice yielded contrasting results on the control of 5-HT/DA networks by 5-HT(2B)Rs, thereby leading to opposite views on the therapeutic potential of 5-HT(2B)R agents for treating the above disorders. These discrepancies may result from anatomo-functional differences related to a different cellular location of 5-HT(2B)Rs in rat and mouse brain. Using immunohistochemistry, we assessed this hypothesis by examining the expression of 5-HT(2B)Rs in 5-HT and GABAergic neurons of rats and mice within different subregions of the dorsal raphe nucleus (DRN), currently considered as the main site of action of 5-HT(2B) agents. Likewise, using in vivo microdialysis, we examined their functional relevance in the control of DRN 5-HT outflow, a surrogate index of 5-HT neuronal activity. In the DRN of both species, 5-HT(2B)Rs are expressed in 5-HT cells expressing tryptophan hydroxylase 2 (TPH(2)), in GABAergic cells expressing glutamic acid decarboxylase 67 (GAD67), and in cells expressing both markers (GAD67 & TPH(2); i.e., GABA-expressing 5-HT neurons). The proportion of 5-HT(2B)R-positive cells expressing only TPH(2) was significantly larger in mouse than in rat DRN, whereas the opposite holds true for the expression in cells expressing GAD67 & TPH(2). No major species differences were found in the dorsal and ventral subregions. In contrast, the lateral subregion exhibited large differences, with a predominant expression of 5-HT(2B)Rs in TPH(2)-positive cells in mice (67.2 vs 19.9 % in rats), associated with a lower expression in GAD67 & TPH(2) cells (7.9 % in mice vs 41.5 % in rats). Intra-DRN (0.1 μM) administration of the preferential 5-HT(2B)R agonist BW 723C86 decreased and increased DRN 5-HT outflow in rats and mice respectively, both effects being prevented by the intra-DRN perfusion of the selective 5-HT(2B)R antagonist RS 127445 (0.1 μM). Altogether, these results show the existence of anatomical differences in the cellular expression of 5-HT(2B)Rs in the rat and mouse DRN, which translate into an opposite control of 5-HT outflow. Also, they highlight the relevance of the subset of GAD67-positive 5-HT neurons as a key factor responsible for the functional differences between rats and mice in terms of 5-HT neuronal activity modulation.

Abstract:
Down syndrome (DS) or Trisomy 21 is the most common genetic cause of mental retardation with severe learning and memory deficits. DS is due to the complete or partial triplication of human chromosome 21 (HSA21) triggering gene overexpression and protein synthesis alterations responsible for a plethora of mental and physical phenotypes. Among the diverse brain target systems that affect hippocampal-dependent learning and memory deficit impairments in DS, the upregulation of the endocannabinoid system (ECS), and notably the overexpression of the cannabinoid type-1 receptor (CB1), seems to play a major role. Combining various protein and gene expression targeted approaches using western blot, qRT-PCR and FISH techniques, we investigated the expression pattern of ECS components in the hippocampus (HPC) of male Ts65Dn mice. Among all the molecules that constitute the ECS, we found that the expression of the CB1 is altered in the HPC of Ts65Dn mice. CB1 distribution is differentially segregated between the dorsal and ventral part of the HPC and within the different cell populations that compose the HPC. CB1 expression is upregulated in GABAergic neurons of Ts65Dn mice whereas it is downregulated in glutamatergic neurons. These results highlight a complex regulation of the CB1 encoding gene (Cnr1) in Ts65Dn mice that could open new therapeutic solutions for this syndrome.

Abstract:
Pregnenolone is a steroid with specific characteristics, being the first steroid to be synthesised from cholesterol at all sites of steroidogenesis, including the brain. For many years, pregnenolone was defined as an inactive precursor of all steroids because no specific target had been discovered. However, over the last decade, it has become a steroid of interest because it has been recognised as being a biomarker for brain-related disorders through the development of metabolomic approaches and advanced analytical methods. In addition, physiological roles for pregnenolone emerged when specific targets were discovered. In this review, we highlight the discovery of the selective interaction of pregnenolone with the type-1 cannabinoid receptor (CB1R). After describing the specific characteristic of CB1Rs, we discuss the newly discovered mechanisms of their regulation by pregnenolone. In particular, we describe the action of pregnenolone as a negative allosteric modulator and a specific signalling inhibitor of the CB1R. These particular characteristics of pregnenolone provide a great strategic opportunity for therapeutic development in CB1-related disorders. Finally, we outline new perspectives using innovative genetic tools for the discovery of original regulatory mechanisms of pregnenolone on CB1-related functions.

Abstract:
Moderate stress increases memory and facilitates adaptation. In contrast, intense stress can induce pathological memories as observed in post-traumatic stress disorders (PTSD). A shift in the balance between the expression of tPA and PAI-1 proteins is responsible for this transition. In conditions of moderate stress, glucocorticoid hormones increase the expression of the tPA protein in the hippocampal brain region which by triggering the Erk1/2(MAPK) signaling cascade strengthens memory. When stress is particularly intense, very high levels of glucocorticoid hormones then increase the production of PAI-1 protein, which by blocking the activity of tPA induces PTSD-like memories. PAI-1 levels after trauma could be a predictive biomarker of the subsequent appearance of PTSD and pharmacological inhibition of PAI-1 activity a new therapeutic approach to this debilitating condition.

Abstract:
Serotonin2B receptor (5-HT2BR) antagonists inhibit cocaine-induced hyperlocomotion independently of changes of accumbal dopamine (DA) release. Given the tight relationship between accumbal DA activity and locomotion, and the inhibitory role of medial prefrontal cortex (mPFC) DA on subcortical DA neurotransmission and DA-dependent behaviors, it has been suggested that the suppressive effect of 5-HT2BR antagonists on cocaine-induced hyperlocomotion may result from an activation of mPFC DA outflow which would subsequently inhibit accumbal DA neurotransmission. Here, we tested this hypothesis by means of the two selective 5-HT2BR antagonists, RS 127445 and LY 266097, using a combination of neurochemical, behavioral and cellular approaches in male rats. The intraperitoneal (i.p.) administration of RS 127445 (0.16 mg/kg) or LY 266097 (0.63 mg/kg) potentiated cocaine (10 mg/kg, i.p.)-induced mPFC DA outflow. The suppressant effect of RS 127445 on cocaine-induced hyperlocomotion was no longer observed in rats with local 6-OHDA lesions in the mPFC. Also, RS 127445 blocked cocaine-induced changes of accumbal glycogen synthase kinase (GSK) 3beta phosphorylation, a postsynaptic cellular marker of DA neurotransmission. Finally, in keeping with the location of 5-HT2BRs on GABAergic interneurons in the dorsal raphe nucleus (DRN), the intra-DRN perfusion of the GABAAR antagonist bicuculline (100 muM) prevented the effect of the systemic or local (1 muM, intra-DRN) administration of RS 127445 on cocaine-induced mPFC DA outflow. Likewise, intra-DRN bicuculline injection (0.1 mug/0.2 mul) prevented the effect of the systemic RS 127445 administration on cocaine-induced hyperlocomotion and GSK3beta phosphorylation. These results show that DRN 5-HT2BR blockade suppresses cocaine-induced hyperlocomotion by potentiation of cocaine-induced DA outflow in the mPFC and the subsequent inhibition of accumbal DA neurotransmission.

Abstract:
BACKGROUND: Phosphorylation by protein kinases is a fundamental molecular process involved in the regulation of signaling activities in living organisms. Understanding this complex network of phosphorylation, especially phosphoproteins, is a necessary step for grasping the basis of cellular pathophysiology. Studying brain intracellular signaling is a particularly complex task due to the heterogeneous complex nature of the brain tissue, which consists of many embedded structures. NEW METHOD: Overcoming this degree of complexity requires a technology with a high throughput and economical in the amount of biological material used, so that a large number of signaling pathways may be analyzed in a large number of samples. We have turned to Alpha (Amplified Luminescent Proximity Homogeneous Assay) technology. COMPARISON WITH EXISTING METHOD: Western blot is certainly the most commonly used method to measure the phosphorylation state of proteins. Even though Western blot is an accurate and reliable method for analyzing modifications of proteins, it is a time-consuming and large amounts of samples are required. Those two parameters are critical when the goal of the research is to comprehend multi-signaling proteic events so as to analyze several targets from small brain areas. RESULT: Here we demonstrate that Alpha technology is particularly suitable for studying brain signaling pathways by allowing rapid, sensitive, reproducible and semi-quantitative detection of phosphoproteins from individual mouse brain tissue homogenates and from cell fractionation and synaptosomal preparations of mouse hippocampus. CONCLUSION: Alpha technology represents a major experimental step forward in unraveling the brain phosphoprotein-related molecular mechanisms involved in brain-related disorders.

Abstract:
Intellectual disability is the most limiting hallmark of Down syndrome, for which there is no gold-standard clinical treatment yet. The endocannabinoid system is a widespread neuromodulatory system involved in multiple functions including learning and memory processes. Alterations of this system contribute to the pathogenesis of several neurological and neurodevelopmental disorders. However, the involvement of the endocannabinoid system in the pathogenesis of Down syndrome has not been explored before. We used the best-characterized preclinical model of Down syndrome, the segmentally trisomic Ts65Dn model. In male Ts65Dn mice, cannabinoid type-1 receptor (CB1R) expression was enhanced and its function increased in hippocampal excitatory terminals. Knockdown of CB1R in the hippocampus of male Ts65Dn mice restored hippocampal-dependent memory. Concomitant with this result, pharmacological inhibition of CB1R restored memory deficits, hippocampal synaptic plasticity and adult neurogenesis in the subgranular zone of the dentate gyrus. Notably, the blockade of CB1R also normalized hippocampal-dependent memory in female Ts65Dn mice. To further investigate the mechanisms involved, we used a second transgenic mouse model overexpressing a single gene candidate for Down syndrome cognitive phenotypes, the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). CB1R pharmacological blockade similarly improved cognitive performance, synaptic plasticity and neurogenesis in transgenic male Dyrk1A mice. Our results identify CB1R as a novel druggable target potentially relevant for the improvement of cognitive deficits associated with Down syndrome.