Structural mechanisms of mitochondrial uncoupling protein 1 regulation in thermogenesis.

In mitochondria, the oxidation of nutrients is coupled to ATP synthesis by the generation of a protonmotive force across the mitochondrial inner membrane. In mammalian brown adipose tissue (BAT), uncoupling protein 1 (UCP1, SLC25A7), a member of the SLC25 mitochondrial carrier family, dissipates the protonmotive force by facilitating the return of protons to the mitochondrial matrix. This process short-circuits the mitochondrion, generating heat for non-shivering thermogenesis. Recent cryo-electron microscopy (cryo-EM) structures of human UCP1 have provided new molecular insights into the inhibition and activation of thermogenesis. Here, we discuss these structures, describing how purine nucleotides lock UCP1 in a proton-impermeable conformation and rationalizing potential conformational changes of this carrier in response to fatty acid activators that enable proton leak for thermogenesis.

Adolescent novelty seeking is associated with greater ventral striatal and prefrontal brain response during evaluation of risk and reward.

Adolescence is a period during which reward sensitivity is heightened. Studies suggest that there are individual differences in adolescent reward-seeking behavior, attributable to a variety of factors, including temperament. This study investigated the neurobiological underpinnings of risk and reward evaluation as they relate to self-reported pleasure derived from novel experiences on the revised Early Adolescent Temperament Questionnaire (EATQ-R). Healthy participants (N = 265, ~50% male), aged 12-17 years, underwent functional magnetic resonance imaging during a modified Wheel of Fortune task, where they evaluated choices with varying probability of winning different monetary rewards. Across all participants, there was increased brain response in salience, reward, and cognitive control circuitry when evaluating choices with larger (compared with moderate) difference in risk/reward. Whole brain and a priori region-of-interest regression analyses revealed that individuals reporting higher novelty seeking had greater activation in bilateral ventral striatum, left middle frontal gyrus, and bilateral posterior cingulate cortex when evaluating the choices for largest difference in risk/reward. These novelty seeking associations with brain response were seen in the absence of temperament-related differences in decision-making behavior. Thus, while heightened novelty seeking in adolescents might be associated with greater neural sensitivity to risk/reward, accompanying increased activation in cognitive control regions might regulate reward-driven risk-taking behavior. More research is needed to determine whether individual differences in brain activation associated with novelty seeking are related to decision making in more ecologically valid settings.

Developmental trajectories of Big Five personality traits among adolescents and young adults: Differences by sex, alcohol use, and marijuana use.

OBJECTIVE: Individual differences in adolescent personality are related to a variety of long-term health outcomes. While previous studies have demonstrated sex differences and non-linear changes in personality development, these results remain equivocal. The current study utilized longitudinal data (n = 831) from the National Consortium on Alcohol and Neurodevelopment in Adolescence to examine sex differences in the development of personality and the association between substance use and personality. METHOD: Participants (ages 12-21 at baseline) completed the Ten-Item Personality Inventory and self-reported past year alcohol and marijuana use at up to 7 yearly visits. Data were analyzed using generalized additive mixed-effects models and linear mixed-effects models. RESULTS: Findings support linear increases in agreeableness and conscientious and decreases in openness with age and inform on timing of sex-specific non-linear development of extraversion and emotional stability. Further, results provide novel information regarding the timing of the association between substance use and personality, and replicate past reporting of differential associations between alcohol and marijuana use and extraversion, and sex-dependent effects of marijuana use on emotional stability. CONCLUSIONS: These findings highlight the importance of modeling sex differences in personality development using flexible non-linear modeling strategies, and accounting for sex- and age-specific effects of alcohol and marijuana use.

Lifetime Alcohol Use Influences the Association Between Future-Oriented Thought and White Matter Microstructure in Adolescents.

AIMS: Future orientation, or the ability to plan ahead and anticipate consequences, is a capacity that develops during adolescence, yet its underlying neurobiology is unknown. Previous independent reports suggest that reduced future orientation and altered white matter microstructure are associated with greater alcohol use in adolescents; however, these effects have not been studied in conjunction. This study investigated the association between future orientation and white matter microstructure as a function of lifetime alcohol use. METHODS: Seventy-seven adolescents (46 female; 15-21 years of age) underwent diffusion weighted imaging (DWI) and completed a fifteen-item Future Orientation Questionnaire. Regression analyses assessed the association between self-reported lifetime alcohol use and future orientation, and the association between future orientation and white matter microstructure, as a function of lifetime alcohol use. RESULTS: Adolescents with more lifetime alcohol use demonstrated lower future orientation. Voxel-wise DWI analyses revealed two regions, bilateral posterior corona radiata (PCR), where greater future orientation was associated with lower mean diffusivity in those with little or no history of alcohol use; however, this association was diminished with increasing rates of lifetime alcohol use. CONCLUSIONS: These findings replicate reports of reduced future orientation as a function of greater lifetime alcohol use and demonstrate an association between future orientation and white matter microstructure, in the PCR, a region containing afferent and efferent fibers connecting the cortex to the brain stem, which depends upon lifetime alcohol use. These findings provide novel information regarding the underlying neurobiology of future-oriented thought and how it relates to alcohol use.

Associations between nucleus accumbens structural connectivity, brain function, and initiation of binge drinking.

Adolescent alcohol use is associated with increased risk for alcohol use disorders later in life; therefore, identifying biomarkers for initiation of heavy alcohol use, such as individual differences in the development of white-matter microstructure, may inform prevention strategies that improve public health. This prospective cohort study included 40 adolescents, ages 14 and 15, without substantial history of alcohol or drug use at baseline. Fractional anisotropy (FA), an index of white-matter microstructure, was assessed in pathways connecting the nucleus accumbens (NAcc) to the rest of the brain using diffusion tensor imaging. Path analyses were conducted voxel-wise within these pathways to examine direct effects of premorbid FA on number of months between baseline assessment and the onset of binge drinking and indirect effects mediated by NAcc activation during decision making assessed using functional magnetic resonance imaging. Adolescents with lower premorbid accumbofrontal FA began binge drinking sooner, an effect which was mediated by greater NAcc activation during decision making involving greater levels of risk and reward (P < .05 corrected). An additional direct effect of FA on duration to onset of binge drinking was observed in white matter near the ventral pallidum, as adolescents with lower premorbid FA in this region began binge drinking sooner (P < .05 corrected). Findings suggest that delayed maturation of prefrontal white matter is associated with less top-down control over striatal sensitivity to reward. These factors, along with individual differences in white matter proximal to ventral pallidum, may represent premorbid risk factors for earlier initiation of heavy alcohol use.

Binge drinking impacts dorsal striatal response during decision making in adolescents.

Adolescence is a time of both increased risk taking and increased vulnerability to the neurotoxic effects of alcohol. However, it is unclear whether brain functioning abnormalities in adolescent binge drinkers are a result of alcohol use itself or whether they represent premorbid risk characteristics. The current study addresses this question by using a modified version of the Wheel of Fortune (WOF) task, during functional magnetic resonance imaging (fMRI), at both baseline, while all subjects were alcohol-naïve, and revisit, when half of the subjects had emerged into regular binge drinking (n=13) and half remained alcohol and substance-naïve (n=13). Region of interest (ROI) analysis revealed that during decision making, there was a significant binge-drinking related reduction in brain activation in the dorsal striatum, an effect associated with degree of recent use. Furthermore, whole-brain analysis revealed a decrease in fronto-parietal brain activation prior to initiation of alcohol use, in adolescents who went on to binge drink. Additionally, there were numerous regions, both cortical and subcortical, in which there was a significant time-related developmental change, across groups. These results demonstrate how abnormalities in decision-making related circuitry might both lead to and perpetuate alcohol drinking behavior. These findings help aid in our ability to disentangle consequences of binge drinking from potential risk markers for future binge drinking, and may help guide future prevention and intervention strategies.

Identification of potent and selective retinoic acid receptor gamma (RARγ) antagonists for the treatment of osteoarthritis pain using structure based drug design.

A series of triaryl pyrazoles were identified as potent pan antagonists for the retinoic acid receptors (RARs) α, ß and γ. X-ray crystallography and structure-based drug design were used to improve selectivity for RARγ by targeting residue differences in the ligand binding pockets of these receptors. This resulted in the discovery of novel antagonists which maintained RARγ potency but were greater than 500-fold selective versus RARα and RARß. The potent and selective RARγ antagonist LY2955303 demonstrated good pharmacokinetic properties and was efficacious in the MIA model of osteoarthritis-like joint pain. This compound demonstrated an improved margin to RARα-mediated adverse effects.

Comparative analysis of hepatitis B virus polymerase sequences required for viral RNA binding, RNA packaging, and protein priming.

Hepatitis B virus replicates a DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific association with a viral RNA signal, termed ε (Hε), located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. HP is made up of four domains, including the terminal protein (TP), the spacer, the reverse transcriptase (RT), and the RNase H domains. A recently developed, Hε-dependent, in vitro protein priming assay was used in this study to demonstrate that almost the entire TP and RT domains and most of the RNase H domain were required for protein priming. Specific residues within TP, RT, and the spacer were identified as being critical for HP-Hε binding and/or protein priming. Comparison of HP sequence requirements for Hε binding, pgRNA packaging, and protein priming allowed the classification of the HP mutants into five groups, each with distinct effects on these complex and related processes. Detailed characterization of HP requirements for these related and essential functions of HP will further elucidate the mechanisms of its multiple functions and aid in the targeting of these functions for antiviral therapy.

Protein-primed terminal transferase activity of hepatitis B virus polymerase.

Hepatitis B virus (HBV) replication requires reverse transcription of an RNA pregenome (pgRNA) by a multifunctional polymerase (HP). HP initiates viral DNA synthesis by using itself as a protein primer and an RNA signal on pgRNA, termed epsilon (Hε), as the obligatory template. We discovered a Mn(2+)-dependent transferase activity of HP in vitro that was independent of Hε but also used HP as a protein primer. This protein-primed transferase activity was completely dependent on the HP polymerase active site. The DNA products of the transferase reaction were linked to HP via a phosphotyrosyl bond, and replacement of the Y63 residue of HP, the priming site for templated DNA synthesis, almost completely eliminated DNA synthesis by the transferase activity, suggesting that Y63 also serves as the predominant priming site for the transferase reaction. For this transferase activity, HP could use all four deoxynucleotide substrates, but TTP was clearly favored for extensive polymerization. The transferase activity was highly distributive, leading to the synthesis of DNA homo- and hetero-oligomeric and -polymeric ladders ranging from 1 nucleotide (nt) to >100 nt in length, with single-nt increments. As with Hε-templated DNA synthesis, the protein-primed transferase reaction was characterized by an initial stage that was resistant to the pyrophosphate analog phosphonoformic acid (PFA) followed by PFA-sensitive DNA synthesis, suggestive of an HP conformational change upon the synthesis of a nascent DNA oligomer. These findings have important implications for HBV replication, pathogenesis, and therapy.

Consortium Profile: The Methylation, Imaging and NeuroDevelopment (MIND) Consortium.

Epigenetic processes, such as DNA methylation, show potential as biological markers and mechanisms underlying gene-environment interplay in the prediction of mental health and other brain-based phenotypes. However, little is known about how peripheral epigenetic patterns relate to individual differences in the brain itself. An increasingly popular approach to address this is by combining epigenetic and neuroimaging data; yet, research in this area is almost entirely comprised of cross-sectional studies in adults. To bridge this gap, we established the Methylation, Imaging and NeuroDevelopment (MIND) Consortium, which aims to bring a developmental focus to the emerging field of Neuroimaging Epigenetics by (i) promoting collaborative, adequately powered developmental research via multi-cohort analyses; (ii) increasing scientific rigor through the establishment of shared pipelines and open science practices; and (iii) advancing our understanding of DNA methylation-brain dynamics at different developmental periods (from birth to emerging adulthood), by leveraging data from prospective, longitudinal pediatric studies. MIND currently integrates 15 cohorts worldwide, comprising (repeated) measures of DNA methylation in peripheral tissues (blood, buccal cells, and saliva) and neuroimaging by magnetic resonance imaging across up to five time points over a period of up to 21 years (Npooled DNAm = 11,299; Npooled neuroimaging = 10,133; Npooled combined = 4,914). By triangulating associations across multiple developmental time points and study types, we hope to generate new insights into the dynamic relationships between peripheral DNA methylation and the brain, and how these ultimately relate to neurodevelopmental and psychiatric phenotypes.

Noncompetitive inhibition of hepatitis B virus reverse transcriptase protein priming and DNA synthesis by the nucleoside analog clevudine.

All currently approved antiviral drugs for the treatment of chronic hepatitis B virus (HBV) infection are nucleos(t)ide reverse transcriptase inhibitors (NRTI), which inhibit the DNA synthesis activity of the HBV polymerase. The polymerase is a unique reverse transcriptase (RT) that has a novel protein priming activity in which HP initiates viral DNA synthesis using itself as a protein primer. We have determined the ability of NRTI-triphosphates (TP) to inhibit HBV protein priming and their mechanisms of action. While entecavir-TP (a dGTP analog) inhibited protein priming initiated specifically with dGTP, clevudine-TP (a TTP analog) was able to inhibit protein priming independently of the deoxynucleoside triphosphate (dNTP) substrate and without being incorporated into DNA. We next investigated the effect of NRTIs on the second stage of protein priming, wherein two dAMP nucleotides are added to the initial deoxyguanosine nucleotide. The obtained results indicated that clevudine-TP as well as tenofovir DF-DP strongly inhibited the second stage of protein priming. Tenofovir DF-DP was incorporated into the viral DNA primer, whereas clevudine-TP inhibited the second stage of priming without being incorporated. Finally, kinetic analyses using the HBV endogenous polymerase assay revealed that clevudine-TP inhibited DNA chain elongation by HP in a noncompetitive manner. Thus, clevudine-TP appears to have the unique ability to inhibit HBV RT via binding to and distorting the HP active site, sharing properties with both NRTIs and nonnucleoside RT inhibitors.

In vitro epsilon RNA-dependent protein priming activity of human hepatitis B virus polymerase.

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. We have purified HP from human cells that retained Hε binding activity in vitro. Furthermore, HP purified as a complex with Hε, but not HP alone, displayed in vitro protein priming activity. While the HP-Hε interaction in vitro and in vivo required the Hε internal bulge, but not its apical loop, and was not significantly affected by the cap-Hε distance, protein priming required both the Hε apical loop and internal bulge, as well as a short distance between the cap and Hε, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-Hε interactions, RNA packaging, and protein priming.

A preliminary study of white matter correlates of a laboratory measure of attention and motor stability in attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) is a complex behavioral disorder, often difficult and time consuming to diagnose. Laboratory assessment of ADHD-related constructs of attention and motor activity may be helpful in elucidating neurobiology; however, neuroimaging studies evaluating laboratory measures of ADHD are lacking. In this preliminary study, we assessed the association between fractional anisotropy (FA), a measure of white matter microstructure, and laboratory measures of attention and motor behavior using the QbTest, a widely used measure thought to improve clinician diagnostic confidence. This is the first look at neural correlates of this widely used measure. The sample included adolescents and young adults (ages 12-20, 35% female) with ADHD (n = 31) and without (n = 52). As expected, ADHD status was associated with motor activity, and cognitive inattention and impulsivity in the laboratory. With regard to MRI findings, laboratory observed motor activity and inattention were associated with greater FA in white matter regions of the primary motor cortex. All three laboratory observations were associated with lower FA in regions subserving fronto-striatal-thalamic and frontoparietal (i.e. superior longitudinal fasciculus) circuitry. Further, FA in white matter regions of the prefrontal cortex appeared to mediate the relationship between ADHD status and motor activity on the QbTest. These findings, while preliminary, suggest that performance on certain laboratory tasks is informative with regard to neurobiological correlates of subdomains of the complex ADHD phenotype. In particular, we provide novel evidence for a relationship between an objective measure of motor hyperactivity and white matter microstructure in motor and attentional networks.

Heightened adolescent emotional reactivity in the brain is associated with lower future distress tolerance and higher depressive symptoms.

Distress tolerance, the ability to persist while experiencing negative psychological states, is essential for regulating emotions and is a transdiagnostic risk/resiliency trait for multiple psychopathologies. Studying distress tolerance during adolescence, a period when emotion regulation is still developing, may help identify early risk and/or protective factors. This study included 40 participants (mean scan age = 17.5 years) and using an emotional Go-NoGo functional magnetic resonance imaging task and voxel-wise regression analysis, examined the association between brain response during emotional face processing and future distress tolerance (two ± 0.5 years), controlling for sex assigned at birth, age, and time between visits. Post-hoc analyses tested the mediating role of distress tolerance on the emotional reactivity and depressive symptom relationship. Whole-brain analysis showed greater inferior occipital gyrus activation was associated with less distress tolerance at follow-up. The mediating role of distress tolerance demonstrated a trend-level indirect effect. Findings suggest that individuals who allocate greater visual resources to emotionally salient information tend to exhibit greater challenges in tolerating distress. Distress tolerance may help to link emotional reactivity neurobiology to future depressive symptoms. Building distress tolerance through emotion regulation strategies may be an appropriate strategy for decreasing depressive symptoms.

Adolescent Substance Use Is Associated With Altered Brain Response During Processing of Negative Emotional Stimuli.

OBJECTIVE: Substance misuse is often associated with emotional dysregulation. Understanding the neurobiology of emotional responsivity and regulation as it relates to substance use in adolescence may be beneficial for preventing future use. METHOD: The present study used a community sample, ages 11-21 years old (N = 130, Mage = 17), to investigate the effects of alcohol and marijuana use on emotional reactivity and regulation using an Emotional Go-NoGo task during functional magnetic resonance imaging. The task consisted of three conditions, where target (Go) stimuli were either happy, scared, or calm faces. Self-report lifetime (and past-90-day) drinking and marijuana use days were provided at all visits. RESULTS: Substance use was not differentially related to task performance based on condition. Whole-brain linear mixed-effects analyses (controlling for age and sex) found that more lifetime drinking occasions was associated with greater neural emotional processing (Go trials) in the right middle cingulate cortex during scared versus calm conditions. In addition, more marijuana use occasions were associated with less neural emotional processing during scared versus calm conditions in the right middle cingulate cortex and right middle and inferior frontal gyri. Substance use was not associated with brain activation during inhibition (NoGo trials). CONCLUSIONS: These findings demonstrate that substance use-related alterations in brain circuitry are important for attention allocation and the integration of emotional processing and motor response when viewing negative emotional stimuli.

Alcohol-induced changes in mesostriatal resting-state functional connectivity are linked to sensation seeking in young adults.

BACKGROUND: Studies in animals and humans suggest that greater levels of sensation seeking and alcohol use are related to individual differences in drug-induced dopamine release. However, it remains unclear whether drug-induced alterations in the functional synchrony between mesostriatal regions are related to sensation seeking and alcohol use. METHODS: In this within-subject masked-design study, 21-year-old participants (n = 34) underwent functional magnetic resonance imaging to measure ventral tegmental area (VTA) resting-state functional connectivity to the striatum after receiving alcohol (target blood alcohol concentration 0.08 g/dL) or placebo. Participants also completed the UPPS-P Impulsive Behavior Scale to assess sensation seeking, the Young Adult Alcohol Consequences Questionnaire, and self-reported patterns of alcohol and drug use. RESULTS: Voxel-wise analyses within the striatum demonstrated that during the alcohol condition (compared with placebo) young adults had less connectivity between the VTA and bilateral caudate (p < 0.05 corrected). However, young adults exhibiting smaller alcohol-induced decreases or increases in VTA-left caudate connectivity reported greater sensation seeking. CONCLUSION: These findings provide novel information about how acute alcohol impacts resting-state connectivity, an effect that may be driven by the complex pre and postsynaptic effects of alcohol on various neurotransmitters including dopamine. Further, alcohol-induced differences in VTA connectivity represent a plausible mechanistic substrate underlying sensation seeking.

Associations between alcohol use and sex-specific maturation of subcortical gray matter morphometry from adolescence to adulthood: Replication across two longitudinal samples.

Subcortical brain morphometry matures across adolescence and young adulthood, a time when many youth engage in escalating levels of alcohol use. Initial cross-sectional studies have shown alcohol use is associated with altered subcortical morphometry. However, longitudinal evidence of sex-specific neuromaturation and associations with alcohol use remains limited. This project used generalized additive mixed models to examine sex-specific development of subcortical volumes and associations with recent alcohol use, using 7 longitudinal waves (n = 804, 51% female, ages 12-21 at baseline) from the National Consortium on Alcohol and Neurodevelopment in Adolescence (NCANDA). A second, independent, longitudinal dataset, with up to four waves of data (n = 467, 43% female, ages 10-18 at baseline), was used to assess replicability. Significant, replicable non-linear normative volumetric changes with age were evident in the caudate, putamen, thalamus, pallidum, amygdala and hippocampus. Significant, replicable negative associations between subcortical volume and alcohol use were found in the hippocampus in all youth, and the caudate and thalamus in female but not male youth, with significant interactions present in the caudate, thalamus and putamen. Findings suggest a structural vulnerability to alcohol use, or a predisposition to drink alcohol based on brain structure, with female youth potentially showing heightened risk, compared to male youth.

Structural basis of purine nucleotide inhibition of human uncoupling protein 1.

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.