168 results on '"Hamilton PJ"'
Search Results
2. The use of an all oral chemotherapy (idarubicin and etoposide) in the treatment of acute myeloid leukaemia in the elderly: a report of toxicity and efficacy
- Author
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Jackson, GH, Taylor, PRA, Iqbal, A, Galloway, MJ, Turner, G, Haynes, A, Hamilton, PJ, Russell, N, and Proctor, SJ
- Published
- 1997
- Full Text
- View/download PDF
3. Acute lymphoblastic leukemia in patients aged 60 years and over: a population-based study of incidence and outcome
- Author
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Taylor, PR, primary, Reid, MM, additional, Bown, N, additional, Hamilton, PJ, additional, and Proctor, SJ, additional
- Published
- 1992
- Full Text
- View/download PDF
4. Autologous bone marrow transplantation for high-grade lymphoid malignancy using melphalan/irradiation conditioning without marrow purging or cryopreservation. The Northern Regional Bone Marrow Transplant Group
- Author
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Carey, PJ, primary, Proctor, SJ, additional, Taylor, P, additional, and Hamilton, PJ, additional
- Published
- 1991
- Full Text
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5. Fatal dissemination of cytomegalovirus after bone marrow transplantation.
- Author
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Craft, AW, Hamilton, PJ, McQuillin, J, Scott, DJ, and Walker, W
- Abstract
The clinical course and necropsy findings are described of an 11-year-old child found to have active cytomegalovirus infection at the time of bone marrow transplantation for acute lymphoblastic leukaemia. Attention is drawn to the presence of primitive mononuclear cells of uncertain origin in the regenerating bone marrow. [ABSTRACT FROM PUBLISHER]
- Published
- 1981
6. Volunteer blood donors who fail the copper sulfate screening test. What does failure mean, and what should be done?
- Author
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Lloyd, H, primary, Collins, A, additional, Walker, W, additional, Fail, B, additional, and Hamilton, PJ, additional
- Published
- 1988
- Full Text
- View/download PDF
7. Cell-Type-Specific Regulation of Cocaine Reward by the E2F3a Transcription Factor in Nucleus Accumbens.
- Author
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Martínez-Rivera FJ, Yim YY, Godino A, Minier-Toribio A, Tofani S, Holt LM, Torres-Berrío A, Futamura R, Browne CJ, Markovic T, Hamilton PJ, Neve RL, and Nestler EJ
- Abstract
The development of drug addiction is characterized by molecular changes in brain reward regions that lead to the transition from recreational to compulsive drug use. These neurobiological processes in brain reward regions, such as the nucleus accumbens (NAc), are orchestrated in large part by transcriptional regulation. Our group recently identified the transcription factor E2F3a as a novel regulator of cocaine's rewarding effects and gene expression regulation in the NAc of male mice. Despite this progress, no information is available about the role of E2F3a in regulating cocaine reward at the sex- and cell-specific levels. Here, we used male and female mice expressing Cre-recombinase in either D1- or D2-type medium spiny neurons (MSNs) combined with viral-mediated gene transfer to bidirectionally control levels of E2F3a in a cell-type-specific manner in the NAc during conditioned place preference (CPP) to cocaine. Our findings show that selective overexpression of E2F3a in D1-MSNs increased cocaine CPP in both male and female mice, whereas opposite effects were observed under knockdown conditions. In contrast, equivalent E2F3a manipulations in D2-MSNs had no significant effects. To further explore the role of E2F3a in sophisticated operant and motivated behaviors, we performed viral manipulations of all NAc neurons in combination with cocaine self-administration and behavioral economics procedures in rats and demonstrated that E2F3a regulates sensitivity aspects of cocaine seeking and taking. These results confirm E2F3a as a central substrate of cocaine reward and demonstrate that this effect is mediated in D1-MSNs, thereby providing increased knowledge of cocaine action at the transcriptional level.
- Published
- 2024
- Full Text
- View/download PDF
8. Histone H1x in mouse ventral hippocampus associates with, but does not cause behavioral adaptations to stress.
- Author
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Kim RK, Truby NL, Silva GM, Picone JA, Miller CS, Baldwin AN, Neve RL, Cui X, and Hamilton PJ
- Subjects
- Animals, Male, Mice, Adaptation, Psychological physiology, Resilience, Psychological, Social Defeat, Anxiety metabolism, Hippocampus metabolism, Stress, Psychological metabolism, Mice, Inbred C57BL, Histones metabolism, Behavior, Animal physiology
- Abstract
Prior research has identified differential protein expression levels of linker histone H1x within the ventral hippocampus (vHipp) of stress-susceptible versus stress-resilient mice. These mice are behaviorally classified based on their divergent responses to chronic social stress. Here, we sought to determine whether elevated vHipp H1x protein levels directly contribute to these diverging behavioral adaptations to stress. First, we demonstrated that stress-susceptible mice uniquely express elevated vHipp H1x protein levels following chronic stress. Given that linker histones coordinate heterochromatin compaction, we hypothesize that elevated levels of H1x in the vHipp may impede pro-resilience transcriptional adaptations and prevent development of the resilient phenotype following social stress. To test this, 8-10-week-old male C57BL/6 J mice were randomly assigned to groups undergoing 10 days of chronic social defeat stress (CSDS) or single housing, respectively. Following CSDS, mice were classified as susceptible versus resilient based on their social interaction behaviors. We synthesized a viral overexpression (OE) vector for H1x and transduced all stressed and single housed mice with either H1x or control GFP within vHipp. Following viral delivery, we conducted social, anxiety-like, and memory-reliant behavior tests on distinct cohorts of mice. We found no behavioral adaptations following H1x OE compared to GFP controls in susceptible, resilient, or single housed mice. In sum, although we confirm elevated vHipp protein levels of H1x associate with susceptibility to social stress, we observe no significant behavioral consequence of H1x OE. Thus, we conclude elevated levels of H1x are associated with, but are not singularly sufficient to drive development of behavioral adaptations to stress., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
9. Sex-Specific Regulation of Stress Susceptibility by the Astrocytic Gene Htra1 .
- Author
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Parise EM, Gyles TM, Godino A, Sial OK, Browne CJ, Parise LF, Torres-Berrío A, Salery M, Durand-de Cuttoli R, Rivera MT, Cardona-Acosta AM, Holt L, Markovic T, van der Zee YY, Lorsch ZS, Cathomas F, Garon JB, Teague C, Issler O, Hamilton PJ, Bolaños-Guzmán CA, Russo SJ, and Nestler EJ
- Abstract
Major depressive disorder (MDD) is linked to impaired structural and synaptic plasticity in limbic brain regions. Astrocytes, which regulate synapses and are influenced by chronic stress, likely contribute to these changes. We analyzed astrocyte gene profiles in the nucleus accumbens (NAc) of humans with MDD and mice exposed to chronic stress. Htra1 , which encodes an astrocyte-secreted protease targeting the extracellular matrix (ECM), was significantly downregulated in the NAc of males but upregulated in females in both species. Manipulating Htra1 in mouse NAc astrocytes bidirectionally controlled stress susceptibility in a sex-specific manner. Such Htra1 manipulations also altered neuronal signaling and ECM structural integrity in NAc. These findings highlight astroglia and the brain's ECM as key mediators of sex-specific stress vulnerability, offering new approaches for MDD therapies.
- Published
- 2024
- Full Text
- View/download PDF
10. A zinc finger transcription factor enables social behaviors while controlling transposable elements and immune response in prefrontal cortex.
- Author
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Truby NL, Kim RK, Silva GM, Qu X, Picone JA, Alemu R, Atiyeh CN, Neve RL, Liu J, Cui X, and Hamilton PJ
- Subjects
- Mice, Animals, Prefrontal Cortex metabolism, Social Behavior, Zinc Fingers genetics, Rodentia genetics, Rodentia metabolism, Immunity, Transcription Factors genetics, DNA Transposable Elements
- Abstract
The neurobiological origins of social behaviors are incompletely understood. Here we utilized synthetic biology approaches to reprogram the function of ZFP189, a transcription factor whose expression and function in rodent prefrontal cortex was previously demonstrated to be protective against stress-induced social deficits. We created novel synthetic ZFP189 transcription factors including ZFP189
VPR , which activates the transcription of target genes and therefore exerts opposite functional control from the endogenous, transcriptionally repressive ZFP189WT . Following viral delivery of these synthetic ZFP189 transcription factors to mouse prefrontal cortex, we observe that ZFP189-mediated transcriptional control promotes mature dendritic spine morphology on transduced pyramidal neurons. Interestingly, inversion of ZFP189-mediated transcription in this brain area, achieved by viral delivery of synthetic ZFP189VPR , precipitates social behavioral deficits in terms of social interaction, motivation, and the cognition necessary for the maintenance of social hierarchy, without other observable behavioral deficits. RNA sequencing of virally manipulated prefrontal cortex tissues reveals that ZFP189 transcription factors of opposing regulatory function (ZFP189WT versus ZFP189VPR ) have opposite influence on the expression of genetic transposable elements as well as genes that participate in adaptive immune functions. Collectively, this work reveals that ZFP189 function in the prefrontal cortex coordinates structural and transcriptional neuroadaptations necessary for complex social behaviors while regulating transposable element-rich regions of DNA and the expression of immune-related genes. Given the evidence for a co-evolution of social behavior and the brain immune response, we posit that ZFP189 may have evolved to augment brain transposon-associated immune function as a way of enhancing an animal's capacity for functioning in social groups., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
11. Neuroepigenetic Editing.
- Author
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Hamilton PJ, Lim CJ, Nestler EJ, and Heller EA
- Subjects
- Animals, Humans, Neurons metabolism, Brain metabolism, Histones metabolism, Chromatin genetics, Chromatin metabolism, CRISPR-Cas Systems, High-Throughput Nucleotide Sequencing methods, Epigenesis, Genetic, Gene Editing methods
- Abstract
Epigenetic regulation is intrinsic to basic neurobiological function as well as neurological disease. Regulation of chromatin-modifying enzymes in the brain is critical during both development and adulthood and in response to external stimuli. Biochemical studies are complemented by numerous next-generation sequencing (NGS) studies that quantify global changes in gene expression, chromatin accessibility, histone and DNA modifications in neurons and glial cells. Neuroepigenetic editing tools are essential to distinguish between the mere presence and functional relevance of histone and DNA modifications to gene transcription in the brain and animal behavior. This review discusses current advances in neuroepigenetic editing, highlighting methodological considerations pertinent to neuroscience, such as delivery methods and the spatiotemporal specificity of editing and it demonstrates the enormous potential of epigenetic editing for basic neurobiological research and therapeutic application., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2024
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- View/download PDF
12. Stereotaxic Surgery as a Method to Deliver Epigenetic Editing Constructs in Rodent Brain.
- Author
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Heller EA and Hamilton PJ
- Subjects
- Animals, Mice, Rats, Genetic Vectors genetics, Genetic Vectors administration & dosage, Gene Editing methods, Brain metabolism, Epigenesis, Genetic, Stereotaxic Techniques, Gene Transfer Techniques
- Abstract
Modern neuroscience research is increasingly discovering that alterations in epigenetic states within key brain cells is correlated with brain diseases. These epigenetic alterations may include changes in histone post-translational modifications and/or DNA modifications, all of which affect transcription and other gene expression programs within the brain cells that comprise central brain regions. However, the exact causal contribution of these epigenome changes to brain disease cannot be elucidated in the absence of direct in vivo manipulations in the implicated brain areas. Combining the design and creation of epigenetic editing constructs, gene delivery strategies, and stereotaxic surgery enables neuroscience researchers to target and manipulate the epigenetic state of the brain cells of laboratory rodents in a locus-specific manner and test its causal contribution to disease-related pathology and behaviors. Here, we describe the surgical protocol utilized by our group and others, which is optimized for herpes simplex virus delivery into the mouse brain, although the protocol outlined herein could be applied for delivery of adeno-associated viruses, lentiviruses, or nonviral gene-delivery methods in both mice and rats. The method allows for the overexpression of engineered DNA-binding proteins for direct and targeted epigenome editing in rodent brain with excellent spatiotemporal control. Nearly any brain region of interest can be targeted in rodents at every stage of postnatal life. Owing to the versatility, reproducibility, and utility of this technique, it is an important method for any laboratory interested in studying the cellular, circuit, and behavioral consequences of manipulating the brain epigenome in laboratory rodents., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2024
- Full Text
- View/download PDF
13. Histone H1x in mouse ventral hippocampus correlates with, but does not cause behavioral adaptations to stress.
- Author
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Kim RK, Truby NL, Silva GM, Picone JA, Miller CS, Neve RL, Cui X, and Hamilton PJ
- Abstract
Prior research has identified differential protein expression levels of linker histone H1x within the ventral hippocampus (vHipp) of stress-susceptible versus stress-resilient mice. These mice are behaviorally classified based on their divergent responses to chronic social stress. Here, we sought to determine whether elevated vHipp H1x protein levels directly contribute to these diverging behavioral adaptations to stress. First, we demonstrate that stress-susceptible mice uniquely express elevated vHipp H1x protein levels following chronic stress. Given that linker histones coordinate heterochromatin compaction, we hypothesize that elevated levels of H1x in the vHipp may impede pro-resilience transcriptional adaptations and prevent development of the resilient phenotype following social stress. To test this, 8-10-week-old male C57BL/6J mice were randomly assigned to stressed and unstressed groups undergoing 10 days of chronic social defeat stress (CSDS) or single housing respectively. Following CSDS, mice were classified as susceptible versus resilient based on their social interaction behaviors. We synthesized a viral overexpression (OE) vector for H1x and transduced experimental mice with either H1x or control GFP within vHipp. Following viral delivery, we conducted social, anxiety-like, and memory-reliant behavior tests on distinct cohorts of mice. We found no behavioral adaptations following H1x OE compared to GFP controls in susceptible, resilient, or unstressed mice. In sum, although we confirm vHipp protein levels of H1x correlate with susceptibility to social stress, we observe no significant behavioral consequence of H1x OE. Thus, we conclude elevated levels of H1x are correlated with, but are not singularly sufficient to drive development of behavioral adaptations to stress.
- Published
- 2023
- Full Text
- View/download PDF
14. Comparison of three DREADD agonists acting on Gq-DREADDs in the ventral tegmental area to alter locomotor activity in tyrosine hydroxylase:Cre male and female rats.
- Author
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Robinson HL, Nicholson KL, Shelton KL, Hamilton PJ, and Banks ML
- Subjects
- Animals, Female, Male, Rats, Dextroamphetamine, Ligands, Locomotion, Rats, Sprague-Dawley, Tyrosine 3-Monooxygenase metabolism, Clozapine pharmacology, Clozapine analogs & derivatives, Imidazoles, Sulfonamides, Thiophenes, Ventral Tegmental Area metabolism
- Abstract
Rationale: Despite the increasingly pervasive use of chemogenetic tools in preclinical neuroscience research, the in vivo pharmacology of DREADD agonists remains poorly understood. The pharmacological effects of any ligand acting at receptors, engineered or endogenous, are influenced by numerous factors including potency, time course, and receptor selectivity. Thus, rigorous comparison of the potency and time course of available DREADD ligands may provide an empirical foundation for ligand selection., Objectives: Compare the behavioral pharmacology of three different DREADD ligands clozapine-N-oxide (CNO), compound 21 (C21), and deschloroclozapine (DCZ) in a locomotor activity assay in tyrosine hydroxylase:cre recombinase (TH:Cre) male and female rats., Methods: Locomotor activity in nine adult TH:Cre Sprague-Dawley rats (5 female, 4 male) was monitored for two hours following administration of d-amphetamine (vehicle, 0.1-3.2 mg/kg, IP), DCZ (vehicle, 0.32-320 µg/kg, IP), CNO (vehicle, 0.32-10 mg/kg), and C21 (vehicle, 0.1-3.2 mg/kg, IP). Behavioral sessions were conducted twice per week prior to and starting three weeks after bilateral intra-VTA hM3Dq DREADD virus injection., Results: d-Amphetamine significantly increased locomotor activity pre- and post-DREADD virus injection. DCZ, CNO, and C21 did not alter locomotor activity pre-DREADD virus injection. There was no significant effect of DCZ, CNO, and C21 on locomotor activity post-DREADD virus injection; however, large individual differences in both behavioral response and receptor expression were observed., Conclusions: Large individual variability was observed in both DREADD agonist behavioral effects and receptor expression. These results suggest further basic research would facilitate the utility of these chemogenetic tools for behavioral neuroscience research., (Copyright © 2023 Elsevier B.V. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
15. Functional connectivity signatures of major depressive disorder: machine learning analysis of two multicenter neuroimaging studies.
- Author
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Gallo S, El-Gazzar A, Zhutovsky P, Thomas RM, Javaheripour N, Li M, Bartova L, Bathula D, Dannlowski U, Davey C, Frodl T, Gotlib I, Grimm S, Grotegerd D, Hahn T, Hamilton PJ, Harrison BJ, Jansen A, Kircher T, Meyer B, Nenadić I, Olbrich S, Paul E, Pezawas L, Sacchet MD, Sämann P, Wagner G, Walter H, Walter M, and van Wingen G
- Subjects
- Humans, Brain Mapping methods, Magnetic Resonance Imaging, Neural Pathways, Brain pathology, Neuroimaging, Depressive Disorder, Major
- Abstract
The promise of machine learning has fueled the hope for developing diagnostic tools for psychiatry. Initial studies showed high accuracy for the identification of major depressive disorder (MDD) with resting-state connectivity, but progress has been hampered by the absence of large datasets. Here we used regular machine learning and advanced deep learning algorithms to differentiate patients with MDD from healthy controls and identify neurophysiological signatures of depression in two of the largest resting-state datasets for MDD. We obtained resting-state functional magnetic resonance imaging data from the REST-meta-MDD (N = 2338) and PsyMRI (N = 1039) consortia. Classification of functional connectivity matrices was done using support vector machines (SVM) and graph convolutional neural networks (GCN), and performance was evaluated using 5-fold cross-validation. Features were visualized using GCN-Explainer, an ablation study and univariate t-testing. The results showed a mean classification accuracy of 61% for MDD versus controls. Mean accuracy for classifying (non-)medicated subgroups was 62%. Sex classification accuracy was substantially better across datasets (73-81%). Visualization of the results showed that classifications were driven by stronger thalamic connections in both datasets, while nearly all other connections were weaker with small univariate effect sizes. These results suggest that whole brain resting-state connectivity is a reliable though poor biomarker for MDD, presumably due to disease heterogeneity as further supported by the higher accuracy for sex classification using the same methods. Deep learning revealed thalamic hyperconnectivity as a prominent neurophysiological signature of depression in both multicenter studies, which may guide the development of biomarkers in future studies., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
16. Transcriptional signatures of heroin intake and relapse throughout the brain reward circuitry in male mice.
- Author
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Browne CJ, Futamura R, Minier-Toribio A, Hicks EM, Ramakrishnan A, Martínez-Rivera FJ, Estill M, Godino A, Parise EM, Torres-Berrío A, Cunningham AM, Hamilton PJ, Walker DM, Huckins LM, Hurd YL, Shen L, and Nestler EJ
- Subjects
- Humans, Mice, Male, Animals, Genome-Wide Association Study, Brain, Reward, Recurrence, Heroin adverse effects, Opioid-Related Disorders
- Abstract
Opioid use disorder (OUD) looms as one of the most severe medical crises facing society. More effective therapeutics will require a deeper understanding of molecular changes supporting drug-taking and relapse. Here, we develop a brain reward circuit-wide atlas of opioid-induced transcriptional regulation by combining RNA sequencing (RNA-seq) and heroin self-administration in male mice modeling multiple OUD-relevant conditions: acute heroin exposure, chronic heroin intake, context-induced drug-seeking following abstinence, and relapse. Bioinformatics analysis of this rich dataset identified numerous patterns of transcriptional regulation, with both region-specific and pan-circuit biological domains affected by heroin. Integration of RNA-seq data with OUD-relevant behavioral outcomes uncovered region-specific molecular changes and biological processes that predispose to OUD vulnerability. Comparisons with human OUD RNA-seq and genome-wide association study data revealed convergent molecular abnormalities and gene candidates with high therapeutic potential. These studies outline molecular reprogramming underlying OUD and provide a foundational resource for future investigations into mechanisms and treatment strategies.
- Published
- 2023
- Full Text
- View/download PDF
17. Transcriptional control of nucleus accumbens neuronal excitability by retinoid X receptor alpha tunes sensitivity to drug rewards.
- Author
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Godino A, Salery M, Durand-de Cuttoli R, Estill MS, Holt LM, Futamura R, Browne CJ, Mews P, Hamilton PJ, Neve RL, Shen L, Russo SJ, and Nestler EJ
- Subjects
- Mice, Male, Female, Animals, Nucleus Accumbens metabolism, Retinoid X Receptor alpha genetics, Retinoid X Receptor alpha metabolism, Neurons physiology, Receptors, Dopamine D1 metabolism, Reward, Mice, Inbred C57BL, Cocaine pharmacology, Mental Disorders metabolism
- Abstract
The complex nature of the transcriptional networks underlying addictive behaviors suggests intricate cooperation between diverse gene regulation mechanisms that go beyond canonical activity-dependent pathways. Here, we implicate in this process a nuclear receptor transcription factor, retinoid X receptor alpha (RXRα), which we initially identified bioinformatically as associated with addiction-like behaviors. In the nucleus accumbens (NAc) of male and female mice, we show that although its own expression remains unaltered after cocaine exposure, RXRα controls plasticity- and addiction-relevant transcriptional programs in both dopamine receptor D1- and D2-expressing medium spiny neurons, which in turn modulate intrinsic excitability and synaptic activity of these NAc cell types. Behaviorally, bidirectional viral and pharmacological manipulation of RXRα regulates drug reward sensitivity in both non-operant and operant paradigms. Together, this study demonstrates a key role for NAc RXRα in promoting drug addiction and paves the way for future studies of rexinoid signaling in psychiatric disease states., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
18. A zinc finger transcription factor tunes social behaviors by controlling transposable elements and immune response in prefrontal cortex.
- Author
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Truby NL, Kim RK, Silva GM, Qu X, Picone JA, Alemu R, Neve RL, Cui X, Liu J, and Hamilton PJ
- Abstract
The neurobiological origins of social behaviors are incompletely understood. Here we utilized synthetic biology approaches to reprogram the function of ZFP189, a transcription factor whose expression and function in the rodent prefrontal cortex was previously determined to be protective against stress-induced social deficits. We created novel synthetic ZFP189 transcription factors including ZFP189
VPR , which activates the transcription of target genes and therefore exerts opposite functional control from the endogenous, transcriptionally repressive ZFP189WT . Upon viral delivery of these synthetic ZFP189 transcription factors to mouse prefrontal cortex, we observe that ZFP189-mediated transcriptional control promotes mature dendritic spine morphology on transduced pyramidal neurons. Interestingly, dysregulation of ZFP189-mediated transcription in this brain area, achieved by delivery of synthetic ZFP189VPR , precipitates social behavioral deficits in terms of social interaction, motivation, and the cognition necessary for the maintenance of social hierarchy, without other observable behavioral deficits. By performing RNA sequencing in virally manipulated prefrontal cortex tissues, we discover that ZFP189 transcription factors of opposing regulatory function have opposite influence on the expression of genetic transposable elements as well as genes that participate in immune functions. Collectively, this work reveals that ZFP189 function in the prefrontal cortex coordinates structural and transcriptional neuroadaptations necessary for social behaviors by binding transposable element-rich regions of DNA to regulate immune-related genes. Given the evidence for a co-evolution of social behavior and the brain immune response, we posit that ZFP189 may have evolved to augment brain transposon-associated immune function as a way of enhancing an animal's capacity for functioning in social groups.- Published
- 2023
- Full Text
- View/download PDF
19. CREB Binding at the Zfp189 Promoter Within Medium Spiny Neuron Subtypes Differentially Regulates Behavioral and Physiological Adaptations Over the Course of Cocaine Use.
- Author
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Teague CD, Picone JA, Wright WJ, Browne CJ, Silva GM, Futamura R, Minier-Toribio A, Estill ME, Ramakrishnan A, Martinez-Rivera FJ, Godino A, Parise EM, Schmidt KH, Pulido NV, Lorsch ZS, Kim JH, Shen L, Neve RL, Dong Y, Nestler EJ, and Hamilton PJ
- Subjects
- Animals, Mice, Mice, Inbred C57BL, Mice, Transgenic, Nucleus Accumbens, Receptors, Dopamine D1 genetics, Receptors, Dopamine D1 metabolism, Adaptation, Physiological genetics, Cocaine pharmacology, Cocaine metabolism, Cocaine-Related Disorders genetics, Medium Spiny Neurons metabolism, Promoter Regions, Genetic, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
Background: Over the course of chronic drug use, brain transcriptional neuroadaptation is thought to contribute to a change in drug use behavior over time. The function of the transcription factor CREB (cAMP response element binding protein) within the nucleus accumbens (NAc) has been well documented in opposing the rewarding properties of many classes of drugs, yet the gene targets through which CREB causally manifests these lasting neuroadaptations remain unknown. Here, we identify zinc finger protein 189 (Zfp189) as a CREB target gene that is transcriptionally responsive to acute and chronic cocaine use within the NAc of mice., Methods: To investigate the role of the CREB-Zfp189 interaction in cocaine use, we virally delivered modified clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 constructs capable of selectively localizing CREB to the Zfp189 gene promoter in the NAc of mice., Results: We observed that CREB binding to the Zfp189 promoter increased Zfp189 expression and diminished the reinforcing responses to cocaine. Furthermore, we showed that NAc Zfp189 expression increased within D1 medium spiny neurons in response to acute cocaine but increased in both D1- and D2-expressing medium spiny neurons in response to chronic cocaine. CREB-mediated induction of Zfp189 potentiated electrophysiological activity of D1- and D2-expressing medium spiny neurons, recapitulating the known effect of CREB on these neurons. Finally, targeting CREB to the Zfp189 promoter within NAc Drd2-expressing neurons, but not Drd1-expressing neurons, was sufficient to diminish cocaine-conditioned behaviors., Conclusions: Together, these findings point to the CREB-Zfp189 interaction within the NAc Drd2+ neurons as a molecular signature of chronic cocaine use that is causal in counteracting the reinforcing effects of cocaine., (Copyright © 2022 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
20. Transcriptional signatures of heroin intake and seeking throughout the brain reward circuit.
- Author
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Browne CJ, Futamura R, Minier-Toribio A, Hicks EM, Ramakrishnan A, Martínez-Rivera F, Estill M, Godino A, Parise EM, Torres-Berrío A, Cunningham AM, Hamilton PJ, Walker DM, Huckins LM, Hurd YL, Shen L, and Nestler EJ
- Abstract
Opioid use disorder (OUD) looms as one of the most severe medical crises currently facing society. More effective therapeutics for OUD requires in-depth understanding of molecular changes supporting drug-taking and relapse. Recent efforts have helped advance these aims, but studies have been limited in number and scope. Here, we develop a brain reward circuit-wide atlas of opioid-induced transcriptional regulation by combining RNA sequencing (RNAseq) and heroin self-administration in male mice modeling multiple OUD-relevant conditions: acute heroin exposure, chronic heroin intake, context-induced drug-seeking following prolonged abstinence, and heroin-primed drug-seeking (i.e., "relapse"). Bioinformatics analysis of this rich dataset identified numerous patterns of molecular changes, transcriptional regulation, brain-region-specific involvement in various aspects of OUD, and both region-specific and pan-circuit biological domains affected by heroin. Integrating RNAseq data with behavioral outcomes using factor analysis to generate an "addiction index" uncovered novel roles for particular brain regions in promoting addiction-relevant behavior, and implicated multi-regional changes in affected genes and biological processes. Comparisons with RNAseq and genome-wide association studies from humans with OUD reveal convergent molecular regulation that are implicated in drug-taking and relapse, and point to novel gene candidates with high therapeutic potential for OUD. These results outline broad molecular reprogramming that may directly promote the development and maintenance of OUD, and provide a foundational resource to the field for future research into OUD mechanisms and treatment strategies.
- Published
- 2023
- Full Text
- View/download PDF
21. Enhancing Endocannabinoid Signaling via β-Catenin in the Nucleus Accumbens Attenuates PTSD- and Depression-like Behavior of Male Rats.
- Author
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Mizrachi Zer-Aviv T, Islami L, Hamilton PJ, Parise EM, Nestler EJ, Sbarski B, and Akirav I
- Abstract
Inhibition of fatty acid amide hydrolase (FAAH), which increases anandamide levels, has been suggested as a potential treatment for stress-related conditions. We examined whether the stress-preventing effects of the FAAH inhibitor URB597 on behavior are mediated via β-catenin in the nucleus accumbens (NAc). Male rats were exposed to the shock and reminders model of PTSD and then treated with URB597 (0.4 mg/kg; i.p.). They were tested for anxiety- (freezing, startle response), depression-like behaviors (despair, social preference, anhedonia), and memory function (T-maze, social recognition). We also tested the involvement of the CB1 receptor (CB1r), β-catenin, and metabotropic glutamate receptor subtype 5 (mGluR5) proteins. URB597 prevented the shock- and reminders-induced increase in anxiety- and depressive-like behaviors, as well as the impaired memory via the CB1r-dependent mechanism. In the NAc, viral-mediated β-catenin overexpression restored the behavior of rats exposed to stress and normalized the alterations in protein levels in the NAc and the prefrontal cortex. Importantly, when NAc β-catenin levels were downregulated by viral-mediated gene transfer, the therapeutic-like effects of URB597 were blocked. We suggest a potentially novel mechanism for the therapeutic-like effects of FAAH inhibition that is dependent on β-catenin activation in the NAc in a PTSD rat model., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
- Published
- 2022
- Full Text
- View/download PDF
22. Gene-Targeted, CREB-Mediated Induction of ΔFosB Controls Distinct Downstream Transcriptional Patterns Within D1 and D2 Medium Spiny Neurons.
- Author
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Lardner CK, van der Zee Y, Estill MS, Kronman HG, Salery M, Cunningham AM, Godino A, Parise EM, Kim JH, Neve RL, Shen L, Hamilton PJ, and Nestler EJ
- Subjects
- Animals, Female, Gene Expression, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Neurons metabolism, Receptors, Dopamine D1 metabolism, Cocaine, Nucleus Accumbens metabolism
- Abstract
Background: The onset and persistence of addiction phenotypes are, in part, mediated by transcriptional mechanisms in the brain that affect gene expression and, subsequently, neural circuitry. ΔFosB is a transcription factor that accumulates in the nucleus accumbens (NAc)-a brain region responsible for coordinating reward and motivation-after exposure to virtually every known rewarding substance, including cocaine and opioids. ΔFosB has also been shown to directly control gene transcription and behavior downstream of both cocaine and opioid exposure, but with potentially different roles in D1 and D2 medium spiny neurons (MSNs) in NAc., Methods: To clarify MSN subtype-specific roles for ΔFosB and investigate how these coordinate the actions of distinct classes of addictive drugs in NAc, we developed a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-based epigenome editing tool to induce endogenous ΔFosB expression in vivo in the absence of drug exposure. After inducing ΔFosB in D1- or D2-MSNs or both, we performed RNA sequencing on bulk male and female NAc tissue (n = 6-8/group)., Results: We found that ΔFosB induction elicits distinct transcriptional profiles in NAc by MSN subtype and by sex, establishing for the first time that ΔFosB mediates different transcriptional effects in males versus females. We also demonstrated that changes in D1-MSNs, but not those in D2-MSNs or both, significantly recapitulate changes in gene expression induced by cocaine self-administration., Conclusions: Together, these findings demonstrate the efficacy of a novel molecular tool for studying cell type-specific transcriptional mechanisms and shed new light on the activity of ΔFosB, a critical transcriptional regulator of drug addiction., (Copyright © 2021 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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23. Chromatin-mediated alternative splicing regulates cocaine-reward behavior.
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Xu SJ, Lombroso SI, Fischer DK, Carpenter MD, Marchione DM, Hamilton PJ, Lim CJ, Neve RL, Garcia BA, Wimmer ME, Pierce RC, and Heller EA
- Subjects
- Alternative Splicing drug effects, Animals, Behavior, Addictive genetics, Behavior, Addictive psychology, Chromatin genetics, Epigenesis, Genetic drug effects, Epigenesis, Genetic physiology, Female, Male, Mice, Mice, Inbred C57BL, Self Administration, Alternative Splicing physiology, Behavior, Addictive metabolism, Chromatin metabolism, Cocaine administration & dosage, Dopamine Uptake Inhibitors administration & dosage, Reward
- Abstract
Neuronal alternative splicing is a key gene regulatory mechanism in the brain. However, the spliceosome machinery is insufficient to fully specify splicing complexity. In considering the role of the epigenome in activity-dependent alternative splicing, we and others find the histone modification H3K36me3 to be a putative splicing regulator. In this study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with the enrichment of H3K36me3 at differentially spliced junctions. Importantly, only targeted epigenetic editing can distinguish between a direct role of H3K36me3 in splicing and an indirect role via regulation of splice factor expression elsewhere on the genome. We targeted Srsf11, which was both alternatively spliced and H3K36me3 enriched in the brain following cocaine self-administration. Epigenetic editing of H3K36me3 at Srsf11 was sufficient to drive its alternative splicing and enhanced cocaine self-administration, establishing the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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24. A network of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) binding sites on the dopamine transporter regulates amphetamine behavior in Drosophila Melanogaster.
- Author
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Belovich AN, Aguilar JI, Mabry SJ, Cheng MH, Zanella D, Hamilton PJ, Stanislowski DJ, Shekar A, Foster JD, Bahar I, Matthies HJG, and Galli A
- Subjects
- Animals, Binding Sites, Drosophila melanogaster, Phosphatidylinositols, Amphetamine pharmacology, Dopamine Plasma Membrane Transport Proteins metabolism
- Abstract
Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. Here, we developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). We have previously shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP
2 ), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. In this study, we demonstrate that the interaction of PIP2 with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. We showed that PIP2 also interacts with intracellular loop 4 at R443. Further, we identified that R443 electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, we determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. We associated this inhibition with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, we showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. We present the first evidence linking PIP2 , DAT, DA efflux, and phosphorylation processes with AMPH reward., (© 2019. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2021
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25. Opioid withdrawal produces sex-specific effects on fentanyl-vs.-food choice and mesolimbic transcription.
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Townsend EA, Kim RK, Robinson HL, Marsh SA, Banks ML, and Hamilton PJ
- Abstract
Background: Opioid withdrawal is a key driver of opioid addiction and an obstacle to recovery. However, withdrawal effects on opioid reinforcement and mesolimbic neuroadaptation are understudied and the role of sex is largely unknown., Methods: Male (n=13) and female (n=12) rats responded under a fentanyl-vs.-food "choice" procedure during daily 2h sessions. In addition to the daily choice sessions, rats were provided extended access to fentanyl during 12h self-administration sessions. After two weeks of this self-administration regimen, the nucleus accumbens (NAc) and ventral tegmental area (VTA) of a subset of rats were subjected to RNA sequencing. In the remaining rats, a third week of this self-administration regimen was conducted, during which methadone effects on fentanyl-vs.-food choice were determined., Results: Prior to opioid dependence, male and female rats similarly allocated responding between fentanyl and food. Abstinence from extended fentanyl access elicited similar increases in somatic withdrawal signs in both sexes. Despite similar withdrawal signs and extended access fentanyl intake, opioid withdrawal was accompanied by a maladaptive increase in fentanyl choice in males, but not females. Behavioral sex differences corresponded with a greater number of differentially expressed genes in the NAc and VTA of opioid-withdrawn females relative to males. Methadone blocked withdrawal-associated increases in fentanyl choice in males, but failed to further decrease fentanyl choice in females., Conclusions: These results provide foundational evidence of sex-specific neuroadaptations to opioid withdrawal, which may be relevant to the female-specific resilience to withdrawal-associated increases in opioid choice and aid in the identification of novel therapeutic targets., Competing Interests: 5. Financial Disclosures The authors report no biomedical financial interests or potential conflicts of interest.
- Published
- 2021
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26. Chronic stress and antidepressant treatment alter purine metabolism and beta oxidation within mouse brain and serum.
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Hamilton PJ, Chen EY, Tolstikov V, Peña CJ, Picone JA, Shah P, Panagopoulos K, Strat AN, Walker DM, Lorsch ZS, Robinson HL, Mervosh NL, Kiraly DD, Sarangarajan R, Narain NR, Kiebish MA, and Nestler EJ
- Subjects
- Animals, Antidepressive Agents, Tricyclic pharmacology, Brain drug effects, Brain pathology, Lipidomics, Male, Mice, Serum metabolism, Brain metabolism, Imipramine pharmacology, Metabolome, Proteome analysis, Purines metabolism, Serum chemistry, Stress, Psychological physiopathology
- Abstract
Major depressive disorder (MDD) is a complex condition with unclear pathophysiology. Molecular disruptions within limbic brain regions and the periphery contribute to depression symptomatology and a more complete understanding the diversity of molecular changes that occur in these tissues may guide the development of more efficacious antidepressant treatments. Here, we utilized a mouse chronic social stress model for the study of MDD and performed metabolomic, lipidomic, and proteomic profiling on serum plus several brain regions (ventral hippocampus, nucleus accumbens, and medial prefrontal cortex) of susceptible, resilient, and unstressed control mice. To identify how commonly used tricyclic antidepressants impact the molecular composition in these tissues, we treated stress-exposed mice with imipramine and repeated our multi-OMIC analyses. Proteomic analysis identified three serum proteins reduced in susceptible animals; lipidomic analysis detected differences in lipid species between resilient and susceptible animals in serum and brain; and metabolomic analysis revealed dysfunction of purine metabolism, beta oxidation, and antioxidants, which were differentially associated with stress susceptibility vs resilience by brain region. Antidepressant treatment ameliorated stress-induced behavioral abnormalities and affected key metabolites within outlined networks, most dramatically in the ventral hippocampus. This work presents a resource for chronic social stress-induced, tissue-specific changes in proteins, lipids, and metabolites and illuminates how molecular dysfunctions contribute to individual differences in stress sensitivity.
- Published
- 2020
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27. Sex-Specific Role for the Long Non-coding RNA LINC00473 in Depression.
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Issler O, van der Zee YY, Ramakrishnan A, Wang J, Tan C, Loh YE, Purushothaman I, Walker DM, Lorsch ZS, Hamilton PJ, Peña CJ, Flaherty E, Hartley BJ, Torres-Berrío A, Parise EM, Kronman H, Duffy JE, Estill MS, Calipari ES, Labonté B, Neve RL, Tamminga CA, Brennand KJ, Dong Y, Shen L, and Nestler EJ
- Subjects
- Adult, Aged, Aged, 80 and over, Animals, Behavior, Animal, Depression genetics, Depression metabolism, Depressive Disorder, Major metabolism, Down-Regulation, Female, Humans, Male, Mice, Mice, Transgenic, Middle Aged, Neurons metabolism, RNA, Long Noncoding metabolism, RNA-Seq, Sex Factors, Stress, Psychological metabolism, Young Adult, Depressive Disorder, Major genetics, Prefrontal Cortex metabolism, RNA, Long Noncoding genetics, Resilience, Psychological, Stress, Psychological genetics
- Abstract
Depression is a common disorder that affects women at twice the rate of men. Here, we report that long non-coding RNAs (lncRNAs), a recently discovered class of regulatory transcripts, represent about one-third of the differentially expressed genes in the brains of depressed humans and display complex region- and sex-specific patterns of regulation. We identified the primate-specific, neuronal-enriched gene LINC00473 as downregulated in prefrontal cortex (PFC) of depressed females but not males. Using viral-mediated gene transfer to express LINC00473 in adult mouse PFC neurons, we mirrored the human sex-specific phenotype by inducing stress resilience solely in female mice. This sex-specific phenotype was accompanied by changes in synaptic function and gene expression selectively in female mice and, along with studies of human neuron-like cells in culture, implicates LINC00473 as a CREB effector. Together, our studies identify LINC00473 as a female-specific driver of stress resilience that is aberrant in female depression., Competing Interests: Declaration of Interests The authors declare no competing interests, (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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28. Silent synapses dictate cocaine memory destabilization and reconsolidation.
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Wright WJ, Graziane NM, Neumann PA, Hamilton PJ, Cates HM, Fuerst L, Spenceley A, MacKinnon-Booth N, Iyer K, Huang YH, Shaham Y, Schlüter OM, Nestler EJ, and Dong Y
- Subjects
- Animals, Male, Rats, Rats, Sprague-Dawley, Cocaine-Related Disorders physiopathology, Drug-Seeking Behavior physiology, Memory Consolidation physiology, Nucleus Accumbens physiopathology, Synapses physiology
- Abstract
Cocaine-associated memories are persistent, but, on retrieval, become temporarily destabilized and vulnerable to disruptions, followed by reconsolidation. To explore the synaptic underpinnings for these memory dynamics, we studied AMPA receptor (AMPAR)-silent excitatory synapses, which are generated in the nucleus accumbens by cocaine self-administration, and subsequently mature after prolonged withdrawal by recruiting AMPARs, echoing acquisition and consolidation of cocaine memories. We show that, on memory retrieval after prolonged withdrawal, the matured silent synapses become AMPAR-silent again, followed by re-maturation ~6 h later, defining the onset and termination of a destabilization window of cocaine memories. These synaptic dynamics are timed by Rac1, with decreased and increased Rac1 activities opening and closing, respectively, the silent synapse-mediated destabilization window. Preventing silent synapse re-maturation within the destabilization window decreases cue-induced cocaine seeking. Thus, cocaine-generated silent synapses constitute a discrete synaptic ensemble dictating the dynamics of cocaine-associated memories and can be targeted for memory disruption.
- Published
- 2020
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29. Epigenetics and addiction.
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Hamilton PJ and Nestler EJ
- Subjects
- Epigenomics, Humans, Reward, Behavior, Addictive, Epigenesis, Genetic, Substance-Related Disorders
- Abstract
As an individual becomes addicted to a drug of abuse, nerve cells within the brain's reward circuitry adapt at the epigenetic level during the course of repeated drug exposure. These drug-induced epigenetic adaptations mediate enduring changes in brain function which contribute to life-long, drug-related behavioral abnormalities that define addiction. Targeting these epigenetic alterations will enhance our understanding of the biological basis of addiction and might even yield more effective anti-addiction therapies. However, the complexity of the neuroepigenetic landscape makes it difficult to determine which drug-induced epigenetic changes causally contribute to the pathogenic mechanisms of drug addiction. In this review, we highlight the evidence that epigenetic modifications, specifically histone modifications, within key brain reward regions are correlated with addiction. We then discuss the emerging field of locus-specific neuroepigenetic editing, which is a promising method for determining the causal epigenetic molecular mechanisms that drive an addicted state. Such approaches will substantially increase the field's ability to establish the precise epigenetic mechanisms underlying drug addiction, and could lead to novel treatments for addictive disorders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2019
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30. Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.
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Lorsch ZS, Hamilton PJ, Ramakrishnan A, Parise EM, Salery M, Wright WJ, Lepack AE, Mews P, Issler O, McKenzie A, Zhou X, Parise LF, Pirpinias ST, Ortiz Torres I, Kronman HG, Montgomery SE, Loh YE, Labonté B, Conkey A, Symonds AE, Neve RL, Turecki G, Maze I, Dong Y, Zhang B, Shen L, Bagot RC, and Nestler EJ
- Subjects
- Animals, Gene Regulatory Networks genetics, Mice, Mice, Inbred C57BL, Prefrontal Cortex metabolism, Transcription, Genetic, Adaptation, Psychological physiology, Stress, Psychological genetics, Zinc Fingers genetics
- Abstract
Understanding the transcriptional changes that are engaged in stress resilience may reveal novel antidepressant targets. Here we use gene co-expression analysis of RNA-sequencing data from brains of resilient mice to identify a gene network that is unique to resilience. Zfp189, which encodes a previously unstudied zinc finger protein, is the highest-ranked key driver gene in the network, and overexpression of Zfp189 in prefrontal cortical neurons preferentially activates this network and promotes behavioral resilience. The transcription factor CREB is a predicted upstream regulator of this network and binds to the Zfp189 promoter. To probe CREB-Zfp189 interactions, we employ CRISPR-mediated locus-specific transcriptional reprogramming to direct CREB or G9a (a repressive histone methyltransferase) to the Zfp189 promoter in prefrontal cortex neurons. Induction of Zfp189 with site-specific CREB is pro-resilient, whereas suppressing Zfp189 expression with G9a increases susceptibility. These findings reveal an essential role for Zfp189 and CREB-Zfp189 interactions in mediating a central transcriptional network of resilience.
- Published
- 2019
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31. Neuroendocrine drivers of risk and resilience: The influence of metabolism & mitochondria.
- Author
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Turkson S, Kloster A, Hamilton PJ, and Neigh GN
- Subjects
- Animals, Humans, Allostasis physiology, Hormones metabolism, Hypothalamo-Hypophyseal System metabolism, Mitochondria metabolism, Resilience, Psychological, Stress, Psychological metabolism
- Abstract
The manifestation of risk versus resilience has been considered from varying perspectives including genetics, epigenetics, early life experiences, and type and intensity of the challenge with which the organism is faced. Although all of these factors are central to determining risk and resilience, the current review focuses on what may be a final common pathway: metabolism. When an organism is faced with a perturbation to the environment, whether internal or external, appropriate energy allocation is essential to resolving the divergence from equilibrium. This review examines the potential role of metabolism in the manifestation of stress-induced neural compromise. In addition, this review details the current state of knowledge on neuroendocrine factors which are poised to set the tone of the metabolic response to a systemic challenge. The goal is to provide an essential framework for understanding stress in a metabolic context and appreciation for key neuroendocrine signals., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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32. Withdrawal from repeated morphine administration augments expression of the RhoA network in the nucleus accumbens to control synaptic structure.
- Author
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Cahill ME, Browne CJ, Wang J, Hamilton PJ, Dong Y, and Nestler EJ
- Subjects
- Animals, Cytoplasm drug effects, Cytoplasm metabolism, Dendritic Spines drug effects, Dendritic Spines metabolism, Male, Mice, Mice, Inbred C57BL, Nucleus Accumbens drug effects, Reward, Signal Transduction drug effects, Synapses drug effects, Synaptosomes drug effects, Synaptosomes metabolism, rho GTP-Binding Proteins genetics, rhoA GTP-Binding Protein, Morphine, Narcotics, Nucleus Accumbens metabolism, Nucleus Accumbens pathology, Substance Withdrawal Syndrome metabolism, Substance Withdrawal Syndrome pathology, Synapses metabolism, Synapses pathology, rho GTP-Binding Proteins biosynthesis
- Abstract
The nucleus accumbens (NAc) is a critical brain reward region that mediates the rewarding effects of drugs of abuse, including those of morphine and other opiates. Drugs of abuse induce widespread alterations in gene transcription and dendritic spine morphology in medium spiny neurons (MSNs) of the NAc that ultimately influence NAc excitability and hence reward-related behavioral responses. Growing evidence indicates that within the NAc small GTPases are common intracellular targets of drugs of abuse where these molecules regulate drug-mediated transcriptional and spine morphogenic effects. The RhoA small GTPase is among the most well-characterized members of the Ras superfamily of small GTPases, and recent work highlights an important role for hippocampal RhoA in morphine-facilitated reward behavior. Despite this, it remains unclear how RhoA pathway signaling in the NAc is affected by withdrawal from morphine. To investigate this question, using subcellular fractionation and subsequent protein profiling we examined the expression of key components of the RhoA pathway in NAc nuclear, cytoplasmic, and synaptosomal compartments during multiple withdrawal periods from repeated morphine administration. Furthermore, using in vivo viral-mediated gene transfer, we determined the consequences of revealed RhoA pathway alterations on NAc MSN dendritic spine morphology. Our findings reveal an important role for RhoA signaling cascades in mediating the effects of long-term morphine withdrawal on NAc MSN dendritic spine elimination., Open Practices: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/., (© 2018 International Society for Neurochemistry.)
- Published
- 2018
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33. Transcriptional and physiological adaptations in nucleus accumbens somatostatin interneurons that regulate behavioral responses to cocaine.
- Author
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Ribeiro EA, Salery M, Scarpa JR, Calipari ES, Hamilton PJ, Ku SM, Kronman H, Purushothaman I, Juarez B, Heshmati M, Doyle M, Lardner C, Burek D, Strat A, Pirpinias S, Mouzon E, Han MH, Neve RL, Bagot RC, Kasarskis A, Koo JW, and Nestler EJ
- Subjects
- Animals, Brain metabolism, Gene Expression Profiling, Gene Expression Regulation drug effects, Gene Transfer Techniques, Locomotion, Male, Mice, Mice, Inbred C57BL, Neurons drug effects, Neurons metabolism, Optogenetics methods, Proto-Oncogene Proteins c-jun genetics, Proto-Oncogene Proteins c-jun metabolism, Reward, Sequence Analysis, RNA, Somatostatin pharmacology, Transcription Factors drug effects, Adaptation, Physiological drug effects, Cocaine pharmacology, Interneurons drug effects, Interneurons metabolism, Nucleus Accumbens drug effects, Nucleus Accumbens metabolism, Somatostatin metabolism, Transcriptome
- Abstract
The role of somatostatin interneurons in nucleus accumbens (NAc), a key brain reward region, remains poorly understood due to the fact that these cells account for < 1% of NAc neurons. Here, we use optogenetics, electrophysiology, and RNA-sequencing to characterize the transcriptome and functioning of NAc somatostatin interneurons after repeated exposure to cocaine. We find that the activity of somatostatin interneurons regulates behavioral responses to cocaine, with repeated cocaine reducing the excitability of these neurons. Repeated cocaine also induces transcriptome-wide changes in gene expression within NAc somatostatin interneurons. We identify the JUND transcription factor as a key regulator of cocaine action and confirmed, by use of viral-mediated gene transfer, that JUND activity in somatostatin interneurons influences behavioral responses to cocaine. Our results identify alterations in NAc induced by cocaine in a sparse population of somatostatin interneurons, and illustrate the value of studying brain diseases using cell type-specific whole transcriptome RNA-sequencing.
- Published
- 2018
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34. Corrigendum: Sex-specific transcriptional signatures in human depression.
- Author
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Labonté B, Engmann O, Purushothaman I, Menard C, Wang J, Tan C, Scarpa JR, Moy G, Loh YE, Cahill M, Lorsch ZS, Hamilton PJ, Calipari ES, Hodes GE, Issler O, Kronman H, Pfau M, Obradovic ALJ, Dong Y, Neve RL, Russo S, Kasarskis A, Tamminga C, Mechawar N, Turecki G, Zhang B, Shen L, and Nestler EJ
- Abstract
This corrects the article DOI: 10.1038/nm.4386.
- Published
- 2018
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35. Estrogen receptor α drives pro-resilient transcription in mouse models of depression.
- Author
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Lorsch ZS, Loh YE, Purushothaman I, Walker DM, Parise EM, Salery M, Cahill ME, Hodes GE, Pfau ML, Kronman H, Hamilton PJ, Issler O, Labonté B, Symonds AE, Zucker M, Zhang TY, Meaney MJ, Russo SJ, Shen L, Bagot RC, and Nestler EJ
- Subjects
- Animals, Estrogen Receptor alpha genetics, Female, Gene Expression Profiling, Male, Mice, Mice, Inbred C57BL, Models, Animal, Sex Factors, Transcriptome genetics, Adaptation, Psychological physiology, Behavior, Animal physiology, Depression physiopathology, Estrogen Receptor alpha metabolism, Nucleus Accumbens metabolism, Stress, Psychological physiopathology
- Abstract
Most people exposed to stress do not develop depression. Animal models have shown that stress resilience is an active state that requires broad transcriptional adaptations, but how this homeostatic process is regulated remains poorly understood. In this study, we analyze upstream regulators of genes differentially expressed after chronic social defeat stress. We identify estrogen receptor α (ERα) as the top regulator of pro-resilient transcriptional changes in the nucleus accumbens (NAc), a key brain reward region implicated in depression. In accordance with these findings, nuclear ERα protein levels are altered by stress in male and female mice. Further, overexpression of ERα in the NAc promotes stress resilience in both sexes. Subsequent RNA-sequencing reveals that ERα overexpression in NAc reproduces the transcriptional signature of resilience in male, but not female, mice. These results indicate that NAc ERα is an important regulator of pro-resilient transcriptional changes, but with sex-specific downstream targets.
- Published
- 2018
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36. Viral Expression of Epigenome Editing Tools in Rodent Brain Using Stereotaxic Surgery Techniques.
- Author
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Hamilton PJ, Lim CJ, Nestler EJ, and Heller EA
- Subjects
- Animals, Gene Expression, Green Fluorescent Proteins genetics, Mice, Rats, Transgenes, Brain metabolism, Brain surgery, Dependovirus genetics, Epigenesis, Genetic, Gene Editing methods, Gene Transfer Techniques, Simplexvirus genetics
- Abstract
Delivery of molecular tools for targeted epigenome editing in rodent brain can be facilitated by the use of viral vector-mediated gene transfer coupled with stereotaxic surgery techniques. Here, we describe the surgical protocol utilized by our group, which is optimized for herpes simplex virus (HSV)-mediated delivery into mouse brain. The protocol outlined herein could also be applied for delivery of adeno-associated viruses (AAV) or lentiviruses in both mice and rats. This method allows for efficient viral transgene expression and subsequent epigenome editing in rodent brain with excellent spatiotemporal control. Nearly any brain region of interest can be targeted in rodents at every stage of postnatal life. Owing to the versatility, reproducibility, and utility of this technique, it is an important method for any laboratory interested in studying the cellular, circuit, and behavioral consequences of in vivo neuroepigenome editing.
- Published
- 2018
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37. Stereotaxic Surgery and Viral Delivery of Zinc-Finger Epigenetic Editing Tools in Rodent Brain.
- Author
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Hamilton PJ, Lim CJ, Nestler EJ, and Heller EA
- Subjects
- Animals, Genetic Vectors administration & dosage, Rodentia, Stereotaxic Techniques, Brain metabolism, Brain surgery, Dependovirus genetics, Epigenomics methods, Gene Editing methods, Gene Transfer Techniques, Zinc Fingers
- Abstract
Delivery of engineered zinc-finger proteins (ZFPs) for targeted epigenetic remodeling in rodent brain can be facilitated by the use of viral vector-mediated gene transfer coupled with stereotaxic surgery techniques. Here we describe the surgical protocol utilized by our group which is optimized for herpes simplex virus (HSV) delivery into mouse brain. The protocol outlined herein could be applied for delivery of adeno-associated viruses (AAV) or lentiviruses in both mice and rats. This method allows for the viral expression of engineered DNA-binding factors, particularly engineered ZFPs, and subsequent epigenome editing in rodent brain with excellent spatiotemporal control. Nearly any brain region of interest can be targeted in rodents at every stage of postnatal life. Owing to the versatility, reproducibility, and utility of this technique, it is an important method for any laboratory interested in studying the cellular, circuit, and behavioral consequences of in vivo neuroepigenetic editing with synthetic ZFP constructs.
- Published
- 2018
- Full Text
- View/download PDF
38. Cell-Type-Specific Epigenetic Editing at the Fosb Gene Controls Susceptibility to Social Defeat Stress.
- Author
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Hamilton PJ, Burek DJ, Lombroso SI, Neve RL, Robison AJ, Nestler EJ, and Heller EA
- Subjects
- Animals, Behavior, Animal physiology, Disease Models, Animal, Disease Susceptibility, Male, Mice, Mice, Transgenic, Phenotype, Depression genetics, Dominance-Subordination, Epigenesis, Genetic genetics, Nucleus Accumbens metabolism, Proto-Oncogene Proteins c-fos genetics, Receptors, Dopamine D1 metabolism, Receptors, Dopamine D2 metabolism, Stress, Psychological genetics
- Abstract
Chronic social defeat stress regulates the expression of Fosb in the nucleus accumbens (NAc) to promote the cell-type-specific accumulation of ΔFosB in the two medium spiny neuron (MSN) subtypes in this region. ΔFosB is selectively induced in D1-MSNs in the NAc of resilient mice, and in D2-MSNs of susceptible mice. However, little is known about the consequences of such selective induction, particularly in D2-MSNs. This study examined how cell-type-specific control of the endogenous Fosb gene in NAc regulates susceptibility to social defeat stress. Histone post-translational modifications (HPTMs) were targeted specifically to Fosb using engineered zinc-finger proteins (ZFPs). Fosb-ZFPs were fused to either the transcriptional repressor, G9a, which promotes histone methylation or the transcriptional activator, p65, which promotes histone acetylation. These ZFPs were expressed in D1- vs D2-MSNs using Cre-dependent viral expression in the NAc of mice transgenic for Cre recombinase in these MSN subtypes. We found that stress susceptibility is oppositely regulated by the specific cell type and HPTM targeted. We report that Fosb-targeted histone acetylation in D2-MSNs or histone methylation in D1-MSNs promotes a stress-susceptible, depressive-like phenotype, while histone methylation in D2-MSNs or histone acetylation in D1-MSNs increases resilience to social stress as quantified by social interaction behavior and sucrose preference. This work presents the first demonstration of cell- and gene-specific targeting of histone modifications, which model naturally occurring transcriptional phenomena that control social defeat stress behavior. This epigenetic-editing approach, which recapitulates physiological changes in gene expression, reveals clear differences in the social defeat phenotype induced by Fosb gene manipulation in MSN subtypes.
- Published
- 2018
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39. Neuroepigenetic Editing.
- Author
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Hamilton PJ, Lim CJ, Nestler EJ, and Heller EA
- Subjects
- Animals, Chromatin genetics, DNA genetics, High-Throughput Nucleotide Sequencing methods, Humans, Mental Disorders genetics, Nervous System Diseases genetics, Neurons pathology, Epigenesis, Genetic, Gene Editing methods, Neurons metabolism
- Abstract
Studies of the mammalian nervous system have revealed widespread epigenetic regulation underlying gene expression intrinsic to basic neurobiological function as well as neurological disease. Over the past decade, a critical role has emerged for the neural regulation of chromatin-modifying enzymes during both development and adulthood, and in response to external stimuli. These biochemical data are complemented by numerous next generation sequencing (NGS) studies that quantify the extent of chromatin and DNA modifications in neurons. Neuroepigenetic editing tools can be applied to distinguish between the mere presence and functional relevance of such modifications to neural transcription and animal behavior. This review discusses current advances in neuroepigenetic editing, highlighting methodological considerations pertinent to neuroscience, such as delivery methods and the spatiotemporal specificity of editing. Although neuroepigenetic editing is a nascent field, the studies presented in this review demonstrate the enormous potential of this approach for basic neurobiological research and therapeutic application.
- Published
- 2018
- Full Text
- View/download PDF
40. Cocaine-Induced Chromatin Modifications Associate With Increased Expression and Three-Dimensional Looping of Auts2.
- Author
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Engmann O, Labonté B, Mitchell A, Bashtrykov P, Calipari ES, Rosenbluh C, Loh YE, Walker DM, Burek D, Hamilton PJ, Issler O, Neve RL, Turecki G, Hurd Y, Chess A, Shen L, Mansuy I, Jeltsch A, Akbarian S, and Nestler EJ
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Animals, Cell Line, Tumor, Cohort Studies, Conditioning, Operant drug effects, Cytoskeletal Proteins, DNA Methylation drug effects, Gene Expression Regulation genetics, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Middle Aged, Models, Molecular, Molecular Conformation, Neuroblastoma pathology, Nuclear Proteins genetics, Nucleus Accumbens drug effects, Nucleus Accumbens metabolism, Rats, Rats, Sprague-Dawley, Receptors, Dopamine D1 genetics, Receptors, Dopamine D1 metabolism, Receptors, Dopamine D2 genetics, Receptors, Dopamine D2 metabolism, Transcription Factors, Young Adult, Chromatin drug effects, Cocaine administration & dosage, Dopamine Uptake Inhibitors administration & dosage, Gene Expression Regulation drug effects, Nuclear Proteins metabolism
- Abstract
Background: Exposure to drugs of abuse alters the epigenetic landscape of the brain's reward regions, such as the nucleus accumbens. We investigated how combinations of chromatin modifications affect genes that regulate responses to cocaine. We focused on Auts2, a gene linked to human evolution and cognitive disorders, which displays strong clustering of cocaine-induced chromatin modifications in this brain region., Methods: We combined chromosome conformation capture, circularized chromosome conformation capture, and related approaches with behavioral paradigms relevant to cocaine phenotypes. Cell type-specific functions were assessed by fluorescence-activated cell sorting and viral-mediated overexpression in Cre-dependent mouse lines., Results: We observed that Auts2 gene expression is increased by repeated cocaine administration specifically in D
2 -type medium spiny neurons in the nucleus accumbens, an effect seen in male but not female mice. Auts2 messenger RNA expression was also upregulated postmortem in the nucleus accumbens of male human cocaine addicts. We obtained evidence that chromosomal looping, bypassing 1524 kb of linear genome, connects Auts2 to the Caln1 gene locus under baseline conditions. This looping was disrupted after repeated cocaine exposure, resulting in increased expression of both genes in D2 -type medium spiny neurons. Cocaine exposure reduces binding of CCCTC-binding factor, a chromosomal scaffolding protein, and increases histone and DNA methylation at the Auts-Caln1 loop base in the nucleus accumbens. Cell type-specific overexpression of Auts2 or Caln1 in D2 -type medium spiny neurons demonstrated that both genes promote cocaine reward., Conclusions: These findings suggest that cocaine-induced alterations of neuronal three-dimensional genome organization destabilize higher order chromatin at specific loci that regulate responses to the drug., (Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)- Published
- 2017
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41. Sex-specific transcriptional signatures in human depression.
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Labonté B, Engmann O, Purushothaman I, Menard C, Wang J, Tan C, Scarpa JR, Moy G, Loh YE, Cahill M, Lorsch ZS, Hamilton PJ, Calipari ES, Hodes GE, Issler O, Kronman H, Pfau M, Obradovic ALJ, Dong Y, Neve RL, Russo S, Kazarskis A, Tamminga C, Mechawar N, Turecki G, Zhang B, Shen L, and Nestler EJ
- Subjects
- Adult, Aged, Animals, Blotting, Western, Case-Control Studies, Cerebral Cortex metabolism, Disease Models, Animal, Down-Regulation, Female, Hippocampus metabolism, Humans, Immunohistochemistry, Male, Mice, Middle Aged, Nucleus Accumbens metabolism, Patch-Clamp Techniques, Prefrontal Cortex metabolism, Pyramidal Cells metabolism, Sequence Analysis, RNA, Sex Characteristics, Sex Factors, Brain metabolism, Depressive Disorder, Major genetics, Stress, Psychological genetics, Transcriptome
- Abstract
Major depressive disorder (MDD) is a leading cause of disease burden worldwide. While the incidence, symptoms and treatment of MDD all point toward major sex differences, the molecular mechanisms underlying this sexual dimorphism remain largely unknown. Here, combining differential expression and gene coexpression network analyses, we provide a comprehensive characterization of male and female transcriptional profiles associated with MDD across six brain regions. We overlap our human profiles with those from a mouse model, chronic variable stress, and capitalize on converging pathways to define molecular and physiological mechanisms underlying the expression of stress susceptibility in males and females. Our results show a major rearrangement of transcriptional patterns in MDD, with limited overlap between males and females, an effect seen in both depressed humans and stressed mice. We identify key regulators of sex-specific gene networks underlying MDD and confirm their sex-specific impact as mediators of stress susceptibility. For example, downregulation of the female-specific hub gene Dusp6 in mouse prefrontal cortex mimicked stress susceptibility in females, but not males, by increasing ERK signaling and pyramidal neuron excitability. Such Dusp6 downregulation also recapitulated the transcriptional remodeling that occurs in prefrontal cortex of depressed females. Together our findings reveal marked sexual dimorphism at the transcriptional level in MDD and highlight the importance of studying sex-specific treatments for this disorder.
- Published
- 2017
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42. Targeted Epigenetic Remodeling of the Cdk5 Gene in Nucleus Accumbens Regulates Cocaine- and Stress-Evoked Behavior.
- Author
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Heller EA, Hamilton PJ, Burek DD, Lombroso SI, Peña CJ, Neve RL, and Nestler EJ
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- Animals, Brain metabolism, Central Nervous System Stimulants pharmacology, Cyclin-Dependent Kinase 5 metabolism, Histones genetics, Histones metabolism, Male, Mice, Mice, Inbred C57BL, Rats, Reward, Zinc Fingers genetics, Behavior, Animal drug effects, Cocaine pharmacology, Cyclin-Dependent Kinase 5 genetics, Epigenesis, Genetic drug effects, Nucleus Accumbens drug effects
- Abstract
Unlabelled: Recent studies have implicated epigenetic remodeling in brain reward regions following psychostimulant or stress exposure. It has only recently become possible to target a given type of epigenetic remodeling to a single gene of interest, and to probe the functional relevance of such regulation to neuropsychiatric disease. We sought to examine the role of histone modifications at the murine Cdk5 (cyclin-dependent kinase 5) locus, given growing evidence of Cdk5 expression in nucleus accumbens (NAc) influencing reward-related behaviors. Viral-mediated delivery of engineered zinc finger proteins (ZFP) targeted histone H3 lysine 9/14 acetylation (H3K9/14ac), a transcriptionally active mark, or histone H3 lysine 9 dimethylation (H3K9me2), which is associated with transcriptional repression, specifically to the Cdk5 locus in NAc in vivo We found that Cdk5-ZFP transcription factors are sufficient to bidirectionally regulate Cdk5 gene expression via enrichment of their respective histone modifications. We examined the behavioral consequences of this epigenetic remodeling and found that Cdk5-targeted H3K9/14ac increased cocaine-induced locomotor behavior, as well as resilience to social stress. Conversely, Cdk5-targeted H3K9me2 attenuated both cocaine-induced locomotor behavior and conditioned place preference, but had no effect on stress-induced social avoidance behavior. The current study provides evidence for the causal role of Cdk5 epigenetic remodeling in NAc in Cdk5 gene expression and in the control of reward and stress responses. Moreover, these data are especially compelling given that previous work demonstrated opposite behavioral phenotypes compared with those reported here upon Cdk5 overexpression or knockdown, demonstrating the importance of targeted epigenetic remodeling tools for studying more subtle molecular changes that contribute to neuropsychiatric disease., Significance Statement: Addiction and depression are highly heritable diseases, yet it has been difficult to identify gene sequence variations that underlie this heritability. Gene regulation via epigenetic remodeling is an additional mechanism contributing to the neurobiological basis of drug and stress exposure. In particular, epigenetic regulation of the Cdk5 gene alters responses to cocaine and stress in mouse and rat models. In this study, we used a novel technology, zinc-finger engineered transcription factors, to remodel histone proteins specifically at the Cdk5 gene. We found that this is sufficient to regulate the expression of Cdk5 and results in altered behavioral responses to cocaine and social stress. These data provide compelling evidence of the significance of epigenetic regulation in the neurobiological basis of reward- and stress-related neuropsychiatric disease., (Copyright © 2016 the authors 0270-6474/16/364690-08$15.00/0.)
- Published
- 2016
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43. Zn(2+) reverses functional deficits in a de novo dopamine transporter variant associated with autism spectrum disorder.
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Hamilton PJ, Shekar A, Belovich AN, Christianson NB, Campbell NG, Sutcliffe JS, Galli A, Matthies HJ, and Erreger K
- Abstract
Our laboratory recently characterized a novel autism spectrum disorder (ASD)-associated de novo missense mutation in the human dopamine transporter (hDAT) gene SLC6A3 (hDAT T356M). This hDAT variant exhibits dysfunctional forward and reverse transport properties that may contribute to DA dysfunction in ASD. Here, we report that Zn(2+) reverses, at least in part, the functional deficits of ASD-associated hDAT variant T356M. These data suggest that the molecular mechanism targeted by Zn(2+) to restore partial function in hDAT T356M may be a novel therapeutic target to rescue functional deficits in hDAT variants associated with ASD.
- Published
- 2015
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44. Rare autism-associated variants implicate syntaxin 1 (STX1 R26Q) phosphorylation and the dopamine transporter (hDAT R51W) in dopamine neurotransmission and behaviors.
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Cartier E, Hamilton PJ, Belovich AN, Shekar A, Campbell NG, Saunders C, Andreassen TF, Gether U, Veenstra-Vanderweele J, Sutcliffe JS, Ulery-Reynolds PG, Erreger K, Matthies HJ, and Galli A
- Abstract
Background: Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA., Methods: We adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD)., Outcomes: Here, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms involve decreased phosphorylation of STX1 at Ser14 mediated by casein kinase 2 as well as a reduction in STX1/DAT interaction. These findings point to STX1/DAT interactions and STX1 phosphorylation as key regulators of DA homeostasis., Interpretation: We determine the molecular identity and the impact of these variants with the intent of defining DA dysfunction and associated behaviors as possible complications of ASD.
- Published
- 2015
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45. SLC6A3 coding variant Ala559Val found in two autism probands alters dopamine transporter function and trafficking.
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Bowton E, Saunders C, Reddy IA, Campbell NG, Hamilton PJ, Henry LK, Coon H, Sakrikar D, Veenstra-VanderWeele JM, Blakely RD, Sutcliffe J, Matthies HJ, Erreger K, and Galli A
- Subjects
- Cell Movement genetics, Dopamine Plasma Membrane Transport Proteins physiology, Humans, Male, Siblings, Autistic Disorder genetics, Dopamine Plasma Membrane Transport Proteins genetics, Mutation genetics, Synaptic Transmission genetics
- Abstract
Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C β (PKCβ) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCβ activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCβ or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission, including ADHD, bipolar disorder, and now ASD. These findings provide valuable insight into a new cellular phenotype (altered hDAT trafficking) supporting dysregulated DA function in these disorders. They also provide a novel potential target (PKCβ) for therapeutic interventions in individuals with ASD.
- Published
- 2014
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46. PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein.
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Hamilton PJ, Belovich AN, Khelashvili G, Saunders C, Erreger K, Javitch JA, Sitte HH, Weinstein H, Matthies HJG, and Galli A
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- Amino Acid Substitution, Animals, Cell Membrane drug effects, Dopamine metabolism, Dopamine Plasma Membrane Transport Proteins chemistry, Dopamine Plasma Membrane Transport Proteins genetics, Drosophila melanogaster physiology, Gene Expression, Humans, Locomotion drug effects, Models, Molecular, Neurons drug effects, Neurons metabolism, Phosphatidylinositol 4,5-Diphosphate pharmacology, Protein Structure, Tertiary, Transgenes, Amphetamine pharmacology, Behavior, Animal drug effects, Central Nervous System Stimulants pharmacology, Dopamine Plasma Membrane Transport Proteins metabolism, Drosophila melanogaster drug effects, Phosphatidylinositol 4,5-Diphosphate metabolism
- Abstract
Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux without altering the hDAT physiological function of DA uptake. We evaluated the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present what is to our knowledge the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.
- Published
- 2014
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47. Missense dopamine transporter mutations associate with adult parkinsonism and ADHD.
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Hansen FH, Skjørringe T, Yasmeen S, Arends NV, Sahai MA, Erreger K, Andreassen TF, Holy M, Hamilton PJ, Neergheen V, Karlsborg M, Newman AH, Pope S, Heales SJ, Friberg L, Law I, Pinborg LH, Sitte HH, Loland C, Shi L, Weinstein H, Galli A, Hjermind LE, Møller LB, and Gether U
- Subjects
- Adult, Amino Acid Sequence, Amino Acid Substitution, Animals, Attention Deficit Disorder with Hyperactivity complications, Brain diagnostic imaging, Brain metabolism, Cohort Studies, DNA Mutational Analysis, Dopamine metabolism, Dopamine Plasma Membrane Transport Proteins chemistry, Female, HEK293 Cells, Humans, Male, Models, Molecular, Molecular Sequence Data, Mutant Proteins chemistry, Oocytes metabolism, Parkinsonian Disorders complications, Pedigree, Positron-Emission Tomography, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Sodium metabolism, Tomography, Emission-Computed, Single-Photon, Xenopus, Attention Deficit Disorder with Hyperactivity genetics, Attention Deficit Disorder with Hyperactivity metabolism, Dopamine Plasma Membrane Transport Proteins genetics, Dopamine Plasma Membrane Transport Proteins metabolism, Mutant Proteins genetics, Mutant Proteins metabolism, Mutation, Missense, Parkinsonian Disorders genetics, Parkinsonian Disorders metabolism
- Abstract
Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we analyzed a cohort of patients with atypical movement disorder and identified 2 DAT coding variants, DAT-Ile312Phe and a presumed de novo mutant DAT-Asp421Asn, in an adult male with early-onset parkinsonism and ADHD. According to DAT single-photon emission computed tomography (DAT-SPECT) scans and a fluoro-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine, and electrophysiological analyses identified a large cation leak that might further perturb dopaminergic neurotransmission. Our results link specific DAT missense mutations to neurodegenerative early-onset parkinsonism. Moreover, the neuropsychiatric comorbidity provides additional support for the idea that DAT missense mutations are an ADHD risk factor and suggests that complex DAT genotype and phenotype correlations contribute to different dopaminergic pathologies.
- Published
- 2014
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48. De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.
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Hamilton PJ, Campbell NG, Sharma S, Erreger K, Herborg Hansen F, Saunders C, Belovich AN, Sahai MA, Cook EH, Gether U, McHaourab HS, Matthies HJ, Sutcliffe JS, and Galli A
- Subjects
- Animals, Child Development Disorders, Pervasive physiopathology, Child, Preschool, Dopaminergic Neurons physiology, Drosophila melanogaster genetics, Homeostasis genetics, Humans, Male, Motor Activity genetics, Mutation, Missense genetics, Risk Factors, Child Development Disorders, Pervasive genetics, Dopamine physiology, Dopamine Plasma Membrane Transport Proteins genetics
- Abstract
De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.
- Published
- 2013
- Full Text
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49. Drosophila melanogaster: a novel animal model for the behavioral characterization of autism-associated mutations in the dopamine transporter gene.
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Hamilton PJ, Campbell NG, Sharma S, Erreger K, Hansen FH, Saunders C, Belovich AN, Sahai MA, Cook EH, Gether U, McHaourab HS, Matthies HJ, Sutcliffe JS, and Galli A
- Subjects
- Animals, Humans, Male, Child Development Disorders, Pervasive genetics, Dopamine physiology, Dopamine Plasma Membrane Transport Proteins genetics
- Published
- 2013
- Full Text
- View/download PDF
50. Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate.
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Buchmayer F, Schicker K, Steinkellner T, Geier P, Stübiger G, Hamilton PJ, Jurik A, Stockner T, Yang JW, Montgomery T, Holy M, Hofmaier T, Kudlacek O, Matthies HJ, Ecker GF, Bochkov V, Galli A, Boehm S, and Sitte HH
- Subjects
- HEK293 Cells, Humans, Second Messenger Systems, Serotonin Plasma Membrane Transport Proteins genetics, Amphetamine pharmacology, Phosphatidylinositol 4,5-Diphosphate metabolism, Serotonin Plasma Membrane Transport Proteins drug effects
- Abstract
Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.
- Published
- 2013
- Full Text
- View/download PDF
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