Antipsychotic-induced epigenomic reorganization in frontal cortex of individuals with schizophrenia.

Genome-wide association studies have revealed >270 loci associated with schizophrenia risk, yet these genetic factors do not seem to be sufficient to fully explain the molecular determinants behind this psychiatric condition. Epigenetic marks such as post-translational histone modifications remain largely plastic during development and adulthood, allowing a dynamic impact of environmental factors, including antipsychotic medications, on access to genes and regulatory elements. However, few studies so far have profiled cell-specific genome-wide histone modifications in postmortem brain samples from schizophrenia subjects, or the effect of antipsychotic treatment on such epigenetic marks. Here, we conducted ChIP-seq analyses focusing on histone marks indicative of active enhancers (H3K27ac) and active promoters (H3K4me3), alongside RNA-seq, using frontal cortex samples from antipsychotic-free (AF) and antipsychotic-treated (AT) individuals with schizophrenia, as well as individually matched controls (n=58). Schizophrenia subjects exhibited thousands of neuronal and non-neuronal epigenetic differences at regions that included several susceptibility genetic loci, such as NRG1, DISC1, and DRD3. By analyzing the AF and AT cohorts separately, we identified schizophrenia-associated alterations in specific transcription factors, their regulatees, and epigenomic and transcriptomic features that were reversed by antipsychotic treatment; as well as those that represented a consequence of antipsychotic medication rather than a hallmark of schizophrenia in postmortem human brain samples. Notably, we also found that the effect of age on epigenomic landscapes was more pronounced in frontal cortex of AT-schizophrenics, as compared to AF-schizophrenics and controls. Together, these data provide important evidence of epigenetic alterations in the frontal cortex of individuals with schizophrenia, and remark for the first time on the impact of age and antipsychotic treatment on chromatin organization.

Glucocorticoid receptor dysregulation underlies 5-HT2AR-dependent synaptic and behavioral deficits in a mouse neurodevelopmental disorder model.

Prenatal environmental insults increase the risk of neurodevelopmental psychiatric conditions in the offspring. Structural modifications of dendritic spines are central to brain development and plasticity. Using maternal immune activation (MIA) as a rodent model of prenatal environmental insult, previous results have reported dendritic structural deficits in the frontal cortex. However, very little is known about the molecular mechanism underlying MIA-induced synaptic structural alterations in the offspring. Using prenatal (E12.5) injection with polyinosinic-polycytidylic acid potassium salt as a mouse MIA model, we show here that upregulation of the serotonin 5-HT2A receptor (5-HT2AR) is at least in part responsible for some of the effects of prenatal insults on frontal cortex dendritic spine structure and sensorimotor gating processes. Mechanistically, we report that this upregulation of frontal cortex 5-HT2AR expression is associated with MIA-induced reduction of nuclear translocation of the glucocorticoid receptor (GR) and, consequently, a decrease in the enrichment of GR at the 5-HT2AR promoter. The translational significance of these preclinical findings is supported by data in postmortem human brain samples suggesting dysregulation of GR translocation in frontal cortex of schizophrenia subjects. We also found that repeated corticosterone administration augmented frontal cortex 5-HT2AR expression and reduced GR binding to the 5-HT2AR promoter. However, virally (adeno-associated virus) mediated augmentation of GR function reduced frontal cortex 5-HT2AR expression and improved sensorimotor gating processes via 5-HT2AR. Together, these data support a negative regulatory relationship between GR signaling and 5-HT2AR expression in the mouse frontal cortex that may carry implications for the pathophysiology underlying 5-HT2AR dysregulation in neurodevelopmental psychiatric disorders.

Sex-specific role for serotonin 5-HT2A receptor in modulation of opioid-induced antinociception and reward in mice.

Opioids are among the most effective analgesics and the mainstay of pain management. However, concerns about safety and abuse liability have challenged their widespread use by the medical community. Opioid-sparing therapies include drugs that in combination with opioids have the ability to enhance analgesia while decreasing opioid requirement as well as their side effects. Sex differences in antinociceptive responses to opioids have received increasing attention in recent years. However, the molecular mechanisms underlying sex differences related to opioid-sparing adjuncts remain largely unexplored. Using warm water tail-withdrawal as a mouse model of acute thermal nociception, our data suggest that adjunctive administration of the serotonin 5-HT2A receptor (5-HT2AR) antagonist volinanserin dose-dependently enhanced potency of the opioid analgesic oxycodone in male, but not female, mice. This antinociceptive-like response induced by oxycodone was also augmented in 5-HT2AR knockout (5-HT2AR-/-) male, but not female mice; an effect that was reversed by Cre-loxP-mediated selective expression of 5-HT2AR in dorsal root ganglion (DRG) neurons of 5-HT2AR-/- littermates. Pharmacological inhibition with volinanserin or genetic deletion in 5-HT2AR-/- animals potentiated the ability of oxycodone to reduce DRG excitability in male mice. Adjunctive volinanserin did not affect oxycodone-induced conditioned place preference (CPP), whereas it reduced oxycodone-induced locomotor sensitization in male and female mice. Together, these results suggest that adjunctive volinanserin augments opioid-induced antinociception, but not abuse-related behavior, through a sex-specific signaling crosstalk mechanism that requires 5-HT2AR expression in mouse DRG neurons. Ultimately, our results may pave the way for the clinical evaluation of volinanserin as a potential sex-specific opioid adjuvant.

Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.

Clinical evidence suggests that rapid and sustained antidepressant action can be attained with a single exposure to psychedelics. However, the biological substrates and key mediators of psychedelics' enduring action remain unknown. Here, we show that a single administration of the psychedelic DOI produces fast-acting effects on frontal cortex dendritic spine structure and acceleration of fear extinction via the 5-HT2A receptor. Additionally, a single dose of DOI leads to changes in chromatin organization, particularly at enhancer regions of genes involved in synaptic assembly that stretch for days after the psychedelic exposure. These DOI-induced alterations in the neuronal epigenome overlap with genetic loci associated with schizophrenia, depression, and attention deficit hyperactivity disorder. Together, these data support that epigenomic-driven changes in synaptic plasticity sustain psychedelics' long-lasting antidepressant action but also warn about potential substrate overlap with genetic risks for certain psychiatric conditions.

Gait Speed Trajectory During the Six-Minute Walk Test in Multiple Sclerosis: A Measure of Walking Endurance.

Background: The six-minute walk (6MW) test is a validated assessment method in Multiple Sclerosis (MS) research. While the total distance covered during six minutes (6MWTD ) is often used as the standard measurement of gait capacity (i.e., the maximum distance one can achieve), we hypothesize that endurance (i.e., ability to maintain speed over a prolonged time) can be inferred by the gait speed trajectory (GST) during the 6MW test (6MWGST ). Objective: To characterize group differences in 6MWGST between MS patients and healthy controls (HCs), and to assess information added by 6MWGST for discerning between MS patients and HCs. Methods: We performed a secondary data analysis on a cross-sectional cohort of 40 MS and 20 HC subjects with three repeated 6MW tests. We modeled 6MWGST using a linear mixed-effects model with time in minutes and replicated walks nested within individuals. We compared the discernibility of 6MWGST with that of conventional metrics using likelihood ratio tests and receiver operating characteristic (ROC) analysis on logistic regression models. Results: MS subjects showed a concave, quadratic GST during 6MW tests, slowing down more than the HC subjects, especially at the beginning of 6MW tests. Despite accelerating at the end of the 6MW, MS subjects were unable to attain or surpass their initial 6MW gait speeds. 6MWGST added useful information (p = 0.002) to the conventional metrics (e.g., 6MWTD ) for discerning between MS and HC subjects, and increased the area under the ROC curve from 0.83 to 0.93 (p = 0.037). Conclusions: The distinctive 6MWGST pattern of MS patients provided increased discernibility compared with currently used gait metrics. Both gait capacity measured by the 6MWTD , and gait endurance measured by parameters of 6MWGST , are significant functional indicators for the MS population.

Psychedelic-like Properties of Quipazine and Its Structural Analogues in Mice.

Known classic psychedelic serotonin 2A receptor (5-HT2AR) agonists retain a tryptamine or phenethylamine at their structural core. However, activation of the 5-HT2AR can be elicited by drugs lacking these fundamental scaffolds. Such is the case of the N-substituted piperazine quipazine. Here, we show that quipazine bound to and activated 5-HT2AR as measured by [3H]ketanserin binding displacement, Ca2+ mobilization, and accumulation of the canonical Gq/11 signaling pathway mediator inositol monophosphate (IP1) in vitro and in vivo. Additionally, quipazine induced via 5-HT2AR an expression pattern of immediate early genes (IEG) in the mouse somatosensory cortex consistent with that of classic psychedelics. In the mouse head-twitch response (HTR) model of psychedelic-like action, quipazine produced a lasting effect with high maximal responses during the peak effect that were successfully blocked by the 5-HT2AR antagonist M100907 and absent in 5-HT2AR knockout (KO) mice. The acute effect of quipazine on HTR appeared to be unaffected by serotonin depletion and was independent from 5-HT3R activation. Interestingly, some of these features were shared by its deaza bioisostere 2-NP, but not by other closely related piperazine congeners, suggesting that quipazine might represent a distinct cluster within the family of psychoactive piperazines. Together, our results add to the mounting evidence that quipazine's profile matches that of classic psychedelic 5-HT2AR agonists at cellular signaling and behavioral pharmacology levels.

Autoantibodies Blocking M3 Muscarinic Receptors Cause Postganglionic Cholinergic Dysautonomia.

A 10-year-old girl presented with ileus, urinary retention, dry mouth, lack of tears, fixed dilated pupils, and diffuse anhidrosis 7 days after a febrile illness. We hypothesized that her syndrome was due to autoimmunity against muscarinic acetylcholine receptors, blocking their activation. Using an indirect enzyme-linked immunosorbent assay for all 5 muscarinic receptors (M1 -M5 ), we identified in the patient's serum antibodies that selectively bound to M3 receptors. In vitro functional studies confirmed that these autoantibodies selectively blocked M3 receptor activation. Thus, autoantibodies against M3 acetylcholine receptors cause acute postganglionic cholinergic dysautonomia. ANN NEUROL 2020;88:1237-1243.

Biased signaling by endogenous opioid peptides.

Opioids, such as morphine and fentanyl, are widely used for the treatment of severe pain; however, prolonged treatment with these drugs leads to the development of tolerance and can lead to opioid use disorder. The "Opioid Epidemic" has generated a drive for a deeper understanding of the fundamental signaling mechanisms of opioid receptors. It is generally thought that the three types of opioid receptors (µ, δ, κ) are activated by endogenous peptides derived from three different precursors: Proopiomelanocortin, proenkephalin, and prodynorphin. Posttranslational processing of these precursors generates >20 peptides with opioid receptor activity, leading to a long-standing question of the significance of this repertoire of peptides. Here, we address some aspects of this question using a technical tour de force approach to systematically evaluate ligand binding and signaling properties ([35S]GTPγS binding and ß-arrestin recruitment) of 22 peptides at each of the three opioid receptors. We show that nearly all tested peptides are able to activate the three opioid receptors, and many of them exhibit agonist-directed receptor signaling (functional selectivity). Our data also challenge the dogma that shorter forms of ß-endorphin do not exhibit receptor activity; we show that they exhibit robust signaling in cultured cells and in an acute brain slice preparation. Collectively, this information lays the groundwork for improved understanding of the endogenous opioid system that will help in developing more effective treatments for pain and addiction.

Adjunctive effect of the serotonin 5-HT2C receptor agonist lorcaserin on opioid-induced antinociception in mice.

Opioid-sparing adjuncts are treatments that aim to reduce the overall dose of opioids needed to achieve analgesia, hence decreasing the burden of side effects through alternative mechanisms of action. Lorcaserin is a serotonin 5-HT2C receptor (5-HT2CR) agonist that has recently been reported to reduce abuse-related effects of the opioid analgesic oxycodone. The goal of our studies was to evaluate the effects of adjunctive lorcaserin on opioid-induced analgesic-like behavior using the tail-flick reflex (TFR) test as a mouse model of acute thermal nociception. We show that whereas subcutaneous (s.c.) administration of lorcaserin alone was inactive on the TFR test, adjunctive lorcaserin (s.c.) significantly increased the potency of oxycodone as an antinociceptive drug. This effect was prevented by the 5-HT2CR antagonist SB242084. A similar lorcaserin (s.c.)-induced adjunctive phenotype was observed upon administration of the opioid analgesics morphine and fentanyl. Remarkably, we also show that, opposite to the effects observed via s.c. administration, intrathecal (i.t.) administration of lorcaserin alone induced antinociceptive TFR behavior, an effect that was not prevented by the opioid receptor antagonist naloxone. This route of administration (i.t.) also led to a significant augmentation of oxycodone-induced antinociception. Lorcaserin (s.c.) did not alter the brain or blood concentrations of oxycodone, which suggests that its adjunctive effects on opioid-induced antinociception do not depend upon changes in opioid metabolism. Together, these data indicate that lorcaserin-mediated activation of the 5-HT2CR may represent a new pharmacological approach to augment opioid-induced antinociception. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

Targeting Cannabinoid 1 and Delta Opioid Receptor Heteromers Alleviates Chemotherapy-Induced Neuropathic Pain.

Cannabinoid 1 (CB1R) and delta opioid receptors (DOR) associate to form heteromers that exhibit distinct pharmacological properties. Not much is known about CB1R-DOR heteromer location or signaling along the pain circuit in either animal models or patients with chemotherapy-induced peripheral neuropathy (CIPN). Here, we use paclitaxel to induce CIPN in mice and confirm the development of mechanical allodynia. Under these conditions, we find significant increases in CB1R-DOR heteromers in the dorsal spinal cord of mice with CIPN as well as in postmortem spinal cords from human subjects with CIPN compared to controls. Next, we investigated receptor signaling in spinal cords of mice with CIPN and found that treatment with a combination of low signaling doses of CB1R and DOR ligands leads to significant enhancement in G-protein activity that could be selectively blocked by the CB1R-DOR antibody. Consistent with this, administration of subthreshold doses of a combination of ligands (CB1R agonist, Hu-210, and DOR agonist, SNC80) leads to significant attenuation of allodynia in mice with CIPN that is not seen with the administration of individual ligands, and this could be blocked by the CB1R-DOR antibody. Together, these results imply that CB1R-DOR heteromers upregulated during CIPN-associated mechanical allodynia could serve as a potential target for treatment of neuropathic pain including CIPN.

The endocannabinoid system in cardiovascular function: novel insights and clinical implications.

RATIONALE: Cardiovascular disease is now recognized as the number one cause of death in the world, and the size of the population at risk continues to increase rapidly. The dysregulation of the endocannabinoid (eCB) system plays a central role in a wide variety of conditions including cardiovascular disorders. Cannabinoid receptors, their endogenous ligands, as well as enzymes conferring their synthesis and degradation, exhibit overlapping distributions in the cardiovascular system. Furthermore, the pharmacological manipulation of the eCB system has effects on blood pressure, cardiac contractility, and endothelial vasomotor control. Growing evidence from animal studies supports the significance of the eCB system in cardiovascular disorders. OBJECTIVE: To summarize the literature surrounding the eCB system in cardiovascular function and disease and the new compounds that may potentially extend the range of available interventions. RESULTS: Drugs targeting CB1R, CB2R, TRPV1 and PPARs are proven effective in animal models mimicking cardiovascular disorders such as hypertension, atherosclerosis and myocardial infarction. Despite the setback of two clinical trials that exhibited unexpected harmful side-effects, preclinical studies are accelerating the development of more selective drugs with promising results devoid of adverse effects. CONCLUSION: Over the last years, increasing evidence from basic and clinical research supports the role of the eCB system in cardiovascular function. Whereas new discoveries are paving the way for the identification of novel drugs and therapeutic targets, the close cooperation of researchers, clinicians and pharmaceutical companies is needed to achieve successful outcomes.

Is biological aging accelerated in drug addiction?

Drug-addiction may trigger early onset of age-related disease, due to drug-induced multi-system toxicity and perilous lifestyle, which remains mostly undetected and untreated. We present the literature on pathophysiological processes that may hasten aging and its relevance to addiction, including: oxidative stress and cellular aging, inflammation in periphery and brain, decline in brain volume and function, and early onset of cardiac, cerebrovascular, kidney, and liver disease. Timely detection of accelerated aging in addiction is crucial for the prevention of premature morbidity and mortality.

Detection of Receptor Heteromerization Using In Situ Proximity Ligation Assay.

Although G protein-coupled receptor (GPCR) heteromerization has been extensively demonstrated in vitro using heterologous cells that overexpress epitope-tagged receptors, their presence in endogenous systems is less well established. This is because a criterion to identify receptor heteromerization is the demonstration that the two interacting receptors are present not only in the same cell but also in the same subcellular compartment in close enough proximity to allow for direct receptor-receptor interaction. This has been difficult to study in native tissues due to a lack of sensitive and selective tools not only capable of detecting low-abundance proteins but also of demonstrating that they are in sufficiently close proximity to interact. The latter can be achieved using a proximity ligation assay (PLA). Detailed in this unit are protocols for demonstrating the presence of GPCR heteromers in endogenous cells as well as animal and human tissues, the controls required for these assays, and troubleshooting tips. © 2016 by John Wiley & Sons, Inc.

Identification of GPR83 as the receptor for the neuroendocrine peptide PEN.

PEN is an abundant peptide in the brain that has been implicated in the regulation of feeding. We identified a receptor for PEN in mouse hypothalamus and Neuro2A cells. PEN bound to and activated GPR83, a G protein (heterotrimeric guanine nucleotide)-binding protein)-coupled receptor (GPCR). Reduction of GPR83 expression in mouse brain and Neuro2A cells reduced PEN binding and signaling, consistent with GPR83 functioning as the major receptor for PEN. In some brain regions, GPR83 colocalized with GPR171, a GPCR that binds the neuropeptide bigLEN, another neuropeptide that is involved in feeding and is generated from the same precursor protein as is PEN. Coexpression of these two receptors in cell lines altered the signaling properties of each receptor, suggesting a functional interaction. Our data established PEN as a neuropeptide that binds GPR83 and suggested that these two ligand-receptor systems-PEN-GPR83 and bigLEN-GPR171-may be functionally coupled in the regulation of feeding.

Head-to-Head Comparison of the Neuropsychiatric Effect of Dopamine Agonists in Parkinson's Disease: A Prospective, Cross-Sectional Study in Non-demented Patients.

INTRODUCTION: Dopaminergic agonists (DAs) are widely used to treat motor symptoms in Parkinson's disease (PD). The differential effect of DAs on neuropsychiatric symptoms of PD has not been accurately studied. MATERIALS AND METHODS: We performed a prospective cross-sectional study of 515 non-demented PD patients receiving treatment with pramipexole [n = 250, monotherapy or with levodopa (L-dopa)], ropinirole (n = 150, monotherapy or with L-dopa), or L-dopa (n = 115, monotherapy); all formulations were immediate release. Neuropsychiatric disturbances were assessed through the Neuropsychiatric Inventory (NPI). Groups were matched in terms of age, education, sex, disease severity (Hoehn and Yahr), disease duration, executive function, total L-dopa daily equivalent dose, and concomitant psychotropic medications (antidepressants, anxiolytics and antipsychotic agents). RESULTS: Patients on pramipexole showed significantly lower total NPI scores than patients on ropinirole (17.2 ± 11 vs. 20.9 ± 13, p = 0.015). Regarding the spectrum of neuropsychiatric symptoms, pramipexole was associated with significantly lower apathy scores than the L-dopa group (1.01 ± 1.7 vs. 1.87 ± 2.93, p = 0.02). The frequency of patients with clinically meaningful symptoms of apathy (NPI apathy scores ≥ 4) was significantly lower in the pramipexole group (11.2 %) than in the ropinirole (20.3 %) and L-dopa (23.8 %) groups (χ (2) 12.49, p = 0.002). No other significant differences were found in NPI subscores between groups. CONCLUSIONS: This is the first head-to-head comparative study of the effect of DAs on neuropsychiatric disturbances in PD that has controlled the sample for the most important confounding factors. In comparable groups of patients, the use of pramipexole seems to be associated with a lower frequency and severity of apathetic symptoms.

Detection of cannabinoid receptors CB1 and CB2 within basal ganglia output neurons in macaques: changes following experimental parkinsonism.

Although type 1 cannabinoid receptors (CB1Rs) are expressed abundantly throughout the brain, the presence of type 2 cannabinoid receptors (CB2Rs) in neurons is still somewhat controversial. Taking advantage of newly designed CB1R and CB2R mRNA riboprobes, we demonstrate by PCR and in situ hybridization that transcripts for both cannabinoid receptors are present within labeled pallidothalamic-projecting neurons of control and MPTP-treated macaques, whereas the expression is markedly reduced in dyskinetic animals. Moreover, an in situ proximity ligation assay was used to qualitatively assess the presence of CB1Rs and CB2Rs, as well as CB1R-CB2R heteromers within basal ganglia output neurons in all animal groups (control, parkinsonian and dyskinetic macaques). A marked reduction in the number of CB1Rs, CB2Rs and CB1R-CB2R heteromers was found in dyskinetic animals, mimicking the observed reduction in CB1R and CB2R mRNA expression levels. The fact that chronic levodopa treatment disrupted CB1R-CB2R heteromeric complexes should be taken into consideration when designing new drugs acting on cannabinoid receptor heteromers.

Calbindin content and differential vulnerability of midbrain efferent dopaminergic neurons in macaques.

Calbindin (CB) is a calcium binding protein reported to protect dopaminergic neurons from degeneration. Although a direct link between CB content and differential vulnerability of dopaminergic neurons has long been accepted, factors other than CB have also been suggested, particularly those related to the dopamine transporter. Indeed, several studies have reported that CB levels are not causally related to the differential vulnerability of dopaminergic neurons against neurotoxins. Here we have used dual stains for tyrosine hydroxylase (TH) and CB in 3 control and 3 MPTP-treated monkeys to visualize dopaminergic neurons in the ventral tegmental area (VTA) and in the dorsal and ventral tiers of the substantia nigra pars compacta (SNcd and SNcv) co-expressing TH and CB. In control animals, the highest percentages of co-localization were found in VTA (58.2%), followed by neurons located in the SNcd (34.7%). As expected, SNcv neurons lacked CB expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA was similar to control monkeys (62.1%), whereas most of the few surviving neurons in the SNcd were CB-ir/TH-ir (88.6%). Next, we have elucidated the presence of CB within identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate nucleus and one injection of cholera toxin (CTB) into the postcommissural putamen, whereas two more monkeys were injected with CTB into the internal division of the globus pallidus (GPi). As expected, all the nigrocaudate- and nigroputamen-projecting neurons were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTP-induced degeneration, our data clearly demonstrated that these neurons are not giving rise to nigrostriatal projections and indeed CB-ir/TH-ir neurons only originate nigroextrastriatal projections.

L-DOPA-treatment in primates disrupts the expression of A(2A) adenosine-CB(1) cannabinoid-D(2) dopamine receptor heteromers in the caudate nucleus.

The molecular basis of priming for L-DOPA-induced dyskinesias in Parkinson's disease (PD), which depends on the indirect pathway of motor control, is not known. In rodents, the indirect pathway contains striatopallidal GABAergic neurons that express heterotrimers composed of A(2A) adenosine, CB(1) cannabinoid and D(2) dopamine receptors that regulate dopaminergic neurotransmission. The present study was designed to investigate the expression of these heteromers in the striatum of a primate model of Parkinson's disease and to determine whether their expression and pharmacological properties are altered upon L-DOPA treatment. By using the recently developed in situ proximity ligation assay and by identification of a biochemical fingerprint, we discovered a regional distribution of A(2A)/CB(1) /D(2) receptor heteromers that predicts differential D(2)-mediated neurotransmission in the caudate-putamen of Macaca fascicularis. Whereas heteromers were abundant in the caudate nucleus of both naïve and MPTP-treated monkeys, L-DOPA treatment blunted the biochemical fingerprint and led to weak heteromer expression. These findings constitute the first evidence of altered receptor heteromer expression in pathological conditions and suggest that drugs targeting A(2A)-CB(1) -D(2) receptor heteromers may be successful to either normalize basal ganglia output or prevent L-DOPA-induced side effects.