Regulation of cannabinoid and opioid receptor levels by endogenous and pharmacological chaperones.

Cannabinoid and opioid receptor activities can be modulated by a variety of posttranslational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB1 receptor (CB1R), delta opioid receptor (DOR), and CB1R-DOR interacting complexes. Focussing on endogenous protein chaperones namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared to other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB1R, DOR, and CB1R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of Cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB1R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking. Significance Statement This study highlights a role for chaperones (endogenous and small membrane-permeable molecules) in modulating levels of CB1R, DOR, and their interacting complexes. These chaperones could be developed as therapeutics for pathologies involving these receptors.

Ad-derived bone marrow transplant induces proinflammatory immune peripheral mechanisms accompanied by decreased neuroplasticity and reduced gut microbiome diversity affecting AD-like phenotype in the absence of Aß neuropathology.

Immune system dysfunction is increasingly recognized as a significant feature that contributes to Alzheimer's disease (AD) pathogenesis, reflected by alterations in central and peripheral responses leading to detrimental mechanisms that can contribute to the worsening of the disease. The damaging alterations in the peripheral immune system may disrupt the peripheral-central immune crosstalk, implicating the gut microbiota in this complex interaction. The central hypothesis posits that the immune signature inherently harbored in bone marrow (BM) cells can be transferred through allogeneic transplantation, influencing the recipient's immune system and modulating peripheral, gut, and brain immune responses. Employing a genetically modified mouse model to develop AD-type pathology we found that recipient wild-type (WT) mice engrafted with AD-derived BM, recapitulated the peripheral immune inflammatory donor phenotype, associated with a significant acceleration of cognitive deterioration in the absence of any overt change in AD-type amyloid neuropathology. Moreover, transcriptomic and phylogenetic 16S microbiome analysis evidence on these animals revealed a significantly impaired expression of genes associated with synaptic plasticity and neurotransmission in the brain and reduced bacteria diversity, respectively, compared to mice engrafted with WT BM. This investigation sheds light on the pivotal role of the peripheral immune system in the brain-gut-periphery axis and its profound potential to shape the trajectory of AD. In summary, this study advances our understanding of the complex interplay among the peripheral immune system, brain functionality, and the gut microbiome, which collectively influence AD onset and progression.

Colony-stimulating factor 2 (CSF2) as a gut microbiome dependent immune factor that alters molecular and behavioral responses to cocaine in male mice.

Cocaine use disorder is a condition that leads to tremendous morbidity and mortality for which there are currently no FDA-approved pharmacotherapies. Previous research has demonstrated an important role for the resident population of bacteria of the large intestine, collectively dubbed the gut microbiome, in modulating brain and behavior in models of cocaine and other substance use disorders. Importantly, previous work has repeatedly shown that depletion of the gut microbiome leads to increased cocaine taking and seeking behaviors in multiple models. While the precise mechanism of these gut-brain signaling pathways in models of cocaine use is not fully clear, and intriguing possibility is through gut microbiome influences on innate immune system function. In this manuscript we identify the cytokine colony stimulating factor 2 (CSF2) as an immune factor that is increased by cocaine in a gut microbiome dependent manner. Peripherally injected CSF2 crosses the blood-brain barrier into the nucleus accumbens, a brain region central to behavioral responses to cocaine. Treatment with peripheral CSF2 reduces acute and sensitized locomotor responses to cocaine as well as reducing cocaine place preference at high doses. On a molecular level, we find that peripheral injections of CSF2 alter the transcriptional response to both acute and repeated cocaine in the nucleus accumbens. Finally, treatment of microbiome depleted mice with CSF2 reverses the behavioral effects of microbiome depletion on the conditioned place preference assay. Taken together, this work identifies an innate immune factor that represents a novel gut-brain signaling cascade in models of cocaine use and lays the foundations for further translational work targeting this pathway.

Acetate supplementation rescues social deficits and alters transcriptional regulation in prefrontal cortex of Shank3 deficient mice.

BACKGROUND: The pathophysiology of autism spectrum disorder (ASD) involves genetic and environmental factors. Mounting evidence demonstrates a role for the gut microbiome in ASD, with signaling via short-chain fatty acids (SCFA) as one mechanism. Here, we utilize mice carrying deletion to exons 4-22 of Shank3 (Shank3KO) to model gene by microbiome interactions in ASD. We identify SCFA acetate as a mediator of gut-brain interactions and show acetate supplementation reverses social deficits concomitant with alterations to medial prefrontal cortex (mPFC) transcriptional regulation independent of microbiome status. METHODS: Shank3KO and wild-type (Wt) littermates were divided into control, Antibiotic (Abx), Acetate and Abx + Acetate groups upon weaning. After six weeks, animals underwent behavioral testing. Molecular analysis including 16S and metagenomic sequencing, metabolomic and transcriptional profiling were conducted. Additionally, targeted serum metabolomic data from Phelan McDermid Syndrome (PMS) patients (who are heterozygous for the Shank3 gene) were leveraged to assess levels of SCFA's relative to ASD clinical measures. RESULTS: Shank3KO mice were found to display social deficits, dysregulated gut microbiome and decreased cecal levels of acetate - effects exacerbated by Abx treatment. RNA-sequencing of mPFC showed unique gene expression signature induced by microbiome depletion in the Shank3KO mice. Oral treatment with acetate reverses social deficits and results in marked changes in gene expression enriched for synaptic signaling, pathways among others, even in Abx treated mice. Clinical data showed sex specific correlations between levels of acetate and hyperactivity scores. CONCLUSION: These results suggest a key role for the gut microbiome and the neuroactive metabolite acetate in regulating ASD-like behaviors.

Dietary polyphenols drive dose-dependent behavioral and molecular alterations to repeated morphine.

Opioid Use Disorder (OUD) is associated with tremendous morbidity and mortality. Despite this burden, current pharmacotherapies for OUD are ineffective or intolerable for many patients. As such, interventions aimed at promoting resilience against OUD are of immense clinical interest. Treatment with a Bioactive Dietary Polyphenol Preparation (BDPP) promotes resilience and adaptive neuroplasticity in multiple models of neuropsychiatric disease. Here, we assessed effects of BDPP treatment on behavioral and molecular responses to repeated morphine treatment in male mice. BDPP pre-treatment alters responses for both locomotor sensitization and conditioned place preference. Most notably, polyphenol treatment consistently reduced formation of preference at low dose (5 mg/kg) morphine but enhanced it at high dose (15 mg/kg). In parallel, we performed transcriptomic profiling of the nucleus accumbens, which again showed a dose × polyphenol interaction. We also profiled microbiome composition and function, as polyphenols are metabolized by the microbiome and can act as prebiotics. The profile revealed polyphenol treatment markedly altered microbiome composition and function. Finally, we investigated involvement of the SIRT1 deacetylase, and the role of polyphenol metabolites in behavioral responses. These results demonstrate polyphenols have robust dose-dependent effects on behavioral and physiological responses to morphine and lay the foundation for future translational work.

Large 22q13.3 deletions perturb peripheral transcriptomic and metabolomic profiles in Phelan-McDermid syndrome.

Phelan-McDermid syndrome (PMS) is a rare neurodevelopmental disorder caused at least in part by haploinsufficiency of the SHANK3 gene, due to sequence variants in SHANK3 or subtelomeric 22q13.3 deletions. Phenotypic differences have been reported between PMS participants carrying small "class I" mutations and large "class II" mutations; however, the molecular perturbations underlying these divergent phenotypes remain obscure. Using peripheral blood transcriptome and serum metabolome profiling, we examined the molecular perturbations in the peripheral circulation associated with a full spectrum of PMS genotypes spanning class I (n = 37) and class II mutations (n = 39). Transcriptomic data revealed 52 genes with blood expression profiles that tightly scale with 22q.13.3 deletion size. Furthermore, we uncover 208 underexpressed genes in PMS participants with class II mutations, which were unchanged in class I mutations. These genes were not linked to 22q13.3 and were strongly enriched for glycosphingolipid metabolism, NCAM1 interactions, and cytotoxic natural killer (NK) immune cell signatures. In silico predictions estimated a reduction in CD56+ CD16- NK cell proportions in class II mutations, which was validated by mass cytometry time of flight. Global metabolomics profiling identified 24 metabolites that were significantly altered in PMS participants with class II mutations and confirmed a general reduction in sphingolipid metabolism. Collectively, these results provide new evidence linking PMS participants carrying class II mutations with decreased expression of cytotoxic cell signatures, reduced relative proportions of NK cells, and lower sphingolipid metabolism. These findings highlight alternative avenues for therapeutic development and offer new mechanistic insights supporting genotype-to-phenotype associations in PMS.

Stress-activated brain-gut circuits disrupt intestinal barrier integrity and social behaviour.

Chronic stress underlies the etiology of both major depressive disorder (MDD) and irritable bowel syndrome (IBS), two highly prevalent and debilitating conditions with high rates of co-morbidity. However, it is not fully understood how the brain and gut bi-directionally communicate during stress to impact intestinal homeostasis and stress-relevant behaviours. Using the chronic social defeat stress (CSDS) model, we find that stressed mice display greater intestinal permeability and circulating levels of the endotoxin lipopolysaccharide (LPS) compared to unstressed control (CON) mice. Interestingly, the microbiota in the colon also exhibit elevated LPS biosynthesis gene expression following CSDS. Additionally, CSDS triggers an increase in pro-inflammatory colonic IFNγ+ Th1 cells and a decrease in IL4+ Th2 cells compared to CON mice, and this gut inflammation contributes to stress-induced intestinal barrier permeability and social avoidance behaviour. We next investigated the role of enteric neurons and identified that noradrenergic dopamine beta-hydroxylase (DBH)+ neurons in the colon are activated by CSDS, and that their ablation protects against gut pathophysiology and disturbances in social behaviour. Retrograde tracing from the colon identified a population of corticotropin-releasing hormone-expressing (CRH+) neurons in the paraventricular nucleus of the hypothalamus (PVH) that innervate the colon and are activated by stress. Chemogenetically activating these PVH CRH+ neurons is sufficient to induce gut inflammation, barrier permeability, and social avoidance behaviour, while inhibiting these cells prevents these effects following exposure to CSDS. Thus, we define a stress-activated brain-to-gut circuit that confers colonic inflammation, leading to impaired intestinal barrier function, and consequent behavioural deficits.

Systematic review of imaging tests to predict the development of rheumatoid arthritis in people with unclassified arthritis.

OBJECTIVE: To estimate and compare the diagnostic accuracy of magnetic resonance imaging (MRI) and ultrasound, for the prediction of rheumatoid arthritis (RA) in unclassified arthritis (UA). METHODS: MEDLINE, Embase and BIOSIS were searched from 1987 to May 2019. Studies evaluating any imaging test in participants with UA were eligible. Reference standards were RA classification criteria or methotrexate initiation. Two authors independently extracted data and assessed validity using QUADAS-2. Sensitivities and specificities were calculated for each imaging characteristic and joint area. Summary estimates with 95% confidence intervals (CI) were estimated where possible. RESULTS: Nineteen studies were included; 13 evaluated MRI (n=1,143; 454 with RA) and 6 evaluated ultrasound (n=531; 205 with RA). Studies were limited by unclear recruitment procedures, inclusion of patients with RA at baseline, differential verification, lack of blinding and consensus grading. Study heterogeneity largely precluded meta-analysis, however summary sensitivity and specificity for MRI synovitis in at least one joint were 93% (95% CI 88%, 96%) and 25% (95% CI 13%, 41%) (3 studies). Specificities may be higher for other MRI characteristics but data are limited. Ultrasound results were difficult to synthesise due to different diagnostic thresholds and reference standards. CONCLUSION: The evidence for MRI or ultrasound as single tests for predicting RA in people with UA is heterogeneous and of variable methodological quality. Larger studies using consensus grading and consistently defined RA diagnosis are needed to identify whether combinations of imaging characteristics, either alone or in combination with other clinical findings, can better predict RA in this population.

Contributions of neuroimmune and gut-brain signaling to vulnerability of developing substance use disorders.

Epidemiology and clinical research indicate that only a subset of people who are exposed to drugs of abuse will go on to develop a substance use disorder. Numerous factors impact individual susceptibility to developing a substance use disorder, including intrinsic biological factors, environmental factors, and interpersonal/social factors. Given the extensive morbidity and mortality that is wrought as a consequence of substance use disorders, a substantial body of research has focused on understanding the risk factors that mediate the shift from initial drug use to pathological drug use. Understanding these risk factors provides a clear path for the development of risk mitigation strategies to help reduce the burden of substance use disorders in the population. Here we will review the rapidly growing body of literature that examines the importance of interactions between the peripheral immune system, the gut microbiome, and the central nervous system (CNS) in mediating the transition to pathological drug use. While these systems had long been viewed as distinct, there is growing evidence that there is bidirectional communication between both the immune system and the gut microbiome that drive changes in neural and behavioral plasticity relevant to substance use disorders. Further, both of these systems are highly sensitive to environmental perturbations and are implicated in numerous neuropsychiatric conditions. While the field of study examining these interactions in substance use disorders is in its relative infancy, clarifying the relationship between gut-immune-brain signaling and substance use disorders has potential to improve our understanding of individual propensity to developing addiction and yield important insight into potential treatment options.

Post-weaning A1/A2 ß-casein milk intake modulates depressive-like behavior, brain µ-opioid receptors, and the metabolome of rats.

The postnatal period is critical for brain and behavioral development and is sensitive to environmental stimuli, such as nutrition. Prevention of weaning from maternal milk was previously shown to cause depressive-like behavior in rats. Additionally, loss of dietary casein was found to act as a developmental trigger for a population of brain opioid receptors. Here, we explore the effect of exposure to milk containing A1 and A2 ß-casein beyond weaning. A1 but not A2 ß-casein milk significantly increased stress-induced immobility in rats, concomitant with an increased abundance of Clostridium histolyticum bacterial group in the caecum and colon of A1 ß-casein fed animals, brain region-specific alterations of µ-opioid and oxytocin receptors, and modifications in urinary biochemical profiles. Moreover, urinary gut microbial metabolites strongly correlated with altered brain metabolites. These findings suggest that consumption of milk containing A1 ß-casein beyond weaning age may affect mood via a possible gut-brain axis mechanism.

Granulocyte colony-stimulating factor (G-CSF) enhances cocaine effects in the nucleus accumbens via a dopamine release-based mechanism.

Cocaine use disorder is associated with alterations in immune function including altered expression of multiple peripheral cytokines in humans-several of which correlate with drug use. Individuals suffering from cocaine use disorder show altered immune system responses to drug-associated cues, highlighting the interaction between the brain and immune system as a critical factor in the development and expression of cocaine use disorder. We have previously demonstrated in animal models that cocaine use upregulates the expression of granulocyte colony-stimulating factor (G-CSF)-a pleiotropic cytokine-in the serum and the nucleus accumbens (NAc). G-CSF signaling has been causally linked to behavioral responses to cocaine across multiple behavioral domains. The goal of this study was to define whether increases in G-CSF alter the pharmacodynamic effects of cocaine on the dopamine system and whether this occurs via direct mechanisms within local NAc microcircuits. We find that systemic G-CSF injection increases cocaine effects on dopamine terminals. The enhanced dopamine levels in the presence of cocaine occur through a release-based mechanism, rather than through effects on the dopamine transporter-as uptake rates were unchanged following G-CSF treatment. Critically, this effect could be recapitulated by acute bath application of G-CSF to dopamine terminals, an effect that was occluded by prior G-CSF treatment, suggesting a similar mechanistic basis for direct and systemic exposures. This work highlights the critical interaction between the immune system and psychostimulant effects that can alter drug responses and may play a role in vulnerability to cocaine use disorder.