<p>Osteoarthritis (OA) is a leading cause of disability globally, with higher incidence in older people and lower socioeconomic status populations. The challenges health care systems face with management of the disease highlights the importance of OA research. Many studies examine possible risk factors of knee and hip OA including obesity, smoking, and alcohol consumption. Findings support that while obesity increases risk of knee OA, smoking is not a major risk factor. These extrinsic factors are, however, associated with lower socioeconomic status, and also with anxiety and depression disorders. Up to 30% of patients with chronic knee OA have described psychological stress and decreased quality of life due to debilitating pain, but the effects of psychological stress on development of knee OA has not been described.</p><p><br></p><p>At the cellular level, mechanosensitive cation channels in cartilage and bone, are involved with OA, but studies looking specifically at synovium and joint capsule are limited. Transient receptor potential (TRP) channels are upregulated in joint capsule in end-stage primary shoulder OA. We were unable to identify any previous studies evaluating Piezo channel expression in musculoskeletal soft tissues, but Piezo channel antagonism reduces chondrocyte death after mechanical injury. These findings suggest channels may help regulate joint responses to repetitive loading during training or work while also contributing to protective mechanisms within the musculoskeletal system. The overall objective of this research was to investigate factors that impact OA development or the disease phenotype. Two studies evaluated the following aims: 1) demonstrate the influence of chronic psychological stress on knee OA and overall systemic health, and 2) characterize the role of mechanosensitive channels in the joint capsule in OA. The first study used a mouse chronic social defeat model paired with destabilization of the medial meniscus (DMM) surgery to create a social stress scenario during OA development. We hypothesized chronic social defeat would exacerbate knee OA structural changes and systemic inflammation. The second study aimed to explore the role of mechanosensitive channels in joint capsule during OA development in the equine. Immunohistochemistry was performed on forelimb fetlock joint capsule from horses with varying degrees of lameness to first identify TRP and Piezo channel expression. Next, fibroblasts were isolated from the tissue to determine channel activity. We hypothesized that TRP and Piezo channels are required for normal homeostasis, but are dysregulated in OA and dysregulation contributes to fibrosis of the joint capsule. Joint capsule fibrosis leads to joint stiffening and reduced range of motion, two of the cardinal signs of OA.</p><p><br></p><p>The results of the first study showed OA was induced to a similar extent in both groups of mice that underwent DMM surgery. While anxiety- and depressive-like behaviors were exhibited by mice that underwent chronic social defeat episodes, unexpectedly, the majority of systemic inflammatory markers were not worse in mice with DMM and chronic social defeat compared to DMM alone. We were also able to show TRP and Piezo channel expression in one normal dorsal and palmar fetlock joint capsule sample, however, COVID-19 prevented further investigation. With our results we were able to conclude that while chronic social stress influences development of OA, in the current experiments, neither systemic inflammation nor structural signs of knee OA were worse with chronic social stress. We hope that exploration of OA through these two studies will help us understand how the disease contributes to overall systemic dysfunction while also providing a baseline for future development of TRP and Piezo channel modulators to prevent joint pathologies.</p>

Chronic stress and dysfunction of the serotonergic system in the brain have been considered two of the major risks for development of depression. In this study, adult Fischer 344 rats were subjected to a regimen of chronic social defeat (CSD). To mimic stressful conditions, some rats were not exposed to CSD, but instead treated with corticosterone (CORT) in oral solution while maintained in their home cage. Protein levels of the serotonin transporter (SERT) in the dorsal raphe nucleus (DRN), hippocampus, frontal cortex, and amygdala were examined by Western blotting or immunofluorescence staining. The results showed that CSD up-regulated SERT protein levels in the DRN, hippocampus, frontal cortex, and amygdala regions. This up-regulation was abolished or prevented by adrenalectomy, or treatment with antagonists of corticosteroid receptors mifepristone and spironolactone, alone or in combination. Similarly, up-regulated SERT protein levels in these brain regions were also observed in rats treated with oral CORT ingestion, which was analogously prevented by treatment with mifepristone and spironolactone. Furthermore, both CSD- and CORT-induced up-regulation of SERT protein levels in the DRN and three brain regions were attenuated by simultaneous treatment with fluoxetine, an antidepressant that specifically inhibits serotonin reuptake. The results indicate that up-regulation in SERT protein levels in the DRN and forebrain limbic structures caused by CSD regimen was mainly motivated by CORT through corticosteroid receptors. The present findings demonstrate that chronic stress is closely correlated with the serotonergic system by acting on the regulation of the SERT expression in the DRN and its projection regions, which may contribute to the development of depression. Chronic stress and dysfunction of the serotonergic system are etiologically related to depression. In an attempt to explore their interaction, we found that chronic social defeat upregulated expression of serotonin transporter in the DRN and the projection regions, which may induce an alteration of serotonin transformation in the brain. This interaction may account for the development of this disease.

Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. However, the molecular link between chronic stress and noradrenergic neurons remains to be elucidated. In the present study adult Fischer 344 rats were subjected to a regimen of chronic social defeat (CSD) for 4 weeks. Measurements by in situ hybridization and Western blotting showed that CSD significantly increased mRNA and protein levels of the norepinephrine transporter (NET) in the LC region and NET protein levels in the hippocampus, frontal cortex and amygdala. CSD-induced increases in NET expression were abolished by adrenalectomy or treatment with corticosteroid receptor antagonists, suggesting the involvement of corticosterone and corticosteroid receptors in this upregulation. Furthermore, protein levels of protein kinase A (PKA), protein kinase C (PKC), and phosphorylated cAMP-response element binding (pCREB) protein were significantly reduced in the LC and its terminal regions by the CSD paradigm. Similarly, these reduced protein levels caused by CSD were prevented by adrenalectomy. However, effects of corticosteroid receptor antagonists on CSD-induced down-regulation of PKA, PKC, and pCREB proteins were not consistent. While mifeprestone and spironolactone, either alone or in combination, totally abrogate CSD effects on these protein levels of PKA, PKC and pCREB in the LC and those in the hippocampus, frontal cortex and amygdala, their effects on PKA and PKC in the hippocampus, frontal cortex and amygdala were region-dependent. The present findings indicate a correlation between chronic stress and activation of the noradrenergic system. This correlation and CSD-induced alteration in signal transduction molecules may account for their critical effects on the development of symptoms of major depression.

Our previous studies demonstrated that chronic social defeat (CSD) up-regulated expression of the serotonin transporter (SERT) and norepinephrine transporter (NET) in the brain, which was mediated by corticosteroid receptors. In the present study we first analyzed the alterations of corticosteroid receptors in different brain regions after the CSD paradigm. The results showed that CSD significantly reduced glucocorticoid receptor (GR) protein levels in the CA1 and dentate gyrus of the hippocampus, as well as in central and basolateral nuclei of the amygdala, which was accompanied by the translocation of GR from cytoplasm to nuclei. CSD also markedly reduced GR mRNA levels and MR immunoreactivity in the CA1, CA3 and dentate gyrus areas of the hippocampus. Conversely, CSD pronouncedly enhanced GR mRNA and protein levels in the dorsal raphe nucleus and locus coeruleus relative to the control. As an extension of our previous studies, in situ hybridization and immunohistochemical staining demonstrated that CSD regimen caused a notable increase of SERT mRNA levels in the dorsal raphe nucleus and increased SERT immunoreactivities in CA1 and CA3 of the hippocampus, as well as those in the basolateral nuclei of the amygdala. Likewise, CSD regimen resulted in an evident enhancement of NET immunoreactivity in the CA1 of the hippocampus and in the basolateral nuclei of the amygdala. Our current findings suggest that GR expressional alterations in response to CSD are complex and brain region-specific, which may correspond to their different functions in these regions.

Les antidépresseurs ISRS (inhibiteurs sélectifs de la recapture de sérotonine) sont parmi les plus prescrits pour traiter les épisodes dépressifs majeurs. Cependant, leur efficacité n’est pas optimale. En effet, ils montrent un long délai d’action, de nombreux effets indésirables et sont inefficaces pour une proportion non négligeable de patients. La dépression étant actuellement un enjeu de santé publique majeur, il est donc nécessaire de développer de nouvelles molécules possédant un meilleur profil thérapeutique. Récemment, un intérêt croissant a été porté sur le récepteur 5-HT3, notamment depuis le développement de la vortioxétine, ISRS de nouvelle génération ayant des propriétés antagonistes pour ce récepteur localisé dans les zones cérébrales contrôlant l’humeur. Le but de ce travail de thèse a été d’étudier le rôle du récepteur 5-HT3 dans la réponse aux antidépresseurs, ainsi que dans la physiopathologie de la dépression. Dans ce cadre, nous avons utilisé une approche génétique, en caractérisant un modèle de souris knockout (KO) dont le gène de la sous-unité 3A du récepteur 5-HT3 a été invalidé. Le phénotype de ces animaux et leurs contrôles de type sauvage (Wild-Type, WT) a tout d’abord été évalué dans des tests comportementaux de screening de molécules anxiolytiques et antidépressives, puis leur réponse à des traitements aigus d’ISRS a été mesurée par ces mêmes approches. L’effet de traitements antidépresseurs chroniques a quant à lui été évalué par une technique d’électrophysiologie in vitro. Enfin, les souris ont été soumises au modèle du stress de défaite sociale chronique (CSDS), afin de déterminer le rôle du récepteur 5-HT3 dans la réponse au stress. En conditions basales, le turn-over de la sérotonine est diminué chez les souris 5-HT3 KO par rapport aux souris WT. Cet effet est accompagné par une augmentation de l’expression génique des récepteurs 5-HT1A et de leur couplage aux protéines G au niveau du noyau raphé dorsal (NRD) des souris KO par rapport aux WT. Au niveau comportemental, les souris KO 5-HT3 montrent un phénotype apparenté à celui induit par un anxiolytique et par un antidépresseur. Lorsqu’elles reçoivent une injection de citalopram, un ISRS sélectif, les souris 5-HT3 KO ne se comportent pas différemment de leurs contrôles WT dans les tests de screening des antidépresseurs. Cependant, dans ces mêmes tests, l’effet de la fluoxétine, ISRS possédant des propriétés antagonistes pour le récepteur 5-HT3, est perdu chez les souris 5-HT3 KO. Le traitement chronique par le citalopram (20 mg/kg/j) induit une désensibilisation similaire des autorécepteurs 5-HT1A localisés sur les neurones sérotoninergiques du NRD chez les animaux WT et KO. Dans les mêmes conditions, mais en utilisant une dose de citalopram plus faible (5 mg/kg/j), la désensibilisation des autorécepteurs 5-HT1A est plus forte chez les animaux KO que chez les WT, confortant ainsi l’effet potentialisateur de l’invalidation des récepteurs 5-HT3 dans l’efficacité thérapeutique des ISRS. Afin de disséquer le rôle des récepteurs 5-HT3 dans la réponse au stress chronique, le CSDS a fait l’objet d’une validation avec l’agomélatine, antidépresseur de nouvelle génération. Ce stress a engendré des altérations de la mémoire à long terme, en lien avec des modifications de l’expression génique de l’exon IV du BDNF et d’enzymes de régulation épigénétique. Ces effets délétères du stress ont été traités efficacement par l’agomélatine (50 mg/kg/j) en injection chronique, mais cette molécule n’a cependant pas modifié les effets du CSDS sur les phénotypes de type anxieux et dépressifs observés après le stress. Nous avons montré que le CSDS augmentait l’expression génique de la sous-unité 3A du récepteur 5-HT3 dans différentes structures cérébrales des souris WT. De plus, les altérations de l’expression génique de CamkIIa et SOD1 induites par le stress dans le cortex préfrontal des souris WT n’ont pas été retrouvées chez les souris 5-HT3 KO (...) / SSRI (selective serotonin reuptake inhibitor) antidepressants are among the most prescribed drugs to treat major depression. However, their efficacy is not optimal yet. Indeed, they possess a long delay of action, various side effects and show not efficacy in some patients. As depression is currently a global burden, there is a great need for new molecules with a better therapeutic efficacy. Recently, an increased attention has been taking to 5-HT3 receptors, notably since the development of vortioxetine, a new generation SSRI that antagonizes this receptor. The aim of the study was to assess the role of 5-HT3 receptor in the antidepressant response and the physiopathology of depression. In this context, we used a genetic approach, by characterizing a knockout (KO) mice model lacking the 3A subunit of the 5-HT3 receptor. Their phenotype and the one of wild-type (WT) control mice was first evaluated in behavioral tests widely used for antidepressant and anxiolytic drugs screening, then following acute SSRI treatments. Effect of chronic SSRI administration was assessed by in vitro electrophysiology. Finally, mice were submitted to the chronic social defeat stress (CSDS) model, to determine the role of 5-HT3 receptor in stress response. In basal conditions, 5-HT turnover was decreased in 5-HT3 KO mice compared with WT mice. This effect was accompanied by an increase in the 5-HT1A receptor gene expression and their coupling to G proteins at the dorsal raphe nucleus (DRN) level. 5-HT3 KO mice displayed anxiolytic-like and antidepressive-like phenotype. When injected with citalopram, a very selective SSRI, 5-HT3 KO mice behaved similarly as WT mice in antidepressant screening tests. However, in the same tests, the effect of fluoxetine, a SSRI that possesses 5-HT3 receptor antagonist properties, was blunted in 5-HT3 KO mice. Chronic treatment with citalopram (20 mg/kg/d) induced in WT and KO mice a similar desensitization of 5-HT1A autoreceptors located on DRN 5-HT neurons. In the same conditions, but using a lower citalopram dose (5 mg/kg/d), 5-HT1A autoreceptor desensitization was higher in 5-HT3 KO mice than in WT mice, thus reinforcing the potentiating effect of the 5-HT3 receptor in the SSRI efficacy. In order to assess the role of 5-HT3 receptor in chronic stress response, CSDS paradigm was validated using agomelatine, a new generation antidepressant drug. This stress model provoked long term memory alterations, linked with modifications in hippocampal mRNA levels of BDNF exon IV and epigenetic modifying enzymes. These deleterious stress effects were prevented by chronic agomelatine treatment (50 mg/kg/d), but this molecule did not modify the stress-induced anxious- and depressive-like phenotypes. We showed that subunit 3A gene expression was increased in various WT mice brain structures subjected to CSDS. Moreover, stress-induced modifications of CamkIIa and SOD1 gene expression in the prefrontal cortex of WT mice were not present in KO mice. Genetic invalidation of 5-HT3 receptor blocked the effects of social stress in some behavioral tests (splash test, saccharine preference test) and on body weight gain. Taken altogether, these data show that 5-HT3 receptor plays an important role in anxiety- and depression-related behaviors. Moreover, invalidation of this receptor increased the effect of a low dose chronic SSRI treatment, and blunted the effect of a SSRI targeting 5-HT3 receptor. These results highlight the interest of this receptor in the development of innovating therapies to treat anxio-depressive disorders. Finally, the reduced sensitivity of 5-HT3 KO mice to chronic stress suggests an involvement of this receptor in stress-related behaviors and depression physiopathology.

Un faible apport alimentaire en acides gras polyinsaturés (AGPI) de la série n-3 a été associé à la prévalence des troubles de l'humeur chez l’Homme. Chez les rongeurs, les approches nutritionnelles visant à modéliser une alimentation pauvre en AGPI n-3 ont largement été développées au siècle dernier. En effet, un régime alimentaire carencé en AGPI n-3 sur une ou plusieurs générations induit chez le rongeur des altérations des comportements émotionnels tels que des comportements de type dépressif ou anxieux. Nous avons montré au laboratoire Nutrineuro que des souris nourries avec un régime déficient en AGPI n-3 présentent des niveaux d’AGPI n-3, en particulier l'acide docosahexaénoïque (DHA, un AGPI n-3) plus faible dans le cortex préfrontal (PFC) et dans le noyau accumbens (NAc) par rapport aux souris contrôle. De plus, nous avons pu mettre en évidence qu’une alimentation déficiente en AGPI n-3 est capable de moduler la plasticité synaptique dépendante du système endocannabinoïde (eCB). De fait, la réduction de DHA dans le CPF et le NAc est accompagnée d'une altération de la dépression à long terme (LTD-eCB) et des voies de signalisation dépendantes du système eCB au niveau du CPF (Lafourcade et al., 2011 ; Larrieu et al, 2012). Nos données indiquent que ces altérations sont dues à un découplage entre le récepteur cannabinoïde 1 (CB1R) et la protéine Gi/o. De plus, les souris déficientes en AGPI n-3 présentent des déficits comportementaux dans plusieurs tests évaluant les comportements émotionnels. Afin de mieux comprendre les mécanismes qui sous-tendent la diminution du DHA dans le CPF et les altérations des comportements émotionnels, nous avons étudié la morphologie neuronale dans le CPF et l’axe hypothalamo-hypophysaire (HPA) chez les souris déficientes en AGPI n-3. Nous avons montré que le régime alimentaire déficient en AGPI n-3 induit une atrophie de l’arborisation dendritique dans les neurones pyramidaux du CPF. L'atrophie dendritique est semblable à celle mesurée chez les souris soumises au régime équilibré en AGPI n-3 et soumises à un stress chronique de défaite sociale (CSDS). Aucun effet additionnel du CSDS sur la morphologie neuronale et le comportement émotionnel n’a été observé chez les souris déficientes en AGPI n-3. Nous avons ensuite étudié le rôle de l’axe HPA dans le développement des altérations comportementales et neurobiologiques chez les souris déficientes en AGPI n-3. Ces souris présentent une diminution de l'expression des récepteurs des glucocorticoïdes (GR) dans le CPF associée à une augmentation des taux circulants de corticostérone. Dans leur ensemble, nos résultats montrent qu’un faible apport alimentaire en AGPI n-3 peut modifier la plasticité synaptique dépendante du système eCB ainsi que l’arborisation dendritique des neurones du CPF. Nous avons également pu montrer que l’élévation des niveaux de corticostérone était impliquée dans l’altération des comportements émotionnels observée chez des souris nourries avec un régime déficient en AGPI n-3. / Low dietary intake of n-3 polyunsaturated fatty acids (PUFAs) has been associated with the prevalence of mood disorders in humans. In rodents, nutritional approaches aiming at modeling poor dietary n-3 PUFAs intake have been extensively developed in the last century. As a result, one- or multi-generation dietary n-3 deficiency induces depressive and anxiety-like behaviors. We have shown in the Nutrineuro lab that mice fed with a diet deficient in n-3 PUFAs exhibit decreased n-3 PUFAs levels, especially docosahexaenoic acid (DHA, a n-3 PUFA) levels in the prefrontal cortex (PFC) and in the nucleus accumbens (NAc). We showed that dietary n-3 PUFA is able to modulate endocannabinoid (eCB) dependent plasticity since DHA reduction in PFC and NAc is accompanied with eCB dependent long term depression (eCB-LTD) and eCB signaling impairment in the PFC (Lafourcade et al., 2011; Larrieu et al., 2012). Our data indicate that LTD alteration results from region-specific uncoupling of CB1 receptor from its effector Gi/o protein. In addition, n-3 deficient mice display behavioral deficits in several tests measuring emotional behavior. To further understand the mechanisms underlying DHA decrease in the PFC and emotional behavior alteration, we thoroughly investigated neuronal morphology and hypothalamic-pituitary-adrenal (HPA) axis in n-3 deficient mice. We showed that n-3 deficient diet induced dendritic atrophy in pyramidal neurons within the PFC. The dendritic atrophy was comparable to the one measured in control diet mice submitted to chronic social defeat stress (CSDS). No additional effect of CSDS on both neuronal morphology and emotional behavior was measured in n-3 deficient mice. We therefore investigated the role of the HPA axis deregulation in the development of behavioral and neurobiological alterations of n-3 deficient mice. We found a decreased expression of glucocorticoid receptor (GR) in the PFC of n-3 deficient mice together with increased circulating levels of corticosterone. Collectively, we unraveled one crucial mechanism underlying n-3 deficiency-induced alterations. Our results show that low dietary n-3 PUFAs can alter eCB-dependent plasticity and neuronal dendritic atrophy within the PFC leading to emotional behavior impairment. Importantly, we further demonstrated that corticosterone elevation in n-3 deficient mice was involved in the n-3 deficiency-induced emotional behavior and dendritic arborization alterations.