Cells and molecules underpinning cannabis-related variations in cortical thickness during adolescence.

During adolescence, cannabis experimentation is common, and its association with inter-individual variations in brain maturation well studied. Cellular and molecular underpinnings of these system-level relationships are, however, unclear. We thus conducted a three-step study. First, we exposed adolescent male mice to Δ-9-tetrahydrocannabinol (THC) or a synthetic cannabinoid WIN 55,212-2 (WIN) and assessed differentially expressed genes (DEGs), spine numbers and dendritic complexity in their frontal cortex. Second, in human (male) adolescents, we examined group differences in cortical thickness in 34 brain regions, using magnetic resonance imaging, between those who experimented with cannabis before age 16 (n=140) and those who did not (n=327). Finally, we correlated spatially these group differences with gene expression of human homologs of the mouse-identified DEGs. The spatial expression of 13 THC-related human homologs of DEGs correlated with cannabis-related variations in cortical thickness, and virtual histology revealed co-expression patterns of these 13 genes with cell-specific markers of astrocytes, microglia and a type of pyramidal cells enriched in dendrite-regulating genes. Similarly, the spatial expression of 18 WIN-related human homologs of DEGs correlated with group differences in cortical thickness, and showed co-expression patterns with the same three cell types. Gene ontology analysis indicated that 37 THC-related human homologs are enriched in neuron-projection development, while 33 WIN-related homologs are enriched in processes associated with learning and memory. In mice, we observed spine loss and lower dendritic complexity in pyramidal cells of THC-exposed animals (vs. controls). Experimentation with cannabis during adolescence may influence cortical thickness by impacting glutamatergic synapses and dendritic arborization.Significance Statement Cells and molecular underpinning of cannabis-related variations in cortical thickness observed in adolescents are poorly understood. We assessed differences in gene expression in the frontal cortex of adolescent mice exposed (or not) to Δ-9-tetra-hydrocannabinol (THC) or WIN 55,212-2 (WIN). THC preferentially targeted glial cells, while WIN affected pyramidal neurons; both modified nuclear-coded subunits in mitochondrial respiratory complexes and excitatory synapse genes. In humans, we evaluated a spatial correlation between group differences in cortical thickness and the expression of human homologs of the cannabinoid-sensitive genes in mice. These genes co-expressed with those specific to astrocytes, microglia and a type of pyramidal cells enriched in dendrite-regulating genes. Experimentation with cannabis during adolescence may influence cortical thickness via synaptic processes and dendritic arborization.

Acan downregulation in parvalbumin GABAergic cells reduces spontaneous recovery of fear memories.

While persistence of fear memories is essential for survival, a failure to inhibit fear in response to harmless stimuli is a feature of anxiety disorders. Extinction training only temporarily suppresses fear memory recovery in adults, but it is highly effective in juvenile rodents. Maturation of GABAergic circuits, in particular of parvalbumin-positive (PV+) cells, restricts plasticity in the adult brain, thus reducing PV+ cell maturation could promote the suppression of fear memories following extinction training in adults. Epigenetic modifications such as histone acetylation control gene accessibility for transcription and help couple synaptic activity to changes in gene expression. Histone deacetylase 2 (Hdac2), in particular, restrains both structural and functional synaptic plasticity. However, whether and how Hdac2 controls the maturation of postnatal PV+ cells is not well understood. Here, we show that PV+- cell specific Hdac2 deletion limits spontaneous fear memory recovery in adult mice, while enhancing PV+ cell bouton remodeling and reducing perineuronal net aggregation around PV+ cells in prefrontal cortex and basolateral amygdala. Prefrontal cortex PV+ cells lacking Hdac2, show reduced expression of Acan, a critical perineuronal net component, which is rescued by Hdac2 re-expression. Pharmacological inhibition of Hdac2 before extinction training is sufficient to reduce both spontaneous fear memory recovery and Acan expression in wild-type adult mice, while these effects are occluded in PV+-cell specific Hdac2 conditional knockout mice. Finally, a brief knock-down of Acan expression mediated by intravenous siRNA delivery before extinction training but after fear memory acquisition is sufficient to reduce spontaneous fear recovery in wild-type mice. Altogether, these data suggest that controlled manipulation of PV+ cells by targeting Hdac2 activity, or the expression of its downstream effector Acan, promotes the long-term efficacy of extinction training in adults.

Treatment of Acute Pain in Adults With Sickle Cell Disease in an Infusion Center Versus the Emergency Department : A Multicenter Prospective Cohort Study.

BACKGROUND: Patients with sickle cell disease (SCD) have vaso-occlusive crises (VOCs). Infusion centers (ICs) are alternatives to emergency department (ED) care and may improve patient outcomes. OBJECTIVE: To assess whether care in ICs or EDs leads to better outcomes for the treatment of uncomplicated VOCs. DESIGN: Prospective cohort. (ClinicalTrials.gov: NCT02411396). SETTING: 4 U.S. sites, with recruitment between April 2015 and December 2016. PARTICIPANTS: Adults with SCD living within 60 miles of a study site. MEASUREMENTS: Participants were followed for 18 months after enrollment. Outcomes of interest were time to first dose of parenteral pain medication, whether pain reassessment was completed within 30 minutes after the first dose, and patient disposition on discharge from the acute care visit. Treatment effects for ICs versus EDs were estimated using a time-varying propensity score adjustment. RESULTS: Researchers enrolled 483 participants; the 269 who had acute care visits on weekdays are included in this report. With inverse probability of treatment-weighted adjustment, the mean time to first dose was 62 minutes in ICs and 132 minutes in EDs; the difference was 70 minutes (95% CI, 54 to 98 minutes; E-value, 2.8). The probability of pain reassessment within 30 minutes of the first dose of parenteral pain medication was 3.8 times greater (CI, 2.63 to 5.64 times greater; E-value, 4.7) in the IC than the ED. The probability that a participant's visit would end in admission to the hospital was smaller by a factor of 4 (0.25 [CI, 0.18 to 0.33]) with treatment in an IC versus an ED. LIMITATION: The study was restricted to participants with uncomplicated VOCs. CONCLUSION: In adults with SCD having a VOC, treatment in an IC is associated with substantially better outcomes than treatment in an ED. PRIMARY FUNDING SOURCE: Patient-Centered Outcomes Research Institute.

The Lived Experiences of Non-Offending Fathers with Children Who Survived Sexual Abuse.

A non-offending father figure plays an integral role in the healing process of a child who survived sexual abuse. However, becoming aware of the sexual abuse can significantly affect non-offending father figures and therefore impact their ability to properly support and care for the survivor. We sought to better understand the non-offending father figures' reactions to the aftermath of sexual abuse of their children. Using an existential-phenomenological approach, we offered a platform for non-offending father figures to share their stories. Through the interviews, we found five major themes, which include: "Guilt, anguish, and stigma", "Hypervigilance and competing demands of fathering", "Who can we trust?", "Refocusing on the family", and "Picking up the pieces". Based on this and previous studies, non-offending father figures experience psychological pain in the aftermath of the disclosure of sexual abuse, they deal with competing demands of various fatherly roles, and they prioritize supporting the family through the healing process. The findings suggest that the psychological well-being of the non-offending father figures can benefit the family. Therefore, mental health treatment protocols addressing father figures' needs can contribute to a sexual abuse treatment model that encourages paternal involvement in the care of children with a sexual abuse history.

Design and construction of conformational biosensors to monitor ion channel activation: A prototype FlAsH/BRET-approach to Kir3 channels.

Ion channels play a vital role in numerous physiological functions and drugs that target them are actively pursued for development of novel therapeutic agents. Here we report a means for monitoring in real time the conformational changes undergone by channel proteins upon exposure to pharmacological stimuli. The approach relies on tracking structural rearrangements by monitoring changes in bioluminescence energy transfer (BRET). To provide proof of principle we have worked with Kir3 neuronal channels producing 10 different constructs which were combined into 17 donor-acceptor BRET pairs. Among these combinations, pairs bearing the donor Nano-Luc (NLuc) at the C-terminal end of Kir3.2 subunits and the FlAsH acceptor at the N-terminal end (NT) or the interfacial helix (N70) of Kir3.1 subunits were identified as potential tools. These pairs displayed significant changes in energy transfer upon activation with direct channel ligands or via stimulation of G protein-coupled receptors. Conformational changes associated with channel activation followed similar kinetics as channel currents. Dose response curves generated by different agonists in FlAsH-BRET assays displayed similar rank order of potency as those obtained with conventional BRET readouts of G protein activation and ion flux assays. Conformational biosensors as the ones reported herein should prove a valuable complement to other methodologies currently used in channel drug discovery.

Endocytic profiles of δ-opioid receptor ligands determine the duration of rapid but not sustained cAMP responses.

Traditional assays that monitor cAMP inhibition by opioid receptor ligands require second-messenger accumulation over periods of 10-20 minutes. Since receptor regulation occurs within a similar time frame, such assays do not discriminate the actual signal from its modulation. Here we used bioluminescence resonance energy transfer to monitor inhibition of cAMP production by δ-opioid receptor (DOR) agonists in real time. cAMP inhibition elicited by different agonists over a period of 15 minutes was biphasic, with response buildup during the first 6 to 7 minutes, followed by a second phase of response decay or of no further increment. The rate at which the cAMP response disappeared was correlated with operational parameters describing ligand efficiency [log(τ/KA)] to promote Gαi activation, as well as with ligand ability to promote internalization during the time course of the assay. Thus, ligands that displayed low signaling efficiency and poor sequestration(SB235863 ([8R-(4bS*,8aα,8aß,12bß)]7,10-dimethyl-1-methoxy-11-(2-ethylpropyl)oxycarbonyl 5,6,7,8,12,12b-hexahydro-(9H)-4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]isoquinoline hydrochloride), morphine) had minimal or no response decay. On the other hand, the decay rate was pronounced for deltorphin II, [d-Pen(2), d-pen(5)]-enkephalin, met-enkephalin, and SNC-80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide), which displayed high signaling efficiency and internalization. Moreover, inhibition of internalization by dynasore reduced or abolished response decay by internalizing ligands. Unlike acute responses, endocytic profiles were not predictive of whether an agonist would induce prolonged cAMP inhibition over sustained (30-120 minutes) DOR stimulation. Taken together, the data indicate that ligand ability to evoke G-protein activation or promote endocytosis was predictive of response duration over short, but not over sustained periods of cAMP inhibition.

Costs and impact of disease in adults with sickle cell disease: a pilot study.

ABSTRACT: We assessed the feasibility to estimate illness burden in adults with SCD, investigated factors associated with health-related quality of life (HRQoL), and estimated societal burden. We recruited 32 participants and collected data on fatigue, HRQoL, and work productivity and activity impairment via patient survey. Health care utilization was abstracted for the 12 months before enrollment using medical chart review. Mean age was 36.7 years; 84.4% of participants had hemoglobin SS or Sßthal0 disease, and 81.3% reported chronic pain (experiencing pain on ≥3 days per week in the past 6 months). Mean EQ-5D-3L visual analogue scale score was 63.4 and the index score was 0.79. The mean fatigue score was 57.9. Higher fatigue score was correlated with lower EQ-5D index score (correlation coefficient r = -0.35; P = .049) and Adult Sickle Cell Quality of Life Measurement Information System (ASCQ-Me) scores, including pain (r = -0.47; P = .006), sleep (r = -0.38; P = .03), and emotion scores (r = -0.79; P < .0001). The number of hospitalizations was negatively correlated with HRQoL (all P < .05). Patients who reported chronic pain had significantly lower mean ASCQ-Me sleep scores (48.3 vs 57.1; P = .04) and EQ-5D index scores (0.72 vs 0.89; P = .002) than those without chronic pain. Mean estimated annual per person costs were $51 779 (median, $36 366) for total costs, $7619 ($0) for indirect costs (estimated from lost earnings of participants), and $44 160 ($31 873) for medical costs. Fatigue, SCD complications, hospitalization, and chronic pain negatively affected HRQoL. This sample experienced a high economic burden, largely from outpatient doctor visits.

5-HT2A receptor signalling through phospholipase D1 associated with its C-terminal tail.

The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF-PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders.

Long-term follow-up after diagnosis resulting from newborn screening: statement of the US Secretary of Health and Human Services' Advisory Committee on Heritable Disorders and Genetic Diseases in Newborns and Children.

The US Secretary of Health and Human Services' Advisory Committee on Heritable Disorders and Genetic Diseases in Newborns and Children provides guidance to reduce the morbidity and mortality associated with heritable disorders, with a special emphasis on those conditions detectable through newborn screening. Although long-term follow-up is necessary to maximize the benefit of diagnosis through newborn screening, such care is variable and inconsistent. To begin to improve long-term follow-up, the Advisory Committee has identified its key features, including the assurance and provision of quality chronic disease management, condition-specific treatment, and age-appropriate preventive care throughout the lifespan of affected individuals. There are four components central to achieving long-term follow-up: care coordination through a medical home, evidence-based treatment, continuous quality improvement, and new knowledge discovery.

Increased acute care utilization in a prospective cohort of adults with sickle cell disease.

The ESCAPED (Examining Sickle Cell Acute Pain in the Emergency vs Day Hospital) trial is an ongoing prospective study comparing outcomes of people with sickle cell disease (SCD) seeking care for acute pain management in either an emergency department or specialty infusion clinic. The objective of this paper is to describe the baseline characteristics and health care utilization of patients in the trial. This is a multicenter study across 4 US cities that enrolled all adults with SCD living within 60 miles (96.6 km) of a study site who were expected to have acute care utilization over the study period. Twenty-one percent of participants had no acute care visits in the first 12 months of follow-up. Using negative binomial regression, we describe subject characteristics that predict acute care utilization. Three hundred ninety-one subjects have completed 12 months of follow-up with a mean age of 34.5 years (standard deviation, 11.4), 60% are female. Fifty-four percent of subjects with hemoglobin SS disease and 46% with hemoglobin SC disease had 3 or more acute visits over the study period. The prevalence of chronic pain in this cohort was 68%. Predictors of higher rates of acute care utilization included being unemployed, having chronic pain, being on chronic transfusion therapy, having a history of stroke, and being on disability or on Medicaid. This is the first prospective cohort in the modern era, and it demonstrates much higher rates of acute care utilization than reported in the Cooperative Study of Sickle Cell Disease.

Role of the conserved NPxxY motif of the 5-HT2A receptor in determining selective interaction with isoforms of ADP-ribosylation factor (ARF).

In this study we have shown that N376 to D mutation in the conserved NPxxY motif within the carboxy terminal tail domain (CT) of the 5-HT2A receptor alters the binding preference of GST-fusion protein constructs of the CT domain from ARF1 to an alternative isoform, ARF6. These findings were corroborated by experiments investigating co-immunoprecipitation of the wild type (WT) and N376D mutant of the 5-HT2A receptor with ARF1 or 6 or dominant negative ARF1/6 constructs co-expressed in COS7 cells. In functional assays of 5-HT-induced phospholipase D (PLD) activation responses of the WT receptor were inhibited by a dominant negative mutant of ARF1 but not ARF6, whereas responses of the N376D mutant were strongly inhibited by negative mutant ARF6. No equivalent effect of the ARF mutants was seen on phospholipase C activation. In experiments assaying 5-HT-induced increases in [35S]GTPgammaS binding to ARF 1/6 immunoprecipitates as a measure of ARF activation, increased ARF6 activation was seen only with the mutant receptor. When cellular PLD responses of other NPxxY- or a DPxxY-containing GPCRs were measured in the presence of dominant negative ARF1/6 constructs, the majority, but not all, fitted the pattern exemplified by the 5-HT2A receptor and its N376D mutant. These data suggest that the presence of the N or a D in this highly conserved motif is an important, but not exclusive, determinant of which ARF isoform interacts with the GPCR.

Attenuated PLD1 association and signalling at the H452Y polymorphic form of the 5-HT(2A) receptor.

The 5-HT2A receptor (5-HT2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT2AR.

Selective interaction of ARF1 with the carboxy-terminal tail domain of the 5-HT2A receptor.

The 5-hydroxytryptamine 2A receptor (5-HT2AR) is a member of the class I family of rhodopsin-related G protein-coupled receptors. The receptor is known to activate phospholipase C via the heterotrimeric G proteins Gq/11, but we showed previously that it can also signal through the phospholipase D (PLD) pathway in an ADP-ribosylation factor (ARF)-dependent manner that seems to be independent of Gq/11 (Mitchell et al., 1998). Both coimmunoprecipitation experiments and the effects of negative mutant ARF constructs on 5-HT2AR-induced PLD activation here suggested that ARF1 may play a greater role than ARF6 in the function of this receptor. Furthermore, we demonstrated using glutathione S-transferase (GST)-fusion proteins of receptor domains that ARF1 and ARF6 bind to the third intracellular loop (i3) and the carboxy terminal tail (ct) of the 5-HT2AR. The association of ARF1 with the ct domain of the receptor was stronger than its interaction with i3, or the interactions of ARF6 with either construct. Experiments using ARF mutants that are deficient in GTP loading, and the in vitro addition of GTPgammaS suggested that GTP loading enhances ARF1 binding to the receptor. The N376PxxY motif in the transmembrane 7 domain of the receptor (rather than a N376DPxxY mutant form) was shown to be essential for ARF-dependent PLD signaling and ARF1 coimmunoprecipitation. In GST-fusion proteins of the 5-HT2AR ct domain, mutation of Asn376 to Asp also markedly reduced ARF1-HA binding, although additional motifs in the Asn376-Asn384 sequence and to a lesser extent elsewhere, seem also to contribute to the interaction.

ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor.

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.