Abstract:
BACKGROUND: Hyperlocomotion induced by psychotomimetic agents is considered a proxy for positive psychotic-like symptoms and is used in antipsychotic drug screenings with rodents. Cannabinoids are psychotomimetic drugs in humans, and low doses of cannabinoid CB(1) receptor (CB1R) agonists induce hyperlocomotion in rodents. However, the cellular and molecular mechanisms underlying this effect remain unexplored so far. METHODS: We used a combination of pharmacological and genetic manipulations in cultured cells and adult male mouse models, together with an intracellular signaling detection method. Our multidisciplinary approach included locomotor activity (n = 10-12 mice/group), alphaLISA technology (n = 4-12 mice/group), transfection of the beta-arrestin 1 protein in STHdh (Q7/Q7) cells (n = 4-6/group) and a viral strategy in transgenic lines to selectively block GSK-3beta (glycogen synthase kinase-3beta) phosphorylation (n = 8-10 mice/group). We also used fluorescence in situ hybridization, real-time quantitative and droplet digital polymerase chain reaction, and tandem mass spectrometry. RESULTS: We showed that the hyperlocomotion induced by a low dose (0.3 mg/kg) of cannabinoid Delta(9)-tetrahydrocannabinol depends on the activation of plasma membrane CB1R located on dopamine D(2) receptor-positive striatopallidal projecting neurons. This activation regulates the beta-arrestin 1/Akt/GSK-3beta signaling pathway, which is required for the hyperlocomotor effect. The signaling-specific CB1R inhibitors, pregnenolone and its clinically developed analog, AEF0117, prevent GSK-3beta-dependent psychomotor stimulation induced by THC. CONCLUSIONS: This work highlights an intracellular mechanism of CB1R expressed by a selective key neuronal pathway mediating the psychotomimetic effect of THC and, by extension, low-dose cannabis consumption. This discovery will likely aid the development of unprecedented therapeutic modalities for the treatment of psychotic conditions.

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:
The emergence of metabolomics and quantification approaches is revealing new biomarkers applied to drug discovery. In this context, tandem mass spectrometry is the method of choice, requiring a specific validation process for preclinical and clinical applications. Research on the two classes of lipid mediators, steroids and cannabinoids, has revealed a potential interaction in cannabis addiction and metabolism-related disorders. Here we present the development of GC-MS/MS and LC-MS/MS methods for routine quantification of targeted steroids and cannabinoids, respectively. The methods were developed using an isotopic approach, including validation for linearity, selectivity, LLOQ determination, matrix effect, carryover, between- and within-run accuracy and precision, and stability tests to measure 11 steroids and seven cannabinoids in human plasma. These methods were satisfactory for most validity conditions, although not all met the acceptance criteria for all analytes. A comparison of calibration curves in biological and surrogate matrices and in methanol showed that the latter condition was more applicable for our quantification of endogenous compounds. In conclusion, the validation of our methods met the criteria for GLP-qualified rather than GLP-validated methods, which can be used for routine analytical studies for dedicated preclinical and clinical purposes, by combining appropriate system suitability testing, including quality controls in the biological matrix.

Abstract:
Steroids and endocannabinoids are part of two modulatory systems and some evidence has shown their interconnections in several functions. Homeostasis is a common steady-state described in the body, which is settled by regulatory systems to counterbalance deregulated or allostatic set points towards an equilibrium. This regulation is of primary significance in the central nervous system for maintaining neuronal plasticity and preventing brain-related disorders. In this context, the recent discovery of the shutdown of the endocannabinoid system (ECS) overload by the neurosteroid pregnenolone has highlighted new endogenous mechanisms of ECS regulation related to cannabis-induced intoxication. These mechanisms involve a regulatory loop mediated by overactivation of the central type-1 cannabinoid receptor (CB1R), which triggers the production of its own regulator, pregnenolone. Therefore, this highlights a new process of regulation of steroidogenesis in the brain. Pregnenolone, long considered an inactive precursor of neurosteroids, can then act as an endogenous negative allosteric modulator of CB1R. The present review aims to shed light on a new framework for the role of ECS in the addictive characteristics of cannabis with the novel endogenous mechanism of ECS involving the neurosteroid pregnenolone. In addition, this new endogenous regulatory loop could provide a relevant therapeutic model in the current context of increasing recreational and medical use of cannabis.

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.