51 results on '"Alexey Kozlenkov"'
Search Results
2. Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation
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Junhao Li, Manoj K. Jaiswal, Jo-Fan Chien, Alexey Kozlenkov, Jinyoung Jung, Ping Zhou, Mahammad Gardashli, Luc J. Pregent, Erica Engelberg-Cook, Dennis W. Dickson, Veronique V. Belzil, Eran A. Mukamel, and Stella Dracheva
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Science - Abstract
Abstract A repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we investigate single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem motor and frontal cortices from C9-ALS, C9-FTD, and control donors. C9-ALS donors present pervasive alterations of gene expression with concordant changes in chromatin accessibility and histone modifications. The greatest alterations occur in upper and deep layer excitatory neurons, as well as in astrocytes. In neurons, the changes imply an increase in proteostasis, metabolism, and protein expression pathways, alongside a decrease in neuronal function. In astrocytes, the alterations suggest activation and structural remodeling. Conversely, C9-FTD donors have fewer high-quality neuronal nuclei in the frontal cortex and numerous gene expression changes in glial cells. These findings highlight a context-dependent molecular disruption in C9-ALS and C9-FTD, indicating unique effects across cell types, brain regions, and diseases.
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- 2023
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3. Author Correction: Brain Cell Type Specific Gene Expression and Co-expression Network Architectures
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Andrew T. McKenzie, Minghui Wang, Mads E. Hauberg, John F. Fullard, Alexey Kozlenkov, Alexandra Keenan, Yasmin L. Hurd, Stella Dracheva, Patrizia Casaccia, Panos Roussos, and Bin Zhang
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Medicine ,Science - Published
- 2021
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4. Neuronal and glial 3D chromatin architecture informs the cellular etiology of brain disorders
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Benxia Hu, Hyejung Won, Won Mah, Royce B. Park, Bibi Kassim, Keeley Spiess, Alexey Kozlenkov, Cheynna A. Crowley, Sirisha Pochareddy, The PsychENCODE Consortium, Yun Li, Stella Dracheva, Nenad Sestan, Schahram Akbarian, and Daniel H. Geschwind
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Science - Abstract
The cellular heterogeneity in brain obscures the identification of robust cellular regulatory networks. Here the authors integrate genome-wide chromosome conformation data from sorted neurons and glia, with transcriptomic and enhancer profiles, to characterize cell-type-specific gene regulatory landscapes in the human brain, and provide insights into cell-type-specific gene regulatory networks in brain disorders.
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- 2021
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5. Common schizophrenia risk variants are enriched in open chromatin regions of human glutamatergic neurons
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Mads E. Hauberg, Jordi Creus-Muncunill, Jaroslav Bendl, Alexey Kozlenkov, Biao Zeng, Chuhyon Corwin, Sarah Chowdhury, Harald Kranz, Yasmin L. Hurd, Michael Wegner, Anders D. Børglum, Stella Dracheva, Michelle E. Ehrlich, John F. Fullard, and Panos Roussos
- Subjects
Science - Abstract
Here, the authors perform ATAC-seq on four distinct cell populations from three different regions of the human brain, finding that chromatin accessibility varies greatly by cell type and less by brain region. This study reveals differences in biological function and gene regulation, as well as overlap of genetic variants associated with schizophrenia and other neuropsychiatric traits.
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- 2020
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6. Brain Cell Type Specific Gene Expression and Co-expression Network Architectures
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Andrew T. McKenzie, Minghui Wang, Mads E. Hauberg, John F. Fullard, Alexey Kozlenkov, Alexandra Keenan, Yasmin L. Hurd, Stella Dracheva, Patrizia Casaccia, Panos Roussos, and Bin Zhang
- Subjects
Medicine ,Science - Abstract
Abstract Elucidating brain cell type specific gene expression patterns is critical towards a better understanding of how cell-cell communications may influence brain functions and dysfunctions. We set out to compare and contrast five human and murine cell type-specific transcriptome-wide RNA expression data sets that were generated within the past several years. We defined three measures of brain cell type-relative expression including specificity, enrichment, and absolute expression and identified corresponding consensus brain cell “signatures,” which were well conserved across data sets. We validated that the relative expression of top cell type markers are associated with proxies for cell type proportions in bulk RNA expression data from postmortem human brain samples. We further validated novel marker genes using an orthogonal ATAC-seq dataset. We performed multiscale coexpression network analysis of the single cell data sets and identified robust cell-specific gene modules. To facilitate the use of the cell type-specific genes for cell type proportion estimation and deconvolution from bulk brain gene expression data, we developed an R package, BRETIGEA. In summary, we identified a set of novel brain cell consensus signatures and robust networks from the integration of multiple datasets and therefore transcend limitations related to technical issues characteristic of each individual study.
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- 2018
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7. Genetic variants near MLST8 and DHX57 affect the epigenetic age of the cerebellum
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Ake T. Lu, Eilis Hannon, Morgan E. Levine, Ke Hao, Eileen M. Crimmins, Katie Lunnon, Alexey Kozlenkov, Jonathan Mill, Stella Dracheva, and Steve Horvath
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Science - Abstract
This genome-wide association study identifies five significant SNPs in two loci which are associated with the epigenetic age of post-mortem cerebellar tissue according to a DNA methylation based biomarker of human aging.
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- 2016
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8. Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons
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Liudmila Lapatsina, Julia A. Jira, Ewan St. J. Smith, Kate Poole, Alexey Kozlenkov, Daniel Bilbao, Gary R. Lewin, and Paul A. Heppenstall
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ion channel ,mechanotransduction ,peripheral sensory neurons ,stomatin-like proteins ,Biology (General) ,QH301-705.5 - Abstract
A complex of stomatin-family proteins and acid-sensing (proton-gated) ion channel (ASIC) family members participate in sensory transduction in invertebrates and vertebrates. Here, we have examined the role of the stomatin-family protein stomatin-like protein-3 (STOML3) in this process. We demonstrate that STOML3 interacts with stomatin and ASIC subunits and that this occurs in a highly mobile vesicle pool in dorsal root ganglia (DRG) neurons and Chinese hamster ovary cells. We identify a hydrophobic region in the N-terminus of STOML3 that is required for vesicular localization of STOML3 and regulates physical and functional interaction with ASICs. We further characterize STOML3-containing vesicles in DRG neurons and show that they are Rab11-positive, but not part of the early-endosomal, lysosomal or Rab14-dependent biosynthetic compartment. Moreover, uncoupling of vesicles from microtubules leads to incorporation of STOML3 into the plasma membrane and increased acid-gated currents. Thus, STOML3 defines a vesicle pool in which it associates with molecules that have critical roles in sensory transduction. We suggest that the molecular features of this vesicular pool may be characteristic of a ‘transducosome’ in sensory neurons.
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- 2012
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9. Supplementary Tables from A Novel Method for Rapid Molecular Subgrouping of Medulloblastoma
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Cinzia Lavarino, Jaume Mora, Jose-Ignacio Martin-Subero, Michael D. Taylor, Ofelia Cruz, Andrés Morales La Madrid, Stefan M. Pfister, David T.W. Jones, Pascal Johann, Volker Hovestadt, Vijay Ramaswamy, Stella Dracheva, Alexey Kozlenkov, Nada Jabado, Mark W. Kieran, Betty Luu, Ángel M. Carcaboso, Sara Pérez-Jaume, Marta Kulis, Carmen de Torres, Mariona Suñol, Isadora Lemos, Laura Garcia-Gerique, Alícia Garrido-Garcia, and Soledad Gómez
- Abstract
Supplementary Table 1 SampleSheet Supplementary Table 2 GEO_IDs Supplementary Table 3 EpiWNT-SHH_data Supplementary Table 4 EpiWNT-SHH_test Supplementary Table 5 EpiG3-G4_data Supplementary Table 6 EpiG3-G4_test
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- 2023
10. Supplementary Appendix from A Novel Method for Rapid Molecular Subgrouping of Medulloblastoma
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Cinzia Lavarino, Jaume Mora, Jose-Ignacio Martin-Subero, Michael D. Taylor, Ofelia Cruz, Andrés Morales La Madrid, Stefan M. Pfister, David T.W. Jones, Pascal Johann, Volker Hovestadt, Vijay Ramaswamy, Stella Dracheva, Alexey Kozlenkov, Nada Jabado, Mark W. Kieran, Betty Luu, Ángel M. Carcaboso, Sara Pérez-Jaume, Marta Kulis, Carmen de Torres, Mariona Suñol, Isadora Lemos, Laura Garcia-Gerique, Alícia Garrido-Garcia, and Soledad Gómez
- Abstract
Supplementary Methods Supplementary Figure 1 Unsupervised DNA methylation analysis of the training cohort. Supplementary Figure 2 Comparison between the Illumina Infinium Human Methylation450K BeadChip and the Methylation EPIC BeadChip 850K array platforms. Supplementary Figure 3 Specificity of the epigenetic biomarkers for medulloblastoma subgroups. Supplementary Figure 4 Assay performance for low-input titration series. Supplementary Figure 5 Validation of the epigenetic classifier Panel EpiWNT-SHH using DNA methylation microarray data of medulloblastoma samples. Supplementary Figure 6 Validation of the methylation pattern of Panel EpiWNT-SHH using pyrosequencing and direct bisulfite sequencing. Supplementary Figure 7 Schematic overview of the experimental strategy applied for identification of the epigenetic biomarkers and the development of the classifier EpiG3-G4. Supplementary Figure 8 Validation of the epigenetic classifier Panel EpiG3-G4 using DNA methylation microarray data.
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- 2023
11. Data from A Novel Method for Rapid Molecular Subgrouping of Medulloblastoma
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Cinzia Lavarino, Jaume Mora, Jose-Ignacio Martin-Subero, Michael D. Taylor, Ofelia Cruz, Andrés Morales La Madrid, Stefan M. Pfister, David T.W. Jones, Pascal Johann, Volker Hovestadt, Vijay Ramaswamy, Stella Dracheva, Alexey Kozlenkov, Nada Jabado, Mark W. Kieran, Betty Luu, Ángel M. Carcaboso, Sara Pérez-Jaume, Marta Kulis, Carmen de Torres, Mariona Suñol, Isadora Lemos, Laura Garcia-Gerique, Alícia Garrido-Garcia, and Soledad Gómez
- Abstract
Purpose: The classification of medulloblastoma into WNT, SHH, group 3, and group 4 subgroups has become of critical importance for patient risk stratification and subgroup-tailored clinical trials. Here, we aimed to develop a simplified, clinically applicable classification approach that can be implemented in the majority of centers treating patients with medulloblastoma.Experimental Design: We analyzed 1,577 samples comprising previously published DNA methylation microarray data (913 medulloblastomas, 457 non-medulloblastoma tumors, 85 normal tissues), and 122 frozen and formalin-fixed paraffin-embedded medulloblastoma samples. Biomarkers were identified applying stringent selection filters and Linear Discriminant Analysis (LDA) method, and validated using DNA methylation microarray data, bisulfite pyrosequencing, and direct-bisulfite sequencing.Results: Using a LDA-based approach, we developed and validated a prediction method (EpiWNT-SHH classifier) based on six epigenetic biomarkers that allowed for rapid classification of medulloblastoma into the clinically relevant subgroups WNT, SHH, and non-WNT/non-SHH with excellent concordance (>99%) with current gold-standard methods, DNA methylation microarray, and gene signature profiling analysis. The EpiWNT-SHH classifier showed high prediction capacity using both frozen and formalin-fixed material, as well as diverse DNA methylation detection methods. Similarly, we developed a classifier specific for group 3 and group 4 tumors, based on five biomarkers (EpiG3-G4) with good discriminatory capacity, allowing for correct assignment of more than 92% of tumors. EpiWNT-SHH and EpiG3-G4 methylation profiles remained stable across tumor primary, metastasis, and relapse samples.Conclusions: The EpiWNT-SHH and EpiG3-G4 classifiers represent a new simplified approach for accurate, rapid, and cost-effective molecular classification of single medulloblastoma DNA samples, using clinically applicable DNA methylation detection methods. Clin Cancer Res; 24(6); 1355–63. ©2018 AACR.
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- 2023
12. Chromatin domain alterations linked to 3D genome organization in a large cohort of schizophrenia and bipolar disorder brains
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Kiran, Girdhar, Gabriel E, Hoffman, Jaroslav, Bendl, Samir, Rahman, Pengfei, Dong, Will, Liao, Mads E, Hauberg, Laura, Sloofman, Leanne, Brown, Olivia, Devillers, Bibi S, Kassim, Jennifer R, Wiseman, Royce, Park, Elizabeth, Zharovsky, Rivky, Jacobov, Elie, Flatow, Alexey, Kozlenkov, Thomas, Gilgenast, Jessica S, Johnson, Lizette, Couto, Mette A, Peters, Jennifer E, Phillips-Cremins, Chang-Gyu, Hahn, Raquel E, Gur, Carol A, Tamminga, David A, Lewis, Vahram, Haroutunian, Stella, Dracheva, Barbara K, Lipska, Stefano, Marenco, Marija, Kundakovic, John F, Fullard, Yan, Jiang, Panos, Roussos, and Schahram, Akbarian
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Adult ,Bipolar Disorder ,Lysine ,General Neuroscience ,Schizophrenia ,Brain ,Humans ,Chromatin - Abstract
Chromosomal organization, scaling from the 147-base pair (bp) nucleosome to megabase-ranging domains encompassing multiple transcriptional units, including heritability loci for psychiatric traits, remains largely unexplored in the human brain. In this study, we constructed promoter- and enhancer-enriched nucleosomal histone modification landscapes for adult prefrontal cortex from H3-lysine 27 acetylation and H3-lysine 4 trimethylation profiles, generated from 388 controls and 351 individuals diagnosed with schizophrenia (SCZ) or bipolar disorder (BD) (n = 739). We mapped thousands of cis-regulatory domains (CRDs), revealing fine-grained, 104-106-bp chromosomal organization, firmly integrated into Hi-C topologically associating domain stratification by open/repressive chromosomal environments and nuclear topography. Large clusters of hyper-acetylated CRDs were enriched for SCZ heritability, with prominent representation of regulatory sequences governing fetal development and glutamatergic neuron signaling. Therefore, SCZ and BD brains show coordinated dysregulation of risk-associated regulatory sequences assembled into kilobase- to megabase-scaling chromosomal domains.
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- 2022
13. Author Correction: Brain Cell Type Specific Gene Expression and Co-expression Network Architectures
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Alexey Kozlenkov, Alexandra B Keenan, Patrizia Casaccia, Mads E. Hauberg, Bin Zhang, John F. Fullard, Andrew M. McKenzie, Stella Dracheva, Minghui Wang, Yasmin L. Hurd, and Panos Roussos
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Genetic Markers ,Male ,Science ,Datasets as Topic ,Brain Cell ,Mice ,Gene expression ,Animals ,Humans ,Gene Regulatory Networks ,Author Correction ,Neurons ,Network architecture ,Multidisciplinary ,Gene Expression Profiling ,Type specific ,Endothelial Cells ,Temporal Lobe ,Cell biology ,Mice, Inbred C57BL ,Expression (architecture) ,Medicine ,Female ,Single-Cell Analysis ,Databases, Nucleic Acid ,Transcriptome ,Neuroglia ,Geology - Abstract
Elucidating brain cell type specific gene expression patterns is critical towards a better understanding of how cell-cell communications may influence brain functions and dysfunctions. We set out to compare and contrast five human and murine cell type-specific transcriptome-wide RNA expression data sets that were generated within the past several years. We defined three measures of brain cell type-relative expression including specificity, enrichment, and absolute expression and identified corresponding consensus brain cell "signatures," which were well conserved across data sets. We validated that the relative expression of top cell type markers are associated with proxies for cell type proportions in bulk RNA expression data from postmortem human brain samples. We further validated novel marker genes using an orthogonal ATAC-seq dataset. We performed multiscale coexpression network analysis of the single cell data sets and identified robust cell-specific gene modules. To facilitate the use of the cell type-specific genes for cell type proportion estimation and deconvolution from bulk brain gene expression data, we developed an R package, BRETIGEA. In summary, we identified a set of novel brain cell consensus signatures and robust networks from the integration of multiple datasets and therefore transcend limitations related to technical issues characteristic of each individual study.
- Published
- 2021
14. Divergent impacts ofC9orf72repeat expansion on neurons and glia in ALS and FTD
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Junhao Li, Manoj K Jaiswal, Jo-Fan Chien, Alexey Kozlenkov, Ping Zhou, Mahammad Gardashli, Luc J. Pregent, Erica Engelberg-Cook, Dennis W. Dickson, Veronique V. Belzil, Eran A Mukamel, and Stella Dracheva
- Abstract
Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), are strongly influenced by inherited genetic variation, but environmental and epigenetic factors also play key roles in the course of these diseases. A hexanucleotide repeat expansion in theC9orf72(C9) gene is the most common genetic cause of ALS and FTD. To determine the cellular alterations associated with the C9 repeat expansion, we performed single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem samples of motor and frontal cortices from C9-ALS and C9-FTD donors. We found pervasive alterations of gene expression across multiple cortical cell types in C9-ALS, with the largest number of affected genes in astrocytes and excitatory neurons. Astrocytes increased expression of markers of activation and pathways associated with structural remodeling. Excitatory neurons in upper and deep layers increased expression of genes related to proteostasis, metabolism, and protein expression, and decreased expression of genes related to neuronal function. Epigenetic analyses revealed concordant changes in chromatin accessibility, histone modifications, and gene expression in specific cell types. C9-FTD patients had a distinct pattern of changes, including loss of neurons in frontal cortex and altered expression of thousands of genes in astrocytes and oligodendrocyte-lineage cells. Overall, these findings demonstrate a context-dependent molecular disruption in C9-ALS and C9-FTD, resulting in distinct effects across cell types, brain regions, and disease phenotypes.One Sentence SummaryC9orf72-associated ALS and FTDshowed a distinct pattern of transcriptome changes, with the largest number of affected genes in C9-ALS in astrocytes and excitatory neurons in upper and deep layers.
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- 2022
15. Evolution of regulatory signatures in primate cortical neurons at cell-type resolution
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John J. Ely, Alexey Kozlenkov, Michael Wegner, Marit W. Vermunt, Ke Hao, Patrick R. Hof, Eugene V. Koonin, Chet C. Sherwood, Jun-Hao Li, Eran A. Mukamel, Stella Dracheva, Pasha Apontes, Menno P. Creyghton, and Developmental Biology
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Epigenomics ,Male ,Autism Spectrum Disorder ,Gene Expression ,Regulatory Sequences, Nucleic Acid ,Epigenesis, Genetic ,Substance Misuse ,Stem Cell Research - Nonembryonic - Human ,Receptors ,2.1 Biological and endogenous factors ,Regulatory Elements, Transcriptional ,Aetiology ,Cerebral Cortex ,Neurons ,Pediatric ,Multidisciplinary ,biology ,Brain ,Human brain ,Biological Sciences ,Middle Aged ,Histone Code ,United States Department of Veterans Affairs ,Rhesus macaque ,medicine.anatomical_structure ,Cerebral cortex ,Neurological ,GABAergic ,Transcriptional ,Mental health ,Female ,Biotechnology ,Primates ,Cell type ,Pan troglodytes ,Evolution ,1.1 Normal biological development and functioning ,Opioid ,glutamatergic neurons ,Evolution, Molecular ,Glutamatergic ,Genetic ,Interneurons ,Underpinning research ,Genetics ,medicine ,Animals ,Humans ,primate evolution ,Aged ,Nucleic Acid ,GABAergic neurons ,Human Genome ,Neurosciences ,Molecular ,Opioid-Related Disorders ,Stem Cell Research ,biology.organism_classification ,Macaca mulatta ,Regulatory Elements ,United States ,Brain Disorders ,Good Health and Well Being ,mu ,H3K27ac histone modification ,regulatory elements ,Neuron ,Transcriptome ,Drug Abuse (NIDA only) ,Regulatory Sequences ,Neuroscience ,Epigenesis ,Genome-Wide Association Study - Abstract
Significance The cerebral cortex of the human brain is a highly complex, heterogeneous tissue that contains many cell types that are exquisitely regulated at the level of gene expression by noncoding regulatory elements, presumably in a cell-type–dependent manner. However, assessing the regulatory elements in individual cell types is technically challenging, and therefore most of the previous studies on gene regulation were performed with bulk brain tissue. Here we analyze two major types of neurons isolated from the cerebral cortex of humans, chimpanzees, and rhesus macaques, and report complex patterns of cell-type–specific evolution of the regulatory elements in numerous genes. Many genes with evolving regulation are implicated in language abilities as well as psychiatric disorders., The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell-type–specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee, and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype–specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder, and drug addiction. We observe preferential evolutionary divergence in neuron subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergoes subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type–dependent gene-expression programs, and provides a resource for further exploration of human brain evolution and function.
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- 2020
16. Common schizophrenia risk variants are enriched in open chromatin regions of human glutamatergic neurons
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John F. Fullard, Mads E. Hauberg, Sarah Chowdhury, Stella Dracheva, Michael Wegner, Jaroslav Bendl, Chuhyon Corwin, Panos Roussos, Alexey Kozlenkov, Biao Zeng, Yasmin L. Hurd, Anders D. Børglum, Jordi Creus-Muncunill, Harald Kranz, and Michelle E. Ehrlich
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Epigenomics ,0301 basic medicine ,General Physics and Astronomy ,Epigenesis, Genetic ,Mice ,0302 clinical medicine ,Risk Factors ,Epigenetics and behaviour ,GABAergic Neurons ,lcsh:Science ,Promoter Regions, Genetic ,Visual Cortex ,Neurons ,Multidisciplinary ,Human brain ,Chromatin ,Oligodendroglia ,medicine.anatomical_structure ,RNA, Long Noncoding ,Microglia ,Clinical epigenetics ,Cell type ,Science ,Prefrontal Cortex ,Mice, Transgenic ,Biology ,Gyrus Cinguli ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Glutamatergic ,medicine ,Animals ,Humans ,Transcription factor ,General Chemistry ,Epigenome ,Dorsolateral prefrontal cortex ,MicroRNAs ,030104 developmental biology ,Visual cortex ,Gene Expression Regulation ,Astrocytes ,Schizophrenia ,lcsh:Q ,Neuroscience ,030217 neurology & neurosurgery ,Transcription Factors - Abstract
The chromatin landscape of human brain cells encompasses key information to understanding brain function. Here we use ATAC-seq to profile the chromatin structure in four distinct populations of cells (glutamatergic neurons, GABAergic neurons, oligodendrocytes, and microglia/astrocytes) from three different brain regions (anterior cingulate cortex, dorsolateral prefrontal cortex, and primary visual cortex) in human postmortem brain samples. We find that chromatin accessibility varies greatly by cell type and, more moderately, by brain region, with glutamatergic neurons showing the largest regional variability. Transcription factor footprinting implicates cell-specific transcriptional regulators and infers cell-specific regulation of protein-coding genes, long intergenic noncoding RNAs and microRNAs. In vivo transgenic mouse experiments validate the cell type specificity of several of these human-derived regulatory sequences. We find that open chromatin regions in glutamatergic neurons are enriched for neuropsychiatric risk variants, particularly those associated with schizophrenia. Integration of cell-specific chromatin data with a bulk tissue study of schizophrenia brains increases statistical power and confirms that glutamatergic neurons are most affected. These findings illustrate the utility of studying the cell-type-specific epigenome in complex tissues like the human brain, and the potential of such approaches to better understand the genetic basis of human brain function., Here, the authors perform ATAC-seq on four distinct cell populations from three different regions of the human brain, finding that chromatin accessibility varies greatly by cell type and less by brain region. This study reveals differences in biological function and gene regulation, as well as overlap of genetic variants associated with schizophrenia and other neuropsychiatric traits.
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- 2020
17. Evolution of regulatory signatures in primate cortical neurons at cell type resolution
- Author
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Ke Hao, Michael Wegner, Marit W. Vermunt, Stella Dracheva, Patrick R. Hof, Pasha Apontes, Eugene V. Koonin, Menno P. Creyghton, Alexey Kozlenkov, Eran A. Mukamel, John J. Ely, Jun-Hao Li, and Chet C. Sherwood
- Subjects
Regulation of gene expression ,Cell type ,biology ,Human brain ,biology.organism_classification ,Rhesus macaque ,medicine.anatomical_structure ,Histone ,Cerebral cortex ,medicine ,biology.protein ,Neuroscience ,Gene ,Epigenomics - Abstract
The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell type-specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype-specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder and drug addiction. We observe preferential evolutionary divergence in neuron-subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergo subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type-dependent gene expression programs, and provides a resource for further exploration of human brain evolution and function.SIGNIFICANCEThe cerebral cortex of the human brain is a highly complex, heterogeneous tissue that contains many cell types which are exquisitely regulated at the level of gene expression by non-coding regulatory elements, presumably, in a cell-type-dependent manner. However, assessing the regulatory elements in individual cell types is technically challenging, and therefore, most of the previous studies on gene regulation were performed with bulk brain tissue. Here we analyze two major types of neurons isolated from the cerebral cortex of humans, chimpanzees and rhesus macaques, and report complex patterns of cell-type-specific evolution of the regulatory elements in numerous genes. Many genes with evolving regulation are implicated in language abilities as well as psychiatric disorders.
- Published
- 2020
18. Neuronal and glial 3D chromatin architecture illustrates cellular etiology of brain disorders
- Author
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Benxia Hu, Daniel H. Geschwind, Cheynna A. Crowley, Bibi Kassim, Alexey Kozlenkov, Schahram Akbarian, Hyejung Won, Stella Dracheva, Royce Park, Keeley Spiess, Yun Li, Sirisha Pochareddy, Won Mah, and Nenad Sestan
- Subjects
Cell type ,Glutamatergic ,medicine.anatomical_structure ,nervous system ,Gene regulatory network ,medicine ,Epigenetics ,Human brain ,Biology ,Enhancer ,Neuroscience ,Gene ,Chromatin - Abstract
Cellular heterogeneity in the human brain obscures the identification of robust cellular regulatory networks. Here we integrated genome-wide chromosome conformation in purified neurons and glia with transcriptomic and enhancer profiles to build the gene regulatory landscape of two major cell classes in the human brain. Within glutamatergic and GABAergic neurons, we were able to link enhancers to their cognate genes via neuronal chromatin interaction profiles. These cell-type-specific regulatory landscapes were then leveraged to gain insight into the cellular etiology of several brain disorders. We found that Alzheimer’s disease (AD)-associated epigenetic dysregulation was linked to neurons and oligodendrocytes, whereas genetic risk factors for AD highlighted microglia as a central cell type, suggesting that different cell types may confer risk to the disease via different genetic mechanisms. Moreover, neuronal subtype-specific annotation of genetic risk factors for schizophrenia and bipolar disorder identified shared (parvalbumin-expressing interneurons) and distinct cellular etiology (upper layer neurons for bipolar and deeper layer projection neurons for schizophrenia) between these two closely related psychiatric illnesses. Collectively, these findings shed new light on cell-type-specific gene regulatory networks in brain disorders.
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- 2020
19. Neuronal and glial 3D chromatin architecture informs the cellular etiology of brain disorders
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Daniel H. Geschwind, Alexey Kozlenkov, Keeley Spiess, Yun Li, Benxia Hu, Schahram Akbarian, Won Mah, Nenad Sestan, Stella Dracheva, Cheynna A. Crowley, Bibi Kassim, Hyejung Won, Royce Park, and Sirisha Pochareddy
- Subjects
0301 basic medicine ,Epigenomics ,Genetics of the nervous system ,Bipolar Disorder ,Gene regulatory network ,General Physics and Astronomy ,Epigenesis, Genetic ,Histones ,0302 clinical medicine ,2.1 Biological and endogenous factors ,Epigenetics in the nervous system ,Aetiology ,GABAergic Neurons ,Promoter Regions, Genetic ,Neurons ,Multidisciplinary ,Acetylation ,Human brain ,Serious Mental Illness ,Chromatin ,Mental Health ,medicine.anatomical_structure ,Enhancer Elements, Genetic ,Neurological ,Chromatin Immunoprecipitation Sequencing ,Epigenetics ,Neuroglia ,Biotechnology ,Cell type ,Enhancer Elements ,1.1 Normal biological development and functioning ,Science ,PsychENCODE Consortium ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,Promoter Regions ,03 medical and health sciences ,Glutamatergic ,Genetic ,Underpinning research ,Alzheimer Disease ,Genetics ,Acquired Cognitive Impairment ,medicine ,Humans ,Enhancer ,Prevention ,Lysine ,Human Genome ,Neurosciences ,General Chemistry ,Brain Disorders ,030104 developmental biology ,nervous system ,Gene Expression Regulation ,Schizophrenia ,Neuroscience ,030217 neurology & neurosurgery ,Epigenesis ,Genome-Wide Association Study - Abstract
Cellular heterogeneity in the human brain obscures the identification of robust cellular regulatory networks, which is necessary to understand the function of non-coding elements and the impact of non-coding genetic variation. Here we integrate genome-wide chromosome conformation data from purified neurons and glia with transcriptomic and enhancer profiles, to characterize the gene regulatory landscape of two major cell classes in the human brain. We then leverage cell-type-specific regulatory landscapes to gain insight into the cellular etiology of several brain disorders. We find that Alzheimer’s disease (AD)-associated epigenetic dysregulation is linked to neurons and oligodendrocytes, whereas genetic risk factors for AD highlighted microglia, suggesting that different cell types may contribute to disease risk, via different mechanisms. Moreover, integration of glutamatergic and GABAergic regulatory maps with genetic risk factors for schizophrenia (SCZ) and bipolar disorder (BD) identifies shared (parvalbumin-expressing interneurons) and distinct cellular etiologies (upper layer neurons for BD, and deeper layer projection neurons for SCZ). Collectively, these findings shed new light on cell-type-specific gene regulatory networks in brain disorders., The cellular heterogeneity in brain obscures the identification of robust cellular regulatory networks. Here the authors integrate genome-wide chromosome conformation data from sorted neurons and glia, with transcriptomic and enhancer profiles, to characterize cell-type-specific gene regulatory landscapes in the human brain, and provide insights into cell-type-specific gene regulatory networks in brain disorders.
- Published
- 2020
20. Molecular windows into the human brain for psychiatric disorders
- Author
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Alexey Kozlenkov, Yasmin L. Hurd, Stella Dracheva, and Gabor Egervari
- Subjects
0301 basic medicine ,Candidate gene ,medicine.medical_specialty ,Genotype ,Genome-wide association study ,Context (language use) ,Biology ,Article ,Epigenesis, Genetic ,Transcriptome ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,medicine ,Humans ,Genetic Predisposition to Disease ,Epigenetics ,Psychiatry ,Molecular Biology ,Epigenesis ,Genome ,Mental Disorders ,Brain ,Human brain ,Epigenome ,3. Good health ,Psychiatry and Mental health ,Phenotype ,030104 developmental biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Autopsy ,030217 neurology & neurosurgery ,Genome-Wide Association Study - Abstract
Delineating the pathophysiology of psychiatric disorders has been extremely challenging but technological advances in recent decades have facilitated a deeper interrogation of molecular processes in the human brain. Initial candidate gene expression studies of the postmortem brain have evolved into genome wide profiling of the transcriptome and the epigenome, a critical regulator of gene expression. Here, we review the potential and challenges of direct molecular characterization of the post-mortem human brain, and provide a brief overview of recent transcriptional and epigenetic studies with respect to neuropsychiatric disorders. Such information can now be leveraged and integrated with the growing number of genome-wide association databases to provide a functional context of trait-associated genetic variants linked to psychiatric illnesses and related phenotypes. While it is clear that the field is still developing and challenges remain to be surmounted, these recent advances nevertheless hold tremendous promise for delineating the neurobiological underpinnings of mental diseases and accelerating the development of novel medication strategies.
- Published
- 2018
21. TH72. THE ROLE OF DNA (HYDROXY) METHYLATION IN EPIGENETIC REGULATION OF EXCITATORY AND INHIBITORY NEURONS IN THE PREFRONTAL CORTEX OF PATIENTS WITH SCHIZOPHRENIA
- Author
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Eran A. Mukamel, Stella Dracheva, Jo-fan Chien, Jun-Hao Li, and Alexey Kozlenkov
- Subjects
Pharmacology ,Methylation ,Biology ,Inhibitory postsynaptic potential ,medicine.disease ,Psychiatry and Mental health ,chemistry.chemical_compound ,Neurology ,chemistry ,Schizophrenia ,medicine ,Excitatory postsynaptic potential ,Pharmacology (medical) ,Neurology (clinical) ,Epigenetics ,Prefrontal cortex ,Neuroscience ,Biological Psychiatry ,DNA - Published
- 2021
22. Cell Type Specificity of Intralocus Interactions Reveals Oligodendrocyte Intrinsic Mechanisms For Multiple Sclerosis
- Author
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Paul J. Tesar, Robert T. Karl, Drew J. Adams, Tyler E. Miller, Ranjan Dutta, Gursimran Dhillon, Olivia Corradin, An T. Hoang, Parker A. Hall, Bruna R. Lima, Sagar Nisraiyya, Alexey Kozlenkov, Mayur Madhavan, Anna M. Barbeau, Christina Volsko, Cynthia F. Bartels, Peter C. Scacheri, Stella Dracheva, Fadi J. Najm, Kathryn E.A. Hazel, Daniel C. Factor, and Zachary S. Nevin
- Subjects
Autoimmune disease ,Cell type ,Myelin ,medicine.anatomical_structure ,Immune system ,T cell ,Multiple sclerosis ,Central nervous system ,medicine ,Biology ,medicine.disease ,Oligodendrocyte ,Cell biology - Abstract
Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients face progressive disability due to failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue and are associated with white matter microstructure. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development.
- Published
- 2019
23. Genetic variants near MLST8 and DHX57 affect the epigenetic age of the cerebellum
- Author
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Stella Dracheva, Ake T. Lu, Alexey Kozlenkov, Eilis Hannon, Jonathan Mill, Morgan E. Levine, Eileen M. Crimmins, Ke Hao, Steve Horvath, and Katie Lunnon
- Subjects
0301 basic medicine ,Cerebellum ,Aging ,Science ,General Physics and Astronomy ,Genome-wide association study ,Single-nucleotide polymorphism ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,Linkage Disequilibrium ,Cell Line ,Epigenesis, Genetic ,03 medical and health sciences ,medicine ,SNP ,Humans ,Epigenetics ,Adaptor Proteins, Signal Transducing ,Genetics ,Multidisciplinary ,mTOR Associated Protein, LST8 Homolog ,dNaM ,Genetic Variation ,General Chemistry ,030104 developmental biology ,medicine.anatomical_structure ,Gene Expression Regulation ,DNA methylation ,Biomarker (medicine) ,Genome-Wide Association Study - Abstract
DNA methylation (DNAm) levels lend themselves for defining an epigenetic biomarker of aging known as the ‘epigenetic clock'. Our genome-wide association study (GWAS) of cerebellar epigenetic age acceleration identifies five significant (P, This genome-wide association study identifies five significant SNPs in two loci which are associated with the epigenetic age of post-mortem cerebellar tissue according to a DNA methylation based biomarker of human aging.
- Published
- 2016
24. NeuN+ neuronal nuclei in non-human primate prefrontal cortex and subcortical white matter after clozapine exposure
- Author
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Aslihan Dincer, Behnam Javidfar, Jennifer Wiseman, Tobias B. Halene, Schahram Akbarian, Panos Roussos, Royce Park, Patrick R. Hof, Yan Jiang, Amanda C. Mitchell, Stella Dracheva, Alexey Kozlenkov, Eustathia Lela Giannaris, Paula L. Croxson, and Scott E. Hemby
- Subjects
Male ,0301 basic medicine ,Administration, Oral ,Cell Count ,Nerve Tissue Proteins ,behavioral disciplines and activities ,Macaque ,Article ,White matter ,Random Allocation ,03 medical and health sciences ,0302 clinical medicine ,Neurochemical ,biology.animal ,mental disorders ,medicine ,Haloperidol ,Animals ,Gray Matter ,Prefrontal cortex ,Clozapine ,Biological Psychiatry ,Neurons ,Neuronal Plasticity ,biology ,Brain ,Organ Size ,Flow Cytometry ,Immunohistochemistry ,Magnetic Resonance Imaging ,White Matter ,Oligodendroglia ,Psychiatry and Mental health ,030104 developmental biology ,medicine.anatomical_structure ,Frontal lobe ,biology.protein ,Macaca ,Female ,NeuN ,Psychology ,Neuroscience ,030217 neurology & neurosurgery ,Antipsychotic Agents ,medicine.drug - Abstract
Increased neuronal densities in subcortical white matter have been reported for some cases with schizophrenia. The underlying cellular and molecular mechanisms remain unresolved. We exposed 26 young adult macaque monkeys for 6 months to either clozapine, haloperidol or placebo and measured by structural MRI frontal gray and white matter volumes before and after treatment, followed by observer-independent, flow-cytometry-based quantification of neuronal and non-neuronal nuclei and molecular fingerprinting of cell-type specific transcripts. After clozapine exposure, the proportion of nuclei expressing the neuronal marker NeuN increased by approximately 50% in subcortical white matter, in conjunction with a more subtle and non-significant increase in overlying gray matter. Numbers and proportions of nuclei expressing the oligodendrocyte lineage marker, OLIG2, and cell-type specific RNA expression patterns, were maintained after antipsychotic drug exposure. Frontal lobe gray and white matter volumes remained indistinguishable between antipsychotic-drug-exposed and control groups. Chronic clozapine exposure increases the proportion of NeuN+ nuclei in frontal subcortical white matter, without alterations in frontal lobe volumes or cell type-specific gene expression. Further exploration of neurochemical plasticity in non-human primate brain exposed to antipsychotic drugs is warranted.
- Published
- 2016
25. Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS
- Author
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Gursimran Dhillon, Cynthia F. Bartels, Ranjan Dutta, Peter C. Scacheri, Stella Dracheva, Lucille R. Hu, Kevin C. Allan, Fadi J. Najm, Michael D. Gallagher, Anna M. Barbeau, Christina Volsko, Mayur Madhavan, Parker A. Hall, Bruna R. Lima, Kathryn E.A. Hazel, Daniel C. Factor, William Phu, Alexandra G. Sibert, Yanwei Song, Robert T. Karl, Olivia Corradin, An T. Hoang, Drew J. Adams, Sagar Nisraiyya, Alexey Kozlenkov, Zachary S. Nevin, Tyler E. Miller, and Paul J. Tesar
- Subjects
Central Nervous System ,Cell type ,Multiple Sclerosis ,Central nervous system ,Cell Communication ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Myelin ,0302 clinical medicine ,Immune system ,Risk Factors ,medicine ,Animals ,Humans ,Disease ,Remyelination ,Myelin Sheath ,030304 developmental biology ,Epigenomics ,Neurons ,0303 health sciences ,Multiple sclerosis ,Brain ,medicine.disease ,Oligodendrocyte ,Cell biology ,Oligodendroglia ,medicine.anatomical_structure ,030217 neurology & neurosurgery ,Demyelinating Diseases - Abstract
Summary Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. Video Abstract
- Published
- 2020
26. A unique role for DNA (hydroxy)methylation in epigenetic regulation of human inhibitory neurons
- Author
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Alexey Kozlenkov, Stella Dracheva, Michael Wegner, Jun-Hao Li, Eran A. Mukamel, Yasmin L. Hurd, Pasha Apontes, William Byne, and Eugene V. Koonin
- Subjects
Adult ,Male ,0301 basic medicine ,Cell type ,Inhibitory postsynaptic potential ,Polymorphism, Single Nucleotide ,Epigenesis, Genetic ,03 medical and health sciences ,Glutamatergic ,Gene expression ,Cadaver ,Humans ,Epigenetics ,Cell Nucleus ,Neurons ,Regulation of gene expression ,Brain Diseases ,Multidisciplinary ,biology ,Brain ,Neural Inhibition ,DNA Methylation ,Cell biology ,030104 developmental biology ,Histone ,nervous system ,DNA methylation ,biology.protein - Abstract
Brain function depends on interaction of diverse cell types whose gene expression and identity are defined, in part, by epigenetic mechanisms. Neuronal DNA contains two major epigenetic modifications, methylcytosine (mC) and hydroxymethylcytosine (hmC), yet their cell type-specific landscapes and relationship with gene expression are poorly understood. We report high-resolution (h)mC analyses, together with transcriptome and histone modification profiling, in three major cell types in human prefrontal cortex: glutamatergic excitatory neurons, medial ganglionic eminence-derived γ-aminobutyric acid (GABA)ergic inhibitory neurons, and oligodendrocytes. We detected a unique association between hmC and gene expression in inhibitory neurons that differed significantly from the pattern in excitatory neurons and oligodendrocytes. We also found that risk loci associated with neuropsychiatric diseases were enriched near regions of reduced hmC in excitatory neurons and reduced mC in inhibitory neurons. Our findings indicate differential roles for mC and hmC in regulation of gene expression in different brain cell types, with implications for the etiology of human brain diseases.
- Published
- 2018
27. Brain Cell Type Specific Gene Expression and Co-expression Network Architectures
- Author
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Mads E. Hauberg, Panos Roussos, Patrizia Casaccia, Alexey Kozlenkov, Yasmin L. Hurd, John F. Fullard, Minghui Wang, Andrew M. McKenzie, Stella Dracheva, Alexandra B Keenan, and Bin Zhang
- Subjects
0301 basic medicine ,Cell type ,Science ,Cell ,DIVERSITY ,Gene regulatory network ,Computational biology ,Biology ,ASTROCYTES ,MICROGLIA ,Transcriptome ,03 medical and health sciences ,Single-cell analysis ,Gene expression ,medicine ,RNA-SEQ ,TRANSCRIPTOME ,DNA METHYLATION ,Gene ,Multidisciplinary ,CENTRAL-NERVOUS-SYSTEM ,ALZHEIMERS-DISEASE ,Gene expression profiling ,030104 developmental biology ,medicine.anatomical_structure ,Medicine ,STEM-CELLS ,RESPONSES - Abstract
Elucidating brain cell type specific gene expression patterns is critical towards a better understanding of how cell-cell communications may influence brain functions and dysfunctions. We set out to compare and contrast five human and murine cell type-specific transcriptome-wide RNA expression data sets that were generated within the past several years. We defined three measures of brain cell type-relative expression including specificity, enrichment, and absolute expression and identified corresponding consensus brain cell “signatures,” which were well conserved across data sets. We validated that the relative expression of top cell type markers are associated with proxies for cell type proportions in bulk RNA expression data from postmortem human brain samples. We further validated novel marker genes using an orthogonal ATAC-seq dataset. We performed multiscale coexpression network analysis of the single cell data sets and identified robust cell-specific gene modules. To facilitate the use of the cell type-specific genes for cell type proportion estimation and deconvolution from bulk brain gene expression data, we developed an R package, BRETIGEA. In summary, we identified a set of novel brain cell consensus signatures and robust networks from the integration of multiple datasets and therefore transcend limitations related to technical issues characteristic of each individual study.
- Published
- 2018
28. A Novel Method for Rapid Molecular Subgrouping of Medulloblastoma
- Author
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Cinzia Lavarino, Alícia Garrido-Garcia, Volker Hovestadt, Andres Morales La Madrid, Ofelia Cruz, Michael D. Taylor, Soledad Gómez, Jose-Ignacio Martin-Subero, Betty Luu, Stella Dracheva, Alexey Kozlenkov, Sara Perez-Jaume, Laura Garcia-Gerique, Pascal Johann, Isadora Lemos, Angel M. Carcaboso, Mark W. Kieran, David T.W. Jones, Mariona Suñol, Carmen de Torres, Vijay Ramaswamy, Marta Kulis, Nada Jabado, Stefan M. Pfister, and Jaume Mora
- Subjects
Epigenomics ,Male ,0301 basic medicine ,Cancer Research ,Microarray ,Biopsy ,Computational biology ,Biology ,Epigenesis, Genetic ,Metastasis ,03 medical and health sciences ,0302 clinical medicine ,Biomarkers, Tumor ,medicine ,Humans ,Genetic Predisposition to Disease ,Cerebellar Neoplasms ,Genetic Association Studies ,Medulloblastoma ,Microarray analysis techniques ,Gene Expression Profiling ,Reproducibility of Results ,Methylation ,DNA Methylation ,Gene signature ,medicine.disease ,Gene expression profiling ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,DNA methylation ,CpG Islands ,Female - Abstract
Purpose: The classification of medulloblastoma into WNT, SHH, group 3, and group 4 subgroups has become of critical importance for patient risk stratification and subgroup-tailored clinical trials. Here, we aimed to develop a simplified, clinically applicable classification approach that can be implemented in the majority of centers treating patients with medulloblastoma. Experimental Design: We analyzed 1,577 samples comprising previously published DNA methylation microarray data (913 medulloblastomas, 457 non-medulloblastoma tumors, 85 normal tissues), and 122 frozen and formalin-fixed paraffin-embedded medulloblastoma samples. Biomarkers were identified applying stringent selection filters and Linear Discriminant Analysis (LDA) method, and validated using DNA methylation microarray data, bisulfite pyrosequencing, and direct-bisulfite sequencing. Results: Using a LDA-based approach, we developed and validated a prediction method (EpiWNT-SHH classifier) based on six epigenetic biomarkers that allowed for rapid classification of medulloblastoma into the clinically relevant subgroups WNT, SHH, and non-WNT/non-SHH with excellent concordance (>99%) with current gold-standard methods, DNA methylation microarray, and gene signature profiling analysis. The EpiWNT-SHH classifier showed high prediction capacity using both frozen and formalin-fixed material, as well as diverse DNA methylation detection methods. Similarly, we developed a classifier specific for group 3 and group 4 tumors, based on five biomarkers (EpiG3-G4) with good discriminatory capacity, allowing for correct assignment of more than 92% of tumors. EpiWNT-SHH and EpiG3-G4 methylation profiles remained stable across tumor primary, metastasis, and relapse samples. Conclusions: The EpiWNT-SHH and EpiG3-G4 classifiers represent a new simplified approach for accurate, rapid, and cost-effective molecular classification of single medulloblastoma DNA samples, using clinically applicable DNA methylation detection methods. Clin Cancer Res; 24(6); 1355–63. ©2018 AACR.
- Published
- 2018
29. Revealing the brain's molecular architecture
- Author
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Alexey Kozlenkov, Elizabeth Zharovsky, Tyler M. Borrman, Annie W. Shieh, Harm van Bakel, Leonardo Collado-Torres, Yi Jiang, Dominic Fitzgerald, Patrick F. Sullivan, Prashant Emani, Yooree Chae, David A. Lewis, Mo Yang, Joo Heon Shin, Zhen Li, A. Jeremy Willsey, Thomas G. Beach, Robert R. Kitchen, Shannon Schreiner, Barbara K. Lipska, Mingming Niu, Gabriel E. Hoffman, Michael J. Purcaro, Alexander W. Charney, Olivia Devillers, Zhiping Weng, Stephen Sanders, Diane DelValle, Adrian Camarena, Lingyun Song, Fernando S. Goes, Becky C. Carlyle, Nenad Sestan, Valeria N. Spitsyna, Nikolay A. Ivanov, Tarik Hadzic, Lijun Cheng, Peter P. Zandi, Rivka Jacobov, Mimi Brown, Flora M. Vaccarino, Ran Tao, Chang-Gyu Hahn, Mark Gerstein, Soraya Scuderi, Adriana Cherskov, Matthew W. State, Thomas M. Hyde, Kevin P. White, Daifeng Wang, Mario Skarica, Maree J. Webster, Marija Kundakovic, Jennifer R Wiseman, Dalila Pinto, Nancy Francoeur, Alexej Abyzov, Xu Shi, Mohana Ray, Eugenio Mattei, Anna Szekely, Jill Moore, Damon Polioudakis, Allison E. Ashley-Koch, Daniel J. Miller, Jing Zhang, Royce B. Park, Tianliuyun Gao, Donna M. Werling, Patrick Sullivan, Gabriel Santpere, Paola Giusti-Rodríguez, Kay Grennan, Chao Chen, Sirisha Pochareddy, Shuang Liu, Sherman M. Weissman, Joel E. Kleiman, Kiran Girdhar, Leanne Brown, Angus C. Nairn, Mingfeng Li, Heather Witt, Anahita Amiri, Judson Belmont, André M. M. Sousa, Elie Flatow, Lara M. Mangravite, Ying Zhu, Joon Yong An, Amanda J. Price, Mette A. Peters, Melanie E. Garrett, Vivek Swarup, Declan Clarke, Jaroslav Bendl, Oleg V. Evgrafov, Luis de la Torre Ubieta, Alexias Safi, Amira Kefi, Gamze Gürsoy, Majd Alsayed, Xusheng Wang, Jin P. Szatkiewicz, Yunjung Kim, Miguel Brown, Michael J. Gandal, Yongjun Wang, Jinmyung Choi, Tonya M. Brunetti, Yucheng T. Yang, Christoper Armoskus, Brooke Sheppard, Gianfilippo Coppola, Tanmoy Roychowdhury, Timothy E. Reddy, Chunyu Liu, Jack Huey, Mads E. Hauberg, Feinan Wu, Evi Hadjimichael, Peggy J. Farnham, Julien Bryois, Henry S. Pratt, Brie Wamsley, Fabio C. P. Navarro, Graham D. Johnson, Jonathan Warrell, Stella Dracheva, Suhn K. Rhie, Min Xu, Gregory A. Wray, Bibi Kassim, Thomas Goodman, Rujia Dai, Hyejung Won, Junmin Peng, Gregory E. Crawford, Ramu Vadukapuram, Andrew E. Jaffe, Emily E. Burke, Daniel H. Geschwind, James A. Knowles, Gina Giase, Jessica Mariani, Yan Xia, Panos Roussos, Mengting Gu, Jiani Yin, Vahram Haroutunian, John F. Fullard, Schahram Akbarian, and Yan Jiang
- Subjects
Multidisciplinary ,Architecture ,Biology ,Neuroscience - Published
- 2018
30. Antipsychotic-induced Hdac2 transcription via NF-κB leads to synaptic and cognitive side effects
- Author
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Yan Jiang, Carolina Muguruza, Daisuke Ibi, Carlos R. Escalante, Maryum K. Ijaz, Daniel J. Christoffel, Schahram Akbarian, Alexey Kozlenkov, Scott J. Russo, Nebojsa Kezunovic, Vishaka Santosh, Jeremy Seto, Javier González-Maeso, Terrell Holloway, Supriya A Gaitonde, Luis F. Callado, J. Javier Meana, Mario de la Fuente Revenga, José L. Moreno, Stella Dracheva, Mitsumasa Kurita, Grace E. Mosley, George W. Huntley, Juan F. López-Giménez, Aintzane García-Bea, Yongchao Ge, Justin M. Saunders, Japan Society for the Promotion of Science, National Institutes of Health (US), Ministerio de Economía y Competitividad (España), European Commission, Eusko Jaurlaritza, Uehara Memorial Foundation for International Students, and Icahn School of Medicine at Mount Sinai
- Subjects
0301 basic medicine ,Male ,Transcriptional Activation ,Mice, 129 Strain ,medicine.medical_treatment ,Repressor ,Histone Deacetylase 2 ,Mice, Transgenic ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Downregulation and upregulation ,medicine ,Animals ,Humans ,Antipsychotic ,Maze Learning ,Mice, Knockout ,General Neuroscience ,HEK 293 cells ,NF-kappa B ,NF-κB ,Frontal Lobe ,Mice, Inbred C57BL ,IκBα ,030104 developmental biology ,HEK293 Cells ,chemistry ,Synaptic plasticity ,Forebrain ,Synapses ,Psychology ,Cognition Disorders ,Neuroscience ,030217 neurology & neurosurgery ,Antipsychotic Agents - Abstract
Ibi, Daisuke et al., Antipsychotic drugs remain the standard for schizophrenia treatment. Despite their effectiveness in treating hallucinations and delusions, prolonged exposure to antipsychotic medications leads to cognitive deficits in both schizophrenia patients and animal models. The molecular mechanisms underlying these negative effects on cognition remain to be elucidated. Here we demonstrate that chronic antipsychotic drug exposure increases nuclear translocation of NF-κB in both mouse and human frontal cortex, a trafficking event triggered via 5-HT-receptor-dependent downregulation of the NF-κB repressor IκBα. This upregulation of NF-κB activity led to its increased binding at the Hdac2 promoter, thereby augmenting Hdac2 transcription. Deletion of HDAC2 in forebrain pyramidal neurons prevented the negative effects of antipsychotic treatment on synaptic remodeling and cognition. Conversely, virally mediated activation of NF-κB signaling decreased cortical synaptic plasticity via HDAC2. Together, these observations may aid in developing therapeutic strategies to improve the outcome of schizophrenia treatment., NIH R01 MH084894 (J.G.M.), NIH R01 MH111940 (J.G.M.), Dainippon Sumitomo Pharma (J.G.M.), NARSAD (J.G.M.), the Japan Society for the Promotion of Science (JSPS) 15H06719 and 16K19786 (D.I.), NIH R01 MH104491 (G.W.H.), NIH R01 MH086509 (S.A.), NIH P50 MH096890 (S.A.), MINECO/ERDF SAF2009-08460 (J.J.M. and L.F.C.), SAF2013-45084R (J.J.M. and L.F.C.), Basque Government IT616-13 (J.J.M.), NIH R21 MH103877 (S.D.) and NIH R01 MH090264 (S.J.R.) participated in the funding of this study. RNA-seq analysis was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai and the NIH infrastructure grant S10OD018522. C.M. and A.G.B. were recipients of a postdoctoral and a predoctoral fellowship from the Basque Government, respectively. D.I. was a recipient of postdoctoral fellowships from JSPS (Young Scientists JSPS 23-3454) and the Uehara Memorial Foundation.
- Published
- 2017
31. A unique gene expression signature associated with serotonin 2C receptor RNA editing in the prefrontal cortex and altered in suicide
- Author
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Panos Roussos, Yasmin L. Hurd, Antonio Fabio Di Narzo, Stella Dracheva, Alexey Kozlenkov, Larry J. Siever, Eugene V. Koonin, Etienne Sibille, Ke Hao, and David A. Lewis
- Subjects
Gene isoform ,Prefrontal Cortex ,Poison control ,Biology ,Neurotransmitter receptor ,Gene expression ,Receptor, Serotonin, 5-HT2C ,Genetics ,Humans ,Gene Regulatory Networks ,Molecular Biology ,Gene ,Genetics (clinical) ,Neurons ,Depressive Disorder, Major ,Gene Expression Profiling ,Articles ,General Medicine ,DNA Methylation ,Gene expression profiling ,Suicide ,RNA editing ,Case-Control Studies ,DNA methylation ,Autopsy ,RNA Editing - Abstract
Editing of the pre-mRNA for the serotonin receptor 2C (5-HT2CR) by site-specific adenosine deamination (A-to-I pre-mRNA editing) substantially increases the functional plasticity of this key neurotransmitter receptor and is thought to contribute to homeostatic mechanisms in neurons. 5-HT2CR mRNA editing generates up to 24 different receptor isoforms. The extent of editing correlates with 5-HT2CR functional activity: more highly edited isoforms exhibit the least function. Altered 5-HT2CR editing has been reported in postmortem brains of suicide victims. We report a comparative analysis of the connections among 5-HT2CR editing, genome-wide gene expression and DNA methylation in suicide victims, individuals with major depressive disorder and non-psychiatric controls. The results confirm previous findings of an overrepresentation of highly edited mRNA variants (which encode hypoactive 5-HT2CR receptors) in the brains of suicide victims. A large set of genes for which the expression level is associated with editing was detected. This signature set of editing-associated genes is significantly enriched for genes that are involved in synaptic transmission, genes that are preferentially expressed in neurons, and genes whose expression is correlated with the level of DNA methylation. Notably, we report that the link between 5-HT2CR editing and gene expression is disrupted in suicide victims. The results suggest that the postulated homeostatic function of 5-HT2CR editing is dysregulated in individuals who committed suicide.
- Published
- 2014
32. Differences in DNA methylation between human neuronal and glial cells are concentrated in enhancers and non-CpG sites
- Author
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Eugene V. Koonin, Antonio Fabio Di Narzo, Yasmin L. Hurd, Mihaela Barbu, Panos Roussos, Sergei Rudchenko, Stella Dracheva, Brandy Klotzle, Marina Bibikova, William Byne, Alisa Timashpolsky, Alexey Kozlenkov, and Rebecca Lyddon
- Subjects
Adult ,Male ,Gene Expression ,Gene Regulation, Chromatin and Epigenetics ,Biology ,Evolution, Molecular ,Mice ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,Animals ,Humans ,Nucleotide Motifs ,Enhancer ,Gene ,030304 developmental biology ,Cell Nucleus ,Neurons ,0303 health sciences ,Binding Sites ,Genome, Human ,Brain ,Promoter ,Methylation ,DNA Methylation ,Molecular biology ,Enhancer Elements, Genetic ,nervous system ,CpG site ,Reduced representation bisulfite sequencing ,DNA methylation ,CpG Islands ,Human genome ,Transcription Initiation Site ,Neuroglia ,030217 neurology & neurosurgery ,Transcription Factors - Abstract
We applied Illumina Human Methylation450K array to perform a genomic-scale single-site resolution DNA methylation analysis in neuronal and nonneuronal (primarily glial) nuclei separated from the orbitofrontal cortex of postmortem human brain. The findings were validated using enhanced reduced representation bisulfite sequencing. We identified thousands of sites differentially methylated (DM) between neuronal and nonneuronal cells. The DM sites were depleted within CpG-island-containing promoters but enriched in predicted enhancers. Classification of the DM sites into those undermethylated in neurons (neuronal type) and those undermethylated in nonneuronal cells (glial type), combined with findings of others that methylation within control elements typically negatively correlates with gene expression, yielded large sets of predicted neuron-specific and non-neuron-specific genes. These sets of predicted genes were in excellent agreement with the available direct measurements of gene expression in human and mouse. We also found a distinct set of DNA methylation patterns that were unique for neuronal cells. In particular, neuronal-type differential methylation was overrepresented in CpG island shores, enriched within gene bodies but not in intergenic regions, and preferentially harbored binding motifs for a distinct set of transcription factors, including neuron-specific activity-dependent factors. Finally, non-CpG methylation was substantially more prevalent in neurons than in nonneuronal cells.
- Published
- 2013
33. The HIV Antiretroviral Drug Efavirenz has LSD-Like Properties
- Author
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Michael B Gatch, Alexey Kozlenkov, Ren-Qi Huang, Wenjuan Yang, Jacques D Nguyen, Javier González-Maeso, Kenner C Rice, Charles P France, Glenn H Dillon, Michael J Forster, and John A Schetz
- Subjects
Cyclopropanes ,Male ,Hallucinogen ,Efavirenz ,Anti-HIV Agents ,Receptors, Cell Surface ,Pharmacology ,Vesicular monoamine transporter 2 ,Rats, Sprague-Dawley ,Mice ,chemistry.chemical_compound ,Discrimination, Psychological ,Conditioning, Psychological ,medicine ,Animals ,Receptor, Serotonin, 5-HT2A ,Serotonin transporter ,Lysergic acid diethylamide ,Dopamine transporter ,Behavior, Animal ,biology ,virus diseases ,Methamphetamine ,Conditioned place preference ,Benzoxazines ,Rats ,Lysergic Acid Diethylamide ,Psychiatry and Mental health ,chemistry ,Alkynes ,Hallucinogens ,biology.protein ,Original Article ,Psychology ,Serotonin 5-HT2 Receptor Agonists ,medicine.drug - Abstract
Anecdotal reports have surfaced concerning misuse of the HIV antiretroviral medication efavirenz ((4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one) by HIV patients and non-infected teens who crush the pills and smoke the powder for its psychoactive effects. Molecular profiling of the receptor pharmacology of efavirenz pinpointed interactions with multiple established sites of action for other known drugs of abuse including catecholamine and indolamine transporters, and GABAA and 5-HT(2A) receptors. In rodents, interaction with the 5-HT(2A) receptor, a primary site of action of lysergic acid diethylamine (LSD), appears to dominate efavirenz's behavioral profile. Both LSD and efavirenz reduce ambulation in a novel open-field environment. Efavirenz occasions drug-lever responding in rats discriminating LSD from saline, and this effect is abolished by selective blockade of the 5-HT(2A) receptor. Similar to LSD, efavirenz induces head-twitch responses in wild-type, but not in 5-HT(2A)-knockout, mice. Despite having GABAA-potentiating effects (like benzodiazepines and barbiturates), and interactions with dopamine transporter, serotonin transporter, and vesicular monoamine transporter 2 (like cocaine and methamphetamine), efavirenz fails to maintain responding in rats that self-administer cocaine, and it fails to produce a conditioned place preference. Although its molecular pharmacology is multifarious, efavirenz's prevailing behavioral effect in rodents is consistent with LSD-like activity mediated via the 5-HT(2A) receptor. This finding correlates, in part, with the subjective experiences in humans who abuse efavirenz and with specific dose-dependent adverse neuropsychiatric events, such as hallucinations and night terrors, reported by HIV patients taking it as a medication.
- Published
- 2013
34. Repressive Epigenetic Changes at themGlu2Promoter in Frontal Cortex of 5-HT2AKnockout Mice
- Author
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Aintzane García-Bea, Eric J. Nestler, James B. Hanks, José L. Moreno, Terrell Holloway, Rachael L. Neve, Mitsumasa Kurita, Giuseppe Mocci, Scott J. Russo, Alexey Kozlenkov, and Javier González-Maeso
- Subjects
Epigenetics in learning and memory ,Receptors, Metabotropic Glutamate ,Epigenesis, Genetic ,Histones ,Mice ,Histone H3 ,Animals ,Receptor, Serotonin, 5-HT2A ,Epigenetics ,Promoter Regions, Genetic ,Transcription factor ,Early Growth Response Protein 1 ,Mice, Knockout ,Pharmacology ,biology ,Promoter ,Articles ,DNA Methylation ,Molecular biology ,Frontal Lobe ,Histone ,DNA methylation ,Knockout mouse ,biology.protein ,Molecular Medicine ,Protein Processing, Post-Translational ,Protein Binding - Abstract
Serotonin 5-HT(2A) and metabotropic glutamate 2 (mGlu2) are G protein-coupled receptors suspected in the pathophysiology of psychiatric disorders, such as schizophrenia, depression, and suicide. Previous findings demonstrate that mGlu2 mRNA expression is down-regulated in brain cortical regions of 5-HT2A knockout (KO) mice. However, the molecular mechanism responsible for this alteration remains unknown. We show here repressive epigenetic changes at the promoter region of the mGlu2 gene in frontal cortex of 5-HT(2A)-KO mice. Disruption of 5-HT(2A) receptor-dependent signaling in mice was associated with decreased acetylation of histone H3 (H3ac) and H4 (H4ac) and increased tri-methylation of histone H3 at lysine 27 (H3K27me3) at the mGlu2 promoter, epigenetic changes that correlate with transcriptional repression. Neither methylation of histone H3 at lysine 4 (H3K4me1/2/3) nor tri-methylation of histone H3 at lysine 9 (H3K9me3) was affected. We found that Egr1, a transcription factor in which promoter activity was positively regulated by the 5-HT(2A) receptor agonist 4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide, binds less to the mGlu2 promoter in frontal cortex of 5-HT(2A)-KO, compared with wild-type mice. Furthermore, expression of mGlu2 was increased by viral-mediated gene transfer of FLAG-tagged Egr1 in mouse frontal cortex. Together, these observations suggest that 5-HT(2A) receptor-dependent signaling epigenetically affects mGlu2 transcription in mouse frontal cortex.
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- 2013
35. Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia
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Graeme Milligan, Patricia Miranda-Azpiazu, Anastasios Georgakopoulos, Meng Cui, Ariel Ben-Ezra, Georgios Voloudakis, Nikolaos K. Robakis, Diomedes E. Logothetis, Aintzane García-Bea, Juan F. López-Giménez, Alexey Kozlenkov, Lia Baki, Javier González-Maeso, Jason Younkin, Yongchao Ge, José L. Moreno, Amanda K. Fakira, Jose A. Morón, J. Javier Meana, Universidad del País Vasco, Medical Research Council (UK), Ministerio de Ciencia e Innovación (España), Eusko Jaurlaritza, Ministerio de Economía y Competitividad (España), and National Institutes of Health (US)
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0301 basic medicine ,Receptor complex ,GTPase-activating protein ,Biology ,GTP-Binding Protein alpha Subunits, Gi-Go ,Receptors, Metabotropic Glutamate ,Biochemistry ,Rhodopsin-like receptors ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Allosteric Regulation ,Heterotrimeric G protein ,Animals ,Humans ,Receptor, Serotonin, 5-HT2A ,Receptor ,Molecular Biology ,G protein-coupled receptor ,Mice, Knockout ,Cell Biology ,Cell biology ,G beta-gamma complex ,030104 developmental biology ,Metabotropic receptor ,HEK293 Cells ,Schizophrenia ,GTP-Binding Protein alpha Subunits, Gq-G11 ,Protein Multimerization ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
PMCID: PMC4819166.-- Moreno et al., Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Previous findings demonstrated that the G-coupled serotonin 5-HTA receptor and the G-coupled metabotropic glutamate 2 (mGlu2) receptor-GPCRs that are involved in signaling alterations associated with psychosis-assemble into a heteromeric complex in the mammalian brain. In single-cell experiments with various mutant versions of the mGlu2 receptor, we showed that stimulation of cells expressing mGlu2-5-HT heteromers with an mGlu2 agonist led to activation of G proteins by the 5-HTA receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to G proteins, and we determined the relative location of the G-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of G, but not G, was reduced in the frontal cortex of 5-HT knockout mice and was reduced in the frontal cortex of postmortem brains from schizophrenic patients. These findings offer structural insights into this important target in molecular psychiatry., This work was supported, in whole or in part, by the NIH grants R01MH084894 and R56MH084894 (to J.G.-M.), R01HL59949 (to D.E.L.), R37AG017926 and R01AG008200 (to N.K.R.), R01DA025036 and R01DA027460 (to J.A.M.), R01NS047229 and P50AG05138 (to A.G.), and S10RR027411 (to M.C.). This work was also supported by Dainippon Sumitomo Pharma (to J.G.-M.), Spanish MINECO/EDR Funds SAF2009-68460 and SAF2013-48586R (to J.J.M.), the Basque Government (to J.J.M.), the Spanish Government SAF2010-15663 grant (MICINN) (to J.F.L.G.), and Medical Research Council (UK) grants MR/L023806/1 and G0900050 (to G.M.). P.M.-A. and A.G.-B. were recipients of predoctoral fellowships from UPV/EHU and the Basque Government in Spain, respectively.
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- 2016
36. A stomatin dimer modulates the activity of acid-sensing ion channels
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Liudmila Lapatsina, Ewan St. John Smith, Joachim Behlke, Alexey Kozlenkov, Oliver Daumke, Kate Poole, David Schwefel, Janko Brand, Gary R. Lewin, and Damir Omerbašić
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General Immunology and Microbiology ,General Neuroscience ,Dimer ,Biology ,Ligand (biochemistry) ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,chemistry.chemical_compound ,Membrane ,Protein structure ,Membrane protein ,chemistry ,Stomatin ,Molecular Biology ,Acid-sensing ion channel ,Ion channel - Abstract
Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, we determined three crystal structures of the conserved stomatin domain of mouse stomatin that assembles into a banana-shaped dimer. We show that dimerization is crucial for the repression of acid-sensing ion channel 3 (ASIC3) activity. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 activity. In one crystal form, stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerization surfaces are also essential for the inhibition of ASIC3-mediated currents. The assembly mode of stomatin uncovered in this study might serve as a model to understand oligomerization processes of related membrane-remodelling proteins, such as flotillin and prohibitin.
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- 2012
37. Reduced Levels of Serotonin 2A Receptors Underlie Resistance of Egr3-Deficient Mice to Locomotor Suppression by Clozapine
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Joseph Shoker, Scott A Janowski, Alexey Kozlenkov, Wendy Marie Ingram, Amelia L. Gallitano, James R Lish, Diana I. Elizalde, Javier González-Maeso, Christy M. Kamel, Jack Resnik, Alison A. Williams, and Sarah Levine
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Male ,Agonist ,medicine.medical_specialty ,Ketanserin ,medicine.drug_class ,5-HT2A receptor ,Mice, Transgenic ,Motor Activity ,Pharmacology ,Mice ,Piperidines ,Neurotransmitter receptor ,Internal medicine ,medicine ,Animals ,Humans ,Hypnotics and Sedatives ,Receptor, Serotonin, 5-HT2A ,Receptor ,Clozapine ,Early Growth Response Protein 3 ,business.industry ,Disease Models, Animal ,Psychiatry and Mental health ,Endocrinology ,Schizophrenia ,Original Article ,Serotonin Antagonists ,Serotonin ,business ,Immediate early gene ,Serotonin 5-HT2 Receptor Agonists ,medicine.drug - Abstract
The immediate-early gene early growth response 3 (Egr3) is associated with schizophrenia and expressed at reduced levels in postmortem patients' brains. We have previously reported that Egr3-deficient (Egr3(-/-)) mice display reduced sensitivity to the sedating effects of clozapine compared with wild-type (WT) littermates, paralleling the heightened tolerance of schizophrenia patients to antipsychotic side effects. In this study, we have used a pharmacological dissection approach to identify a neurotransmitter receptor defect in Egr3(-/-) mice that may mediate their resistance to the locomotor suppressive effects of clozapine. We report that this response is specific to second-generation antipsychotic agents (SGAs), as first-generation medications suppress the locomotor activity of Egr3(-/-) and WT mice to a similar degree. Further, in contrast to the leading theory that sedation by clozapine results from anti-histaminergic effects, we show that H1 histamine receptors are not responsible for this effect in C57BL/6 mice. Instead, selective serotonin 2A receptor (5HT(2A)R) antagonists ketanserin and MDL-11939 replicate the effect of SGAs, repressing the activity in WT mice at a dosage that fails to suppress the activity of Egr3(-/-) mice. Radioligand binding revealed nearly 70% reduction in 5HT(2A)R expression in the prefrontal cortex of Egr3(-/-) mice compared with controls. Egr3(-/-) mice also exhibit a decreased head-twitch response to 5HT(2A)R agonist 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane (DOI). These findings provide a mechanism to explain the reduced sensitivity of Egr3(-/-) mice to the locomotor suppressive effects of SGAs, and suggest that 5HT(2A)Rs may also contribute to the sedating properties of these medications in humans. Moreover, as the deficit in cortical 5HT(2A)R in Egr3(-/-) mice aligns with numerous studies reporting decreased 5HT(2A)R levels in the brains of schizophrenia patients, and the gene encoding the 5HT(2A)R is itself a leading schizophrenia candidate gene, these findings suggest a potential mechanism by which putative dysfunction in EGR3 in humans may influence risk for schizophrenia.
- Published
- 2012
38. The PsychENCODE project
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Marija Kundakovic, Jonathan Mill, Allison E. Ashley-Koch, Soraya Scuderi, Lijing Yao, Gregory E. Crawford, Shyam Prabhakar, Matthew W. State, Jeremie Poschmann, Nenad Sestan, Dalila Pinto, Nancy Francoeur, Emily Wu, Neelroop N. Parikshak, Angus C. Nairn, Gerhard A. Coetzee, Thomas Lehner, Wenjie Sun, Amanda J. Price, Daifeng Wang, Mario Skarica, Lara M. Mangravite, Anahita Amiri, Gianfilippo Coppola, Andrew E. Jaffe, Alexej Abyzov, Vivek Swarup, Amber Thomas, Chris Armoskus, Shannon Schreiner, Hyejung Won, Andrea Beckel-Mitchener, Yan Jiang, James A. Knowles, Daniel H. Geschwind, Timothy E. Reddy, Pamela Sklar, Annie W. Shieh, Gregory A. Wray, Eirene Markenscoff-Papadimitriou, Chunyu Liu, Eugenio Mattei, Zhiping Weng, Michael J. Purcaro, Alexey Kozlenkov, Sirisha Pochareddy, Donna M. Werling, Shuang Liu, Nicole North, Junko Tsuji, Sherman M. Weissman, Kay Grennan, Mette A. Peters, Harm van Bakel, Heather Witt, Geetha Senthil, David M. Panchision, Yongjun Wang, Robert Sebra, Schahram Akbarian, Flora M. Vaccarino, Mark Gerstein, Menachem Fromer, Mikihito Shibata, Nikolay A. Ivanov, Chloe C. Y. Wong, Patrick F. Sullivan, Larsson Omberg, Kevin P. White, Fabio C. P. Navarro, Robert R. Kitchen, Jennifer Herstein, Benjamin P. Berman, David H. Kavanagh, Kai Wang, Mingfeng Li, Xuming Xu, Zhen Li, A. Jeremy Willsey, Stella Dracheva, Taejeong Bae, Robert Gao, Panos Roussos, and Peggy J. Farnham
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Intellectual and Developmental Disabilities (IDD) ,Autism ,1.1 Normal biological development and functioning ,Disease ,PsychENCODE Consortium ,Biology ,Genome ,Article ,Epigenesis, Genetic ,Genetic ,Underpinning research ,medicine ,Genetics ,2.1 Biological and endogenous factors ,Psychology ,Animals ,Humans ,Bipolar disorder ,Aetiology ,Gene ,Epigenomics ,Regulation of gene expression ,Neurology & Neurosurgery ,General Neuroscience ,Mental Disorders ,Human Genome ,Neurosciences ,Brain ,Chromosome Mapping ,medicine.disease ,Serious Mental Illness ,Brain Disorders ,Mental Health ,Autism spectrum disorder ,Schizophrenia ,Genetic Code ,Cognitive Sciences ,Transcriptome ,Neuroscience ,Epigenesis ,Biotechnology - Abstract
Recent research on disparate psychiatric disorders has implicated rare variants in genes involved in global gene regulation and chromatin modification, as well as many common variants located primarily in regulatory regions of the genome. Understanding precisely how these variants contribute to disease will require a deeper appreciation for the mechanisms of gene regulation in the developing and adult human brain. The PsychENCODE project aims to produce a public resource of multidimensional genomic data using tissue- and cell type–specific samples from approximately 1,000 phenotypically well-characterized, high-quality healthy and disease-affected human post-mortem brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. We are beginning with a focus on autism spectrum disorder, bipolar disorder and schizophrenia, and expect that this knowledge will apply to a wide variety of psychiatric disorders. This paper outlines the motivation and design of PsychENCODE.
- Published
- 2015
39. Substantial DNA methylation differences between two major neuronal subtypes in human brain
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Andrew J. Dwork, Michael Wegner, Sergei Rudchenko, Minghui Wang, Yasmin L. Hurd, Mihaela Barbu, Panos Roussos, Bin Zhang, Stella Dracheva, Brandy Klotzle, Alexey Kozlenkov, Eugene V. Koonin, and Marina Bibikova
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0301 basic medicine ,Adult ,Male ,Glutamic Acid ,Prefrontal Cortex ,Biology ,gamma-Aminobutyric acid ,Epigenesis, Genetic ,03 medical and health sciences ,Risk Factors ,Gene expression ,Genetics ,medicine ,Humans ,Epigenetics ,GABAergic Neurons ,Neurons ,Brain Mapping ,Gene regulation, Chromatin and Epigenetics ,Glutamate receptor ,Methylation ,Human brain ,Microtomy ,DNA Methylation ,Middle Aged ,030104 developmental biology ,medicine.anatomical_structure ,CpG site ,nervous system ,Genetic Loci ,Organ Specificity ,DNA methylation ,Schizophrenia ,CpG Islands ,Autopsy ,medicine.drug - Abstract
The brain is built from a large number of cell types which have been historically classified using location, morphology and molecular markers. Recent research suggests an important role of epigenetics in shaping and maintaining cell identity in the brain. To elucidate the role of DNA methylation in neuronal differentiation, we developed a new protocol for separation of nuclei from the two major populations of human prefrontal cortex neurons—GABAergic interneurons and glutamatergic (GLU) projection neurons. Major differences between the neuronal subtypes were revealed in CpG, non-CpG and hydroxymethylation (hCpG). A dramatically greater number of undermethylated CpG sites in GLU versus GABA neurons were identified. These differences did not directly translate into differences in gene expression and did not stem from the differences in hCpG methylation, as more hCpG methylation was detected in GLU versus GABA neurons. Notably, a comparable number of undermethylated non-CpG sites were identified in GLU and GABA neurons, and non-CpG methylation was a better predictor of subtype-specific gene expression compared to CpG methylation. Regions that are differentially methylated in GABA and GLU neurons were significantly enriched for schizophrenia risk loci. Collectively, our findings suggest that functional differences between neuronal subtypes are linked to their epigenetic specification.
- Published
- 2015
40. Decrease of mRNA Editing after Spinal Cord Injury is Caused by Down-regulation of ADAR2 that is Triggered by Inflammatory Response
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Christopher Cardozo, Zacnicte May, Leo Sanelli, David J. Bennett, Alexey Kozlenkov, Karim Fouad, Stella Dracheva, Bin Zhang, Eugene V. Koonin, Antonio Fabio Di Narzo, Yongchao Ge, and Yanqing Li
- Subjects
Adenosine Deaminase ,Central nervous system ,Down-Regulation ,Bioinformatics ,Article ,Transcriptome ,Rats, Sprague-Dawley ,Adenosine deaminase ,Gene expression ,medicine ,Receptor, Serotonin, 5-HT2C ,Animals ,Receptor, Serotonin, 5-HT2A ,Spasticity ,RNA, Messenger ,Spinal cord injury ,Spinal Cord Injuries ,Multidisciplinary ,biology ,Microglia ,business.industry ,Sequence Analysis, RNA ,Bayes Theorem ,medicine.disease ,Rats ,medicine.anatomical_structure ,RNA editing ,biology.protein ,Female ,RNA Editing ,medicine.symptom ,business ,Kv1.1 Potassium Channel ,Neuroscience - Abstract
We recently showed that spinal cord injury (SCI) leads to a decrease in mRNA editing of serotonin receptor 2C (5-HT2CR) contributing to post-SCI spasticity. Here we study post-SCI mRNA editing and global gene expression using massively parallel sequencing. Evidence is presented that the decrease in 5-HT2CR editing is caused by down-regulation of adenosine deaminase ADAR2 and that editing of at least one other ADAR2 target, potassium channel Kv1.1, is decreased after SCI. Bayesian network analysis of genome-wide transcriptome data indicates that down-regulation of ADAR2 (1) is triggered by persistent inflammatory response to SCI that is associated with activation of microglia and (2) results in changes in neuronal gene expression that are likely to contribute both to post-SCI restoration of neuronal excitability and muscle spasms. These findings have broad implications for other diseases of the Central Nervous System and could open new avenues for developing efficacious antispastic treatments.
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- 2015
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41. Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)
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Alexey Kozlenkov, Charlotte Wennberg, Nils Fröhlander, José Luis Millán, Sonia Di Mauro, Lars Beckman, and Marc Hoylaerts
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chemistry.chemical_classification ,ALPL ,Biology ,Molecular biology ,Isozyme ,chemistry.chemical_compound ,genomic DNA ,Enzyme ,Placental alkaline phosphatase ,chemistry ,Biochemistry ,embryonic structures ,Genetics ,Alkaline phosphatase ,Peptide sequence ,Genetics (clinical) ,DNA - Abstract
The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D alle ...
- Published
- 2002
42. DNA Methylation Profiling of Human Prefrontal Cortex Neurons in Heroin Users Shows Significant Difference between Genomic Contexts of Hyper- and Hypomethylation and a Younger Epigenetic Age
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William Byne, Yasmin L. Hurd, Steve Horvath, Sergei Rudchenko, Andrew E. Jaffe, Pasha Apontes, Stella Dracheva, Alisa Timashpolsky, Alexey Kozlenkov, and Mihaela Barbu
- Subjects
0301 basic medicine ,drug addiction ,lcsh:QH426-470 ,brain ,neurons ,Context (language use) ,Biology ,Axonogenesis ,Article ,Substance Misuse ,03 medical and health sciences ,0302 clinical medicine ,mental disorders ,Genetics ,human ,Epigenetics ,suicide ,Genetics (clinical) ,DNA methylation ,heroin ,Human Genome ,Neurosciences ,dNaM ,Promoter ,16. Peace & justice ,lcsh:Genetics ,Mental Health ,Good Health and Well Being ,030104 developmental biology ,CpG site ,Orbitofrontal cortex ,Drug Abuse (NIDA only) ,030217 neurology & neurosurgery - Abstract
We employed Illumina 450 K Infinium microarrays to profile DNA methylation (DNAm) in neuronal nuclei separated by fluorescence-activated sorting from the postmortem orbitofrontal cortex (OFC) of heroin users who died from heroin overdose (N = 37), suicide completers (N = 22) with no evidence of heroin use and from control subjects who did not abuse illicit drugs and died of non-suicide causes (N = 28). We identified 1298 differentially methylated CpG sites (DMSs) between heroin users and controls, and 454 DMSs between suicide completers and controls (p < 0.001). DMSs and corresponding genes (DMGs) in heroin users showed significant differences in the preferential context of hyper and hypo DM. HyperDMSs were enriched in gene bodies and exons but depleted in promoters, whereas hypoDMSs were enriched in promoters and enhancers. In addition, hyperDMGs showed preference for genes expressed specifically by glutamatergic as opposed to GABAergic neurons and enrichment for axonogenesis- and synaptic-related gene ontology categories, whereas hypoDMGs were enriched for transcription factor activity- and gene expression regulation-related terms. Finally, we found that the DNAm-based "epigenetic age" of neurons from heroin users was younger than that in controls. Suicide-related results were more difficult to interpret. Collectively, these findings suggest that the observed DNAm differences could represent functionally significant marks of heroin-associated plasticity in the OFC.
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- 2017
43. The putative common mechanism for inactivation of alkaline phosphatase isoenzymes
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Martin F. Chaplin, O. M. Poltorak, Alexey Kozlenkov, M.D. Trevan, and E. S. Chukhrai
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chemistry.chemical_classification ,Structural similarity ,Process Chemistry and Technology ,Phosphatase ,Bioengineering ,medicine.disease_cause ,Biochemistry ,Isozyme ,Catalysis ,Amino acid ,Enzyme ,chemistry ,medicine ,Alkaline phosphatase ,Denaturation (biochemistry) ,Escherichia coli - Abstract
Alkaline phosphatase (E.C. 3.1.3.1) is a family of dimeric metalloenzymes with a complex inactivation mechanism that still remains to be elucidated. We have put forward a novel mechanism of Escherichia coli alkaline phosphatase inactivation, based on experimental as well as structural data for this isoenzyme. It suggests several stages of disruption of the intersubunit contact before the loss of enzyme activity. Here we present initial evidence that the mechanism could also be valid for mammalian isoenzymes. The evidence includes thermal inactivation kinetics and the structural similarity of different alkaline phosphatases inferred from the alignment of their amino acid sequences. The suggested inactivation mechanism of alkaline phosphatases is supported by recent experimental data showing an important role of three intersubunit contact areas in determining the stability of alkaline phosphatase isoenzymes.
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- 1999
44. Subunit-specific inhibition of acid sensing ion channels by stomatin-like protein 1
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Alexey, Kozlenkov, Liudmila, Lapatsina, Gary R, Lewin, and Ewan St John, Smith
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Neurons ,Action Potentials ,Nerve Tissue Proteins ,CHO Cells ,Hydrogen-Ion Concentration ,Neuroscience: Cellular/Molecular ,Acid Sensing Ion Channels ,Mice, Inbred C57BL ,Mice ,Protein Subunits ,Cricetulus ,Cricetinae ,Ganglia, Spinal ,Animals ,Cells, Cultured - Abstract
There are five mammalian stomatin-domain genes, all of which encode peripheral membrane proteins that can modulate ion channel function. Here we examined the ability of stomatin-like protein 1 (STOML1) to modulate the proton-sensitive members of the acid-sensing ion channel (ASIC) family. STOML1 profoundly inhibits ASIC1a, but has no effect on the splice variant ASIC1b. The inactivation time constant of ASIC3 is also accelerated by STOML1. We examined STOML1 null mutant mice with a β-galactosidase-neomycin cassette gene-trap reporter driven from the STOML1 gene locus, which indicated that STOML1 is expressed in at least 50% of dorsal root ganglion (DRG) neurones. Patch clamp recordings from mouse DRG neurones identified a trend for larger proton-gated currents in neurones lacking STOML1, which was due to a contribution of effects upon both transient and sustained currents, at different pH, a finding consistent with an endogenous inhibitory function for STOML1.
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- 2013
45. Animal Models and Hallucinogenic Drugs
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Alexey Kozlenkov and Javier González-Maeso
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Hallucinogen ,Psychosis ,Mechanism (biology) ,business.industry ,Mescaline ,medicine.disease ,Psilocybin ,Schizophrenia ,medicine ,Serotonin ,business ,Neuroscience ,medicine.drug ,Lysergic acid diethylamide - Abstract
The neuropsychological effects of naturally occurring psychoactive substances have been recognized for millennia. Hallucinogens, which include naturally occurring chemicals, such as mescaline and psilocybin, as well as synthetic compounds, such as lysergic acid diethylamide (LSD), induce profound alterations of human consciousness, emotion, and cognition. The discovery of the hallucinogenic effects of LSD, and the observations that LSD and the endogenous neurotransmitter serotonin share chemical and pharmacological profiles, led to the suggestion that biogenic amines like serotonin were involved in the psychosis of mental disorders such as schizophrenia. Understanding the mechanism by which hallucinogens elicit unique neurobehavioral effects may open up new avenues in drug abuse research, as well as contributing to the understanding of the endogenous psychosis of psychiatric diseases. Here we summarize recent advances in our understanding of the molecular mechanism of action of hallucinogenic drugs, as well as findings obtained in animal models.
- Published
- 2012
46. Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons
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Paul A. Heppenstall, Alexey Kozlenkov, Liudmila Lapatsina, Ewan St. John Smith, Julia A. Jira, Kate Poole, Daniel Bilbao, and Gary R. Lewin
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Genetics and Molecular Biology (all) ,Sensory Receptor Cells ,Mechanotransduction ,Immunology ,Peripheral sensory neurons ,Nerve Tissue Proteins ,Sensory system ,CHO Cells ,Biology ,Biochemistry ,General Biochemistry, Genetics and Molecular Biology ,Mice ,Cricetulus ,Microtubule ,Cricetinae ,Ganglia, Spinal ,Animals ,Compartment (development) ,Transport Vesicles ,lcsh:QH301-705.5 ,Ion channel ,Mice, Knockout ,Neuroscience (all) ,Research ,General Neuroscience ,Vesicle ,Chinese hamster ovary cell ,Stomatin-like proteins ,Biochemistry, Genetics and Molecular Biology (all) ,Membrane Proteins ,Blood Proteins ,Protein Structure, Tertiary ,Cell biology ,Acid Sensing Ion Channels ,lcsh:Biology (General) ,rab GTP-Binding Proteins ,Multiprotein Complexes ,Stomatin ,Research Article - Abstract
A complex of stomatin-family proteins and acid-sensing (proton-gated) ion channel (ASIC) family members participate in sensory transduction in invertebrates and vertebrates. Here, we have examined the role of the stomatin-family protein stomatin-like protein-3 (STOML3) in this process. We demonstrate that STOML3 interacts with stomatin and ASIC subunits and that this occurs in a highly mobile vesicle pool in dorsal root ganglia (DRG) neurons and Chinese hamster ovary cells. We identify a hydrophobic region in the N-terminus of STOML3 that is required for vesicular localization of STOML3 and regulates physical and functional interaction with ASICs. We further characterize STOML3-containing vesicles in DRG neurons and show that they are Rab11-positive, but not part of the early-endosomal, lysosomal or Rab14-dependent biosynthetic compartment. Moreover, uncoupling of vesicles from microtubules leads to incorporation of STOML3 into the plasma membrane and increased acid-gated currents. Thus, STOML3 defines a vesicle pool in which it associates with molecules that have critical roles in sensory transduction. We suggest that the molecular features of this vesicular pool may be characteristic of a ‘transducosome’ in sensory neurons.
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- 2012
47. A stomatin-domain protein essential for touch sensation in the mouse
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Ombretta Caspani, Paul A. Heppenstall, Andreas Eilers, Gary R. Lewin, Jing Hu, Dieter Riethmacher, Lena Harder, Bettina Erdmann, Anne G. Benckendorff, Dominika Labuz, Halina Machelska, Rabih Moshourab, Christiane Wetzel, and Alexey Kozlenkov
- Subjects
Male ,Protein domain ,Sensory system ,Nerve Tissue Proteins ,Biology ,Sodium Channels ,Mice ,Ganglia, Spinal ,medicine ,Animals ,Acid-sensing ion channel ,Afferent Pathways ,Multidisciplinary ,Mechanosensation ,Electric Conductivity ,Membrane Proteins ,Anatomy ,Sensory neuron ,Protein Structure, Tertiary ,Rats ,Mechanoreceptor ,Acid Sensing Ion Channels ,medicine.anatomical_structure ,Touch ,Mutation ,Mechanosensitive channels ,Female ,Stomatin ,Neuroscience ,Mechanoreceptors - Abstract
Touch and mechanical pain are first detected at our largest sensory surface, the skin. The cell bodies of sensory neurons that detect such stimuli are located in the dorsal root ganglia, and subtypes of these neurons are specialized to detect specific modalities of mechanical stimuli. Molecules have been identified that are necessary for mechanosensation in invertebrates but so far not in mammals. In Caenorhabditis elegans, mec-2 is one of several genes identified in a screen for touch insensitivity and encodes an integral membrane protein with a stomatin homology domain. Here we show that about 35% of skin mechanoreceptors do not respond to mechanical stimuli in mice with a mutation in stomatin-like protein 3 (SLP3, also called Stoml3), a mammalian mec-2 homologue that is expressed in sensory neurons. In addition, mechanosensitive ion channels found in many sensory neurons do not function without SLP3. Tactile-driven behaviours are also impaired in SLP3 mutant mice, including touch-evoked pain caused by neuropathic injury. SLP3 is therefore indispensable for the function of a subset of cutaneous mechanoreceptors, and our data support the idea that this protein is an essential subunit of a mammalian mechanotransducer.
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- 2006
48. Residues determining the binding specificity of uncompetitive inhibitors to tissue-nonspecific alkaline phosphatase
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Alexey Kozlenkov, Marie Hélène Le Du, Tor Ny, Philippe Cuniasse, José Luis Millán, and Marc Hoylaerts
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Models, Molecular ,Phosphoric monoester hydrolases ,Endocrinology, Diabetes and Metabolism ,Phenylalanine ,Placenta ,Mice, Transgenic ,Plasma protein binding ,Biology ,Binding, Competitive ,Bone and Bones ,Mice ,Theophylline ,Animals ,Humans ,Protein Isoforms ,Orthopedics and Sports Medicine ,Enzyme kinetics ,Binding site ,Enzyme Inhibitors ,Site-directed mutagenesis ,Binding selectivity ,chemistry.chemical_classification ,Binding Sites ,Models, Theoretical ,Alkaline Phosphatase ,Molecular biology ,Homoarginine ,Kinetics ,Enzyme ,chemistry ,Biochemistry ,Levamisole ,Models, Chemical ,Mutagenesis ,Drug Design ,Mutation ,Mutagenesis, Site-Directed ,Alkaline phosphatase ,Software ,Protein Binding - Abstract
Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalities. Here, we used mutagenesis, kinetic analysis, and computer modeling to identify the residues important for the binding of known ALP inhibitors to the TNALP active site. These data will enable drug design efforts aimed at developing improved specific TNALP inhibitors for therapeutic use.We have shown previously that the genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) function leads to amelioration of soft-tissue ossification in mouse models of osteoarthritis and ankylosis (i.e., Enpp1-/- and ank/ank mutant mice). We surmise that the pharmacologic inhibition of TNALP activity represents a viable therapeutic approach for these diseases. As a first step toward developing suitable TNALP therapeutics, we have now clarified the residues involved in binding well-known uncompetitive inhibitors to the TNALP active site.We compared the modeled 3D structure of TNALP with the 3D structure of human placental alkaline phosphatase (PLALP) and identified the residues that differ between these isozymes within a 12 A radius of the active site, because these isozymes differ significantly in inhibitor specificity. We then used site-directed mutagenesis to substitute TNALP residues to their respective homolog in PLALP. In addition, we mutagenized most of these residues in TNALP to Ala and the corresponding residues in PLALP to their TNALP homolog. All mutants were characterized for their sensitivity toward the uncompetitive inhibitors l-homoarginine (L-hArg), levamisole, theophylline, and l-phenylalanine.We found that the identity of residue 108 in TNALP largely determines the specificity of inhibition by L-hArg. The conserved Tyr-371 is also necessary for binding of L-hArg. In contrast, the binding of levamisole to TNALP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. The main determinant of sensitivity to theophylline is His-434. Thus, we have clarified the location of the binding sites for all three TNALP inhibitors, and we have also been able to exchange inhibitor specificities between TNALP and PLALP. These data will enable drug design efforts aimed at developing improved, selective, and drug-like TNALP inhibitors for therapeutic use.
- Published
- 2004
49. Kinetic characterization of hypophosphatasia mutations with physiological substrates
- Author
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João Martins Pizauro, Lovisa Hessle, José Luis Millán, Thomas Manes, Marc F. Hoylaerts, Sonia Di Mauro, Alexey Kozlenkov, Burnham Inst, Universidade Estadual Paulista (Unesp), Univ Leuven, and Umea Univ
- Subjects
Models, Molecular ,Phosphoric monoester hydrolases ,Endocrinology, Diabetes and Metabolism ,Mutant ,Mutagenesis (molecular biology technique) ,Hypophosphatasia ,Biology ,medicine.disease_cause ,chemistry.chemical_compound ,natural substrates ,missense mutations ,genetic disease ,catalytic efficiency ,medicine ,Humans ,Orthopedics and Sports Medicine ,Pyridoxal phosphate ,Pyrophosphatases ,DNA Primers ,chemistry.chemical_classification ,Mutation ,Base Sequence ,Hydrogen-Ion Concentration ,medicine.disease ,Alkaline Phosphatase ,Kinetics ,Enzyme ,chemistry ,Biochemistry ,Pyridoxal Phosphate ,Alkaline phosphatase ,alkaline phosphatase - Abstract
Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:14:04Z No. of bitstreams: 1 WOS000177048900006.pdf: 2047145 bytes, checksum: 9174288b291249f98e8703eca9bd5c55 (MD5) Made available in DSpace on 2014-02-26T17:14:04Z (GMT). No. of bitstreams: 1 WOS000177048900006.pdf: 2047145 bytes, checksum: 9174288b291249f98e8703eca9bd5c55 (MD5) Previous issue date: 2002-08-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:28:06Z No. of bitstreams: 1 WOS000177048900006.pdf: 2047145 bytes, checksum: 9174288b291249f98e8703eca9bd5c55 (MD5) Made available in DSpace on 2014-05-20T15:28:06Z (GMT). No. of bitstreams: 1 WOS000177048900006.pdf: 2047145 bytes, checksum: 9174288b291249f98e8703eca9bd5c55 (MD5) Previous issue date: 2002-08-01 We have analyzed 16 missense mutations of the tissue-nonspecific AP (TNAP) gene found in patients with hypophosphatasia. These mutations span the phenotypic spectrum of the disease, from the lethal perinatal/infantile forms to the less severe adult and odontohypophosphatasia. Site-directed mutagenesis was used to introduce a sequence tag into the TNAP cDNA and eliminate the glycosylphosphatidylinositol (GPI)-anchor recognition sequence to produce a secreted epitope-tagged TNAP (setTNAP). The properties of GPI-anchored TNAP (gpiTNAP) and setTNAP were found comparable. After introducing each single hypophosphatasia mutation, the setTNAP and mutant TNAP cDNAs were expressed in COS-1 cells and the recombinant flagged enzymes were affinity purified. We characterized the kinetic behavior, inhibition, and heat stability properties of each mutant using the artificial substrate p-nitrophenylphosphate (pNPP) at pH 9.8. We also determined the ability of the mutants to metabolize two natural substrates of TNAP, that is, pyridoxal-5'-phosphate (PLP) and inorganic pyrophosphate (PPi), at physiological pH. Six of the mutant enzymes were completely devoid of catalytic activity (R54C, R54P, A94T, R206W, G317D, and V365I), and 10 others (A16V, A115V, A160T, A162T, E174K, E174G, D277A, E281K, D361V, and G439R) showed various levels of residual activity. The A160T substitution was found to decrease the catalytic efficiency of the mutant enzyme toward pNPP to retain normal activity toward PPi and to display increased activity toward PLP. The A162T substitution caused a considerable reduction in the pNPPase, PPiase, and PLPase activities of the mutant enzyme. The D277A mutant was found to maintain high catalytic efficiency toward pNPP as substrate but not against PLP or PPi. Three mutations ( E174G, E174K, and E281K) were found to retain normal or slightly subnormal catalytic efficiency toward pNPP and PPi but not against PLP. Because abnormalities in PLP metabolism have been shown to cause epileptic seizures in mice null for the TNAP gene, these kinetic data help explain the variable expressivity of epileptic seizures in hypophosphatasia patients. Burnham Inst, La Jolla, CA 92037 USA UNESP, Fac Ciências Agrarias & Vet, São Paulo, Brazil Univ Leuven, Ctr Mol & Vasc Biol, Louvain, Belgium Umea Univ, Dept Med Genet, Umea, Sweden UNESP, Fac Ciências Agrarias & Vet, São Paulo, Brazil
- Published
- 2002
50. Function assignment to conserved residues in mammalian alkaline phosphatases
- Author
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Alexey Kozlenkov, Thomas Manes, José Luis Millán, and Marc Hoylaerts
- Subjects
Models, Molecular ,Time Factors ,Phenylalanine ,Phosphatase ,Biology ,Ligands ,Biochemistry ,Conserved sequence ,Epitopes ,Protein structure ,Leucine ,Animals ,Humans ,Histidine ,Magnesium ,Cysteine ,Disulfides ,Binding site ,Molecular Biology ,Conserved Sequence ,Binding Sites ,Dose-Response Relationship, Drug ,Cell Biology ,Alkaline Phosphatase ,Recombinant Proteins ,Protein Structure, Tertiary ,Kinetics ,Zinc ,Placental alkaline phosphatase ,Spectrometry, Fluorescence ,embryonic structures ,Mutation ,Mutagenesis, Site-Directed ,Alkaline phosphatase ,Tyrosine ,Software ,Protein Binding - Abstract
We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP). Mutations were introduced in wild-type PLAP, i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP. We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP. Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased k(cat) and increased K(m) when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP. All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxyl-terminally located Cys-467-Cys-474 bond plays a lesser structural role. The substitution of the free Cys-101 did not significantly affect the properties of the enzyme. A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit. We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429. The mutations led to a 2-fold decrease in k(cat), a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors l-Phe and l-Leu. Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.
- Published
- 2002
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