Detecting G protein-coupled receptor complexes in postmortem human brain with proximity ligation assay and a Bayesian classifier

Despite the controversy regarding the existence and physiological relevance of class A G protein-coupled receptor dimerization, there is substantial evidence for functional interactions between the dopamine D2 receptor (D2R) and the adenosine A2A receptor (A2AR). A2AR-D2R complexes have been detected in rodent brains by proximity ligation assay; however, their existence in the human brain has not been demonstrated. In this study, we used Brightfield proximity ligation assay, combined with a systematic sampling and a parameter-free naive Bayesian classifier, and demonstrated proximity between the D2R and the A2AR in the adult human ventral striatum, consistent with their colocalization within complexes and the possible existence of D2R-A2AR heteromers. These methods are applicable to the relative quantification of proximity of two proteins, as well as the expression levels of individual proteins

Elevated cerebrospinal fluid 5-hydroxyindoleacetic acid in macaques following early life stress and inverse association with hippocampal volume: preliminary implications for serotonin-related function in mood and anxiety disorders

Background: Early life stress (ELS) is cited as a risk for mood and anxiety disorders, potentially through altered serotonin neurotransmission. We examined the effects of ELS, utilizing the variable foraging demand (VFD) macaque model, on adolescent monoamine metabolites. We sought to replicate an increase in cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) observed in two previous VFD cohorts. We hypothesized that elevated cisternal 5-HIAA was associated with reduced neurotrophic effects, conceivably due to excessive negative feedback at somatodendritic 5-HT1A autoreceptors. A putatively decreased serotonin neurotransmission would be reflected by reductions in hippocampal volume and white matter (WM) fractional anisotropy (FA).Methods: When infants were 2-6 months of age, bonnet macaque mothers were exposed to VFD. We employed cisternal CSF taps to measure monoamine metabolites in VFD (N = 22) and non-VFD (N = 14) offspring (mean age = 2.61 years). Metabolites were correlated with hippocampal volume obtained by MRI and WM FA by diffusion tensor imaging in young adulthood in 17 males [10 VFD (mean age = 4.57 years)].Results: VFD subjects exhibited increased CSF 5-HIAA compared to non-VFD controls. An inverse correlation between right hippocampal volume and 5-HIAA was noted in VFD- but not controls. CSF HVA and MHPG correlated inversely with hippocampal volume only in VFD. CSF 5-HIAA correlated inversely with FA of the VVM tracts of the anterior limb of the internal capsule (ALIC) only in VFD.Conclusions: Elevated cisternal 5-HIAA in VFD may reflect increased dorsal raphe serotonin, potentially inducing excessive autoreceptor activation, inducing a putative serotonin deficit in terminal fields. Resultant reductions in neurotrophic activity are reflected by smaller right hippocampal volume. Convergent evidence of reduced neurotrophic activity in association with high CSF 5-HIAA in VFD was reflected by reduced FA of the ALIC.NIMHSuny Downstate Med Ctr, Nonhuman Primate Lab, Dept Psychiat & Behav Sci, Brooklyn, NY 11203 USANew York State Psychiat Inst & Hosp, New York, NY 10032 USASuny Downstate Med Ctr, Coll Med, Brooklyn, NY 11203 USAUniversidade Federal de São Paulo, Dept Psychiat & Neuroradiol, São Paulo, BrazilFranklin Beha Hlh Consultants, Bronx, NY USANew York State Psychiat Inst & Hosp, Dept Mol Imaging & Neuropathol, New York, NY 10032 USAIcahn Sch Med Mt Sinai, Dept Psychiat, New York, NY 10029 USAIcahn Sch Med Mt Sinai, Dept Neurosci, New York, NY 10029 USAIcahn Sch Med Mt Sinai, Dept Radiol, New York, NY 10029 USAIcahn Sch Med Mt Sinai, Fishberg Dept Neurosci, New York, NY 10029 USAIcahn Sch Med Mt Sinai, Friedman Brain Inst, New York, NY 10029 USAYale Univ, Sch Med, Dept Psychiat, New Haven, CT USAMichael E Debakey VA Med Ctr, Mental Hlth Care Line, Houston, TX USABaylor Coll Med, Menninger Dept Psychiat & Behav Sci, Houston, TX 77030 USAYale Univ, Sch Med, Ctr Child Study, New Haven, CT 06510 USAUniversidade Federal de São Paulo, Dept Psychiat & Neuroradiol, São Paulo, BrazilNIMH: R21MH066748NIMH: R01 MH059990Web of Scienc

Case Study: Predictive Fairness to Reduce Misdemeanor Recidivism Through Social Service Interventions

The criminal justice system is currently ill-equipped to improve outcomes of individuals who cycle in and out of the system with a series of misdemeanor offenses. Often due to constraints of caseload and poor record linkage, prior interactions with an individual may not be considered when an individual comes back into the system, let alone in a proactive manner through the application of diversion programs. The Los Angeles City Attorney's Office recently created a new Recidivism Reduction and Drug Diversion unit (R2D2) tasked with reducing recidivism in this population. Here we describe a collaboration with this new unit as a case study for the incorporation of predictive equity into machine learning based decision making in a resource-constrained setting. The program seeks to improve outcomes by developing individually-tailored social service interventions (i.e., diversions, conditional plea agreements, stayed sentencing, or other favorable case disposition based on appropriate social service linkage rather than traditional sentencing methods) for individuals likely to experience subsequent interactions with the criminal justice system, a time and resource-intensive undertaking that necessitates an ability to focus resources on individuals most likely to be involved in a future case. Seeking to achieve both efficiency (through predictive accuracy) and equity (improving outcomes in traditionally under-served communities and working to mitigate existing disparities in criminal justice outcomes), we discuss the equity outcomes we seek to achieve, describe the corresponding choice of a metric for measuring predictive fairness in this context, and explore a set of options for balancing equity and efficiency when building and selecting machine learning models in an operational public policy setting.Comment: 12 pages, 4 figures, 1 algorithm. The definitive Version of Record will be published in the proceedings of the Conference on Fairness, Accountability, and Transparency (FAT* '20), January 27-30, 2020, Barcelona, Spai

Global epigenomic reconfiguration during mammalian brain development

DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity

Genome-Wide Divergence of DNA Methylation Marks in Cerebral and Cerebellar Cortices

Emerging evidence suggests that DNA methylation plays an expansive role in the central nervous system (CNS). Large-scale whole genome DNA methylation profiling of the normal human brain offers tremendous potential in understanding the role of DNA methylation in brain development and function.Using methylation-sensitive SNP chip analysis (MSNP), we performed whole genome DNA methylation profiling of the prefrontal, occipital, and temporal regions of cerebral cortex, as well as cerebellum. These data provide an unbiased representation of CpG sites comprising 377,509 CpG dinucleotides within both the genic and intergenic euchromatic region of the genome. Our large-scale genome DNA methylation profiling reveals that the prefrontal, occipital, and temporal regions of the cerebral cortex compared to cerebellum have markedly different DNA methylation signatures, with the cerebral cortex being hypermethylated and cerebellum being hypomethylated. Such differences were observed in distinct genomic regions, including genes involved in CNS function. The MSNP data were validated for a subset of these genes, by performing bisulfite cloning and sequencing and confirming that prefrontal, occipital, and temporal cortices are significantly more methylated as compared to the cerebellum.These findings are consistent with known developmental differences in nucleosome repeat lengths in cerebral and cerebellar cortices, with cerebrum exhibiting shorter repeat lengths than cerebellum. Our observed differences in DNA methylation profiles in these regions underscores the potential role of DNA methylation in chromatin structure and organization in CNS, reflecting functional specialization within cortical regions

Early life stress and macaque annygdala hypertrophy: preliminary evidence for a role for the serotonin transporter gene

Background: Children exposed to early life stress (ELS) exhibit enlarged amygdala volume in comparison to controls. the primary goal of this study was to examine amygdala volumes in bonnet macaques subjected to maternal variable foraging demand (VFD) rearing, a well-established model of ELS. Preliminary analyses examined the interaction of ELS and the serotonin transporter gene on amygdala volume. Secondary analyses were conducted to examine the association between amygdala volume and other stress-related variables previously found to distinguish VFD and non-VFD reared animals.Methods: Twelve VFD-reared and nine normally reared monkeys completed MRI scans on a 3T system (mean age = 5.2 years).Results: Left amygdala volume was larger in VFD vs. control macaques. Larger amygdala volume was associated with: high cerebrospinal fluid concentrations of corticotropin releasing-factor (CRF) determined when the animals were in adolescence (mean age = 2.7 years); reduced fractional anisotropy (FA) of the anterior limb of the internal capsule (ALIC) during young adulthood (mean age = 5.2 years) and timid anxiety-like responses to an intruder during full adulthood (mean age = 8.4 years). Right amygdala volume varied inversely with left hippocampal neurogenesis assessed in late adulthood (mean age = 8.7 years). Exploratory analyses also showed a gene-by-environment effect, with VFD-reared macaques with a single short allele of the serotonin transporter gene exhibiting larger amygdala volume compared to VFD-reared subjects with only the long allele and normally reared controls.Conclusion: These data suggest that the left amygdala exhibits hypertrophy after ELS, particularly in association with the serotonin transporter gene, and that amygdala volume variation occurs in concert with other key stress-related behavioral and neurobiological parameters observed across the lifecycle. Future research is required to understand the mechanisms underlying these diverse and persistent changes associated with ELS and amygdala volume.National Institute for Mental HealthNIMHNARSAD Mid-investigator AwardSuny Downstate Med Ctr, Dept Psychiat & Behav Sci, Brooklyn, NY 11203 USAUniversidade Federal de São Paulo, Dept Psiquiatria, São Paulo, BrazilMt Sinai Sch Med, Dept Psychiat, New York, NY USAMt Sinai Sch Med, Dept Neurosci, New York, NY USAMt Sinai Sch Med, Dept Radiol, New York, NY USANew York State Psychiat Inst & Hosp, New York, NY 10032 USAMichael E Debakey VA Med Ctr, Mental Hlth Care Line, Houston, TX USABaylor Coll Med, Menninger Dept Psychiat & Behav Sci, Houston, TX 77030 USAYale Univ, Sch Med, Dept Psychiat, New Haven, CT USANatl Ctr PTSD, Clin Neurosci Div, West Haven, CT USANew York State Psychiat Inst & Hosp, Dept Mol Imaging & Neuropathol, New York, NY 10032 USAColumbia Univ, Coll Phys & Surg, Dept Psychiat, New York, NY USAColumbia Univ, Coll Phys & Surg, Dept Pathol & Cell Biol, New York, NY USAComprehensive NeuroSci Corp, Westchester, NY USAUniv Miami Hlth Sytems, Dept Psychiat & Behav Sci, Miami, FL USAEmory Univ, Sch Med, Dept Psychiat & Behav Sci, Emory, GA USAUniversidade Federal de São Paulo, Dept Psiquiatria, São Paulo, BrazilNational Institute for Mental Health: R01MH65519-01National Institute for Mental Health: R01MH098073NIMH: R21MH066748NIMH: R01MH59990AWeb of Scienc

Necessity of Hippocampal Neurogenesis for the Therapeutic Action of Antidepressants in Adult Nonhuman Primates

Rodent studies show that neurogenesis is necessary for mediating the salutary effects of antidepressants. Nonhuman primate (NHP) studies may bridge important rodent findings to the clinical realm since NHP-depression shares significant homology with human depression and kinetics of primate neurogenesis differ from those in rodents. After demonstrating that antidepressants can stimulate neurogenesis in NHPs, our present study examines whether neurogenesis is required for antidepressant efficacy in NHPs. MATERIALS/METHODOLOGY: Adult female bonnets were randomized to three social pens (N = 6 each). Pen-1 subjects were exposed to control-conditions for 15 weeks with half receiving the antidepressant fluoxetine and the rest receiving saline-placebo. Pen-2 subjects were exposed to 15 weeks of separation-stress with half receiving fluoxetine and half receiving placebo. Pen-3 subjects 2 weeks of irradiation (N = 4) or sham-irradiation (N = 2) and then exposed to 15 weeks of stress and fluoxetine. Dependent measures were weekly behavioral observations and postmortem neurogenesis levels.Exposing NHPs to repeated separation stress resulted in depression-like behaviors (anhedonia and subordinance) accompanied by reduced hippocampal neurogenesis. Treatment with fluoxetine stimulated neurogenesis and prevented the emergence of depression-like behaviors. Ablation of neurogenesis with irradiation abolished the therapeutic effects of fluoxetine. Non-stressed controls had normative behaviors although the fluoxetine-treated controls had higher neurogenesis rates. Across all groups, depression-like behaviors were associated with decreased rates of neurogenesis but this inverse correlation was only significant for new neurons in the anterior dentate gyrus that were at the threshold of completing maturation.We provide evidence that induction of neurogenesis is integral to the therapeutic effects of fluoxetine in NHPs. Given the similarity between monkeys and humans, hippocampal neurogenesis likely plays a similar role in the treatment of clinical depression. Future studies will examine several outstanding questions such as whether neuro-suppression is sufficient for producing depression and whether therapeutic neuroplastic effects of fluoxetine are specific to antidepressants

Proteomics Research in Schizophrenia

Despite intense scientific efforts, the neuropathology and pathophysiology of schizophrenia are poorly understood. Proteomic studies, by testing large numbers of proteins for associations with disease, may contribute to the understanding of the molecular mechanisms of schizophrenia. They may also indicate the types and locations of cells most likely to harbor pathological alterations. Investigations using proteomic approaches have already provided much information on quantitative and qualitative protein patterns in postmortem brain tissue, peripheral tissues and body fluids. Different proteomic technologies such as 2-D PAGE, 2-D DIGE, SELDI-TOF, shotgun proteomics with label-based (ICAT) and label-free (MSE) quantification have been applied to the study of schizophrenia for the past 15 years. In this article, we compare proteomic studies of schizophrenia with reports of quantitative studies of cell-type specific expression. This review summarizes the results, mostly from brain but also from other tissues and bodily fluids, of proteomics studies in schizophrenia. Emphasis is given to proteomics platforms, varying sources of material, proposed candidate biomarkers emerging from comparative proteomics studies, and the specificity of the putative markers in terms of other mental illnesses. We also compare proteins altered in schizophrenia with reports of protein or mRNA sequences that are relatively enriched in specific cell types. While proteomic studies of schizophrenia find abnormalities in the expression of many proteins that are not cell type-specific, there appears to be a disproportionate representation of proteins whose synthesis and localization are highly enriched in one or more brain cell type compared with other types of brain cells. Two of the three proteins most commonly altered in schizophrenia are aldolase C and glial fibrillary acidic protein, astrocytic proteins with entirely different functions, but the studies are approximately evenly divided with regard to the direction of the differences and the concordance or discordance between the two proteins. Alterations of common myelin-associated proteins were also frequently observed, and in four studies that identified alterations in at least two, all differences were downwards in schizophrenia, consistent with earlier studies examining RNA or targeting myelin-associated proteins

Detection of G Protein-Coupled Receptor Complexes in Postmortem Human Brain by Proximity Ligation Assay

Combining immunological and molecular biological methods, the antibody-based proximity ligation assay (PLA) has been used for more than a decade to detect and quantify protein-protein interactions, protein modification, and protein expression in situ, including in brain tissue. However, the transfer of this technology to human brain samples requires a number of precautions due to the nature of the specimens and their specific processing. Here, we used the PLA brightfield detection technique to assess the expression of dopamine D2 receptor and adenosine A2A receptor and their proximity in human postmortem brains, and we developed a systematic random sampling method to help quantify the PLA signals. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Sample preparation and sectioning for PLA_BF Basic Protocol 2: PLA_BF staining of brain tissue Basic Protocol 3: Image acquisition and result analysis Support Protocol: Luxol fast blue/cresyl violet staining