- Email: [email protected]
7. Altered MAP2 Phosphorylation and Dendritic Spine Density in Schizophrenia
Biological Psychiatry
Thursday Abstracts
ORAL SESSION Genetic and Molecular Mechanisms of Psychosis Thursday, May 18, 2017, 12:30 PM - 2:30 PM Aqua 310 AB Chair: Thomas Lehner
6. Altered RNA Editing and Behavior in Prenatally Stressed Mice are Reversed by Clozapine Greg Bristow2, Evelyn Nwabuisi-Heath2, Erbo Dong2, Alessandro Guidotti2, and Monsheel Sodhi1 1
Stritch School of Medicine/Loyola University Chicago, University of Illinois at Chicago
2
5. Mitochondrial DNA Haplogroups are Associated with Psychiatric Disease: A Nation-Wide Study of 74,763 Danes Michael Christiansen1, Jonas Bybjerg-Grauholm1, Christian Hagen1, Vanessa Gonçalves2, Marie Bækvad-Hansen1, Christine Hansen1, Paula Hedley1, Jørgen Kanters3, Jimmi Nielsen4, Michael Theisen1, Ole Mors5, James Kennedy6, Thomas Werge7, Merete Nordentoft7, Anders Børglum5, Preben Mortensen5, and David Hougaard1 Statens Serum Institut, 2Centre for Addiction and Mental Health, University of Toronto, 3University of Copenhagen, 4 Aalborg University Hospital, 5Aarhus University, 6CAMH, University of Toronto, 7Mental Health Centre, Capital Region
1
Background: Disturbed mitochondrial function has been implicated in psychiatric disease. Mitochondria contain a maternally inherited DNA (mtDNA) of 16.6 kb. Through evolution and genetic drift haplogroups (hgs) with a characteristic geno-geographical distribution as well as functional and pathological associations, have become fixed. We examined the association between psychiatric disease and mtDNA hgs in the Danish population. Methods: DNA from 50,567 Danish psychiatric patients and 24,196 controls collected as part of the Neonatal Screening program from 1981 - 2005 was genotyped for 418 mtDNA SNPs on the PsychChip (Illumina). For each subject the mtDNA hg was established using signature SNPs from Phylotree. Clinical information was obtained from electronic patient files. Results: Hg M was associated with affective disorder (n 5 17260) with an OR of 0.47 (p 5 1*10E-14), ADHD (n 5 13395) with an OR of 0.50 (p 5 2*10E-11). Among patients belonging to the macro-hg N, patients with schizophrenia (n 5 2589), had a high proportion of hg A (n 5 45), OR: 4.52(p 5 1.2*10E12). In a mitoGWAS, three mitoSNPs were highly associated with affective disorder (p-values: 2*10E-25 – 2*10E-21), ADHD (p-values: 2*10E-11 – 1*10E-10), and anorexia (p-values: 6*10E-8 - 6*10E-7), and another mitoSNP with schizophrenia (p 5 2*10E-11). Conclusions: Psychiatric disease seems to be a bi-genomic disease. Haplogroup A is a risk factor for schizophrenia and haplogroup M is a protective factor for ADHD and affective disorder. However, future research is needed to define the molecular mechanism. The demonstrated association should be included in the genetic dissection of psychiatric disease. Supported By: iPSYCH, Lundbeck Foundation, Denmark Keywords: Schiizophrenia, ADHD, Affective Disorders, Mitochondrial dysfunction, Mitochondrial haplogroup
Background: RNA editing that is catalyzed by ADAR enzymes is a post-transcriptional process and occurs in numerous messenger (m)RNAs that are important for neuronal function, including mRNAs that encode glutamate receptors, calcium and potassium channels. RNA editing has a profound impact on brain development and behavior. Accumulating data reveal that RNA editing plays a role in developmental disorders, mood disorders and inflammation. Methods: Male mice exposed to prenatal stress (PRS) were tested for social interaction behavior and locomotor activity in adulthood (PND75). The effects of haloperidol and clozapine on behavior in PRS and control mice were also measured. The hippocampus was tested for altered RNA editing by QPCR of RNA editing enzymes (ADARs1-3), and targeted next-generation sequencing analysis of the RNA editing levels of 24 genes. We have also performed these tests in the hippocampus of subjects with major depression and subjects without a history of psychiatric illness. Results: We report that mice exposed to PRS had reduced social interaction behavior and reduced hippocampal RNA editing of the AMPAR subunits GluA2-4, the potassium channel Kv1.1, and 5-HT2CR. Social interaction behavior was increased by clozapine, and this behavioral improvement was correlated with the RNA editing of GluA2 in the hippocampus but not in the frontal cortex. No other mRNAs tested had altered RNA editing in the clozapine-treated PRS mice. Analyses of human postmortem subjects are still in progress. Conclusions: Our current data indicate that PRS is associated with many RNA editing alterations in the hippocampal epitranscriptome. Only clozapine alleviated the behavioral deficits induced by PRS. These effects of clozapine, although correlated with ADAR expression, were associated with increased hippocampal RNA editing of GluA2 but no other mRNA targets. Supported By: Chicago Biomedical Consortium Award Keywords: Antipsychotics, Glutamate, Next Generation Sequencing, Hippocampus, Animal Behavior
7. Altered MAP2 Phosphorylation and Dendritic Spine Density in Schizophrenia Megan Garver1, Matthew MacDonald1, Harris Bell Temin1, Ying Ding1, Jason Newman1, David Lewis2, Nathan Yates1, and Robert Sweet1 1 University of Pittsburgh, 2University of Pittsburgh School of Medicine
Background: Lower immunoreactivity for the dendritic microtubule associated protein, MAP2, in the cerebral cortex has been reported in 60% of individuals with schizophrenia. We recently demonstrated that in auditory cortex of subjects with schizophrenia lower MAP2: 1) was associated with dendritic spine
Biological Psychiatry May 15, 2017; 81:S1–S139 www.sobp.org/journal
S3
Biological Psychiatry
Thursday Abstracts
reductions and 2) was not due to reduction in MAP2 protein levels. Hyperphosphorylation of the highly homologous microtubule associated protein, Tau, is known to alter its immunoreactivity and induce dendritic pathology. We therefore hypothesized that MAP2 is abnormally phosphorylated in schizophrenia, and phosphoMAP2 levels correlate with dendritic spine loss. Methods: Whole gray matter was dissected from primary auditory cortex of individuals with schizophrenia and normal comparison subjects matched for age, sex, and postmortem interval, and total protein extracted. Proteins were digested with trypsin and phosphorylated peptides captured with TiO2 and analyzed by nano-flow liquid chromatography tandem mass spectrometry. Extracted ion chromatograms for all peptides were manually inspected and quantified. Temporal and parietal cortex from a cohort of monkeys chronically exposed to haloperidol, olanzapine, or sham were similarly processed and analyzed. Results: Four MAP2 phosphopeptides (of 19 identified) were increased and one decreased in schizophrenia subjects (all p,0.05, Bonferroni corrected). Levels of these phosphopeptides were not altered by antipsychotic exposure in monkeys. Phosphorylations occurred at sites homologous to pathologic phosphorylations in Tau and were significantly correlated with dendritic spine reductions. Conclusions: These findings demonstrate altered MAP2 phosphorylation in schizophrenia. The homology of identified phosphosites with Tau protein provide links to possible mechanisms of dendritic spine loss conserved in schizophrenia and tauopathies. Supported By: MH 071533 and K01 MH 107756 from NIMH; Young Investigator Award from The Brain & Behavior Research Foundation Keywords: Schizophrenia, Human Postmortem Brain, Dendritic spines, cytoskeleton, mass spectrometry
8. CRISPR/Cas9 Genome-Editing of the RERE SuperEnhancer Alters Expression of Genes in Independent Schizophrenia GWAS Regions Cathy Barr, Yu Feng, Aidan Dineen, Karen Wigg, and Ambalika Sarkar Krembil Research Institute Background: The majority of associated markers for psychiatric disorders reside in gene regulatory regions, particularly enhancers and super-enhancers. Enhancers can reside megabases from the gene they regulate (target gene) and their targets are often not the nearest gene. Thus, the assumption that the gene nearest a GWAS-significant marker will be the risk gene will in many cases be incorrect. Methods: To identify the target genes of enhancers with GWAS significant markers, we analyzed Capture-HiC data selecting enhancers for functional studies using CRISPR/Cas9 in human neural precursor cells (hNPCs). The impact on expression was measured by RT-qPCR and RNA-seq in the edited versus the mock-transfected cells. Results: We selected the super-enhancer spanning the 3’ end of the RERE gene for study. Capture-HiC data indicate interactions with RERE (retinoic acid signaling co-repressor/co-activator),
S4
PARK7 (protects neurons from oxidative stress) and PER3. Using CRISPR/Cas9, we deleted a 2kb region in hNPCs. RTqPCR showed RERE and PER3 were reduced in expression however PARK7 was upregulated. RERE, PER3 and PARK7 regulate gene expression thus we used RNA-seq to examine transcriptome changes. 107 genes were differentially expressed, including 14 regulated by retinoic acid. Importantly, 3 of these are located in independent GWAS regions for schizophrenia. Conclusions: Capture-HiC provides important new leads in pinpointing the target genes of enhancer-mediated regulation emanating from the GWAS findings and functional studies confirm altered expression of interacting genes. The finding of altered expression of genes in independent GWAS regions is an important new lead in understanding the regulation of schizophrenia risk genes. Supported By: Krembil Foundation, Ontario Mental Health Foundation Keywords: CRISPR, transcriptome, chromosomal conformation, Schizophrenia, genome-wide association study
9. Polygenic Risk Profile Scores Should Predict Cognition in Schizophrenia but, for Individuals with a Large Premorbid/current IQ Difference, They Don’t Dwight Dickinson1, Sofia Zaidman2, Evan Giangrande2, Daniel Weinberger3, and Karen F. Berman4 National Institutes of Health, 2CTNB, NIMH, NIH, 3Leiber Institute for Brain Development, 4Section on Integrative Neuroimaging, Clinical & Translational Neuroscience Branch, National Institute of Mental Health, Bethesda, MD
1
Background: Prior work indicates that cognitive and schizophrenia genetics overlap in affected individuals and families. We tested whether “cognitive developmental trajectory” subgroups, defined by different patterns of premorbid versus current IQ, showed different associations of schizophrenia polygenic risk profile scores (RPS) with cognitive performance. Methods: Cluster analyses in 550 schizophrenia cases, using premorbid (WRAT) and current (WAIS) IQ as indicators, yielded three groups: 42% with high WRAT and IQ (SzHH), 36% with high WRAT and low IQ (SzHL), and 22% with two low scores (SzLL). Subgroup assignments were applied to 239 unaffected siblings (one per family). Schizophrenia RPS were calculated at 10 p-value thresholds using variants identified by the Psychiatric Genetics Consortium. We tested the association of RPS with general cognitive ability (“g”) by subgroup, controlling for age, sex, and population stratification. Results: Cognitive performance was impaired and RPS scores were elevated in schizophrenia groups relative to controls. Siblings were intermediate. The SzHH showed significant negative associations between RPS and “g” across the more inclusive RPS thresholds (e.g., at RPS_0.05 n5173; p5.002; R250.051). Their siblings showed the same pattern (SibHH at RPS_0.05 n5118; p5.028; R250.042), as did the smaller SzLL subgroup, though non-significantly. The SzHL showed no evidence of RPS association with “g” at any threshold. Conclusions: For many schizophrenia cases and unaffected siblings, RPS are inversely associated with cognition. For
Biological Psychiatry May 15, 2017; 81:S1–S139 www.sobp.org/journal
Thursday Abstracts
ORAL SESSION Genetic and Molecular Mechanisms of Psychosis Thursday, May 18, 2017, 12:30 PM - 2:30 PM Aqua 310 AB Chair: Thomas Lehner
6. Altered RNA Editing and Behavior in Prenatally Stressed Mice are Reversed by Clozapine Greg Bristow2, Evelyn Nwabuisi-Heath2, Erbo Dong2, Alessandro Guidotti2, and Monsheel Sodhi1 1
Stritch School of Medicine/Loyola University Chicago, University of Illinois at Chicago
2
5. Mitochondrial DNA Haplogroups are Associated with Psychiatric Disease: A Nation-Wide Study of 74,763 Danes Michael Christiansen1, Jonas Bybjerg-Grauholm1, Christian Hagen1, Vanessa Gonçalves2, Marie Bækvad-Hansen1, Christine Hansen1, Paula Hedley1, Jørgen Kanters3, Jimmi Nielsen4, Michael Theisen1, Ole Mors5, James Kennedy6, Thomas Werge7, Merete Nordentoft7, Anders Børglum5, Preben Mortensen5, and David Hougaard1 Statens Serum Institut, 2Centre for Addiction and Mental Health, University of Toronto, 3University of Copenhagen, 4 Aalborg University Hospital, 5Aarhus University, 6CAMH, University of Toronto, 7Mental Health Centre, Capital Region
1
Background: Disturbed mitochondrial function has been implicated in psychiatric disease. Mitochondria contain a maternally inherited DNA (mtDNA) of 16.6 kb. Through evolution and genetic drift haplogroups (hgs) with a characteristic geno-geographical distribution as well as functional and pathological associations, have become fixed. We examined the association between psychiatric disease and mtDNA hgs in the Danish population. Methods: DNA from 50,567 Danish psychiatric patients and 24,196 controls collected as part of the Neonatal Screening program from 1981 - 2005 was genotyped for 418 mtDNA SNPs on the PsychChip (Illumina). For each subject the mtDNA hg was established using signature SNPs from Phylotree. Clinical information was obtained from electronic patient files. Results: Hg M was associated with affective disorder (n 5 17260) with an OR of 0.47 (p 5 1*10E-14), ADHD (n 5 13395) with an OR of 0.50 (p 5 2*10E-11). Among patients belonging to the macro-hg N, patients with schizophrenia (n 5 2589), had a high proportion of hg A (n 5 45), OR: 4.52(p 5 1.2*10E12). In a mitoGWAS, three mitoSNPs were highly associated with affective disorder (p-values: 2*10E-25 – 2*10E-21), ADHD (p-values: 2*10E-11 – 1*10E-10), and anorexia (p-values: 6*10E-8 - 6*10E-7), and another mitoSNP with schizophrenia (p 5 2*10E-11). Conclusions: Psychiatric disease seems to be a bi-genomic disease. Haplogroup A is a risk factor for schizophrenia and haplogroup M is a protective factor for ADHD and affective disorder. However, future research is needed to define the molecular mechanism. The demonstrated association should be included in the genetic dissection of psychiatric disease. Supported By: iPSYCH, Lundbeck Foundation, Denmark Keywords: Schiizophrenia, ADHD, Affective Disorders, Mitochondrial dysfunction, Mitochondrial haplogroup
Background: RNA editing that is catalyzed by ADAR enzymes is a post-transcriptional process and occurs in numerous messenger (m)RNAs that are important for neuronal function, including mRNAs that encode glutamate receptors, calcium and potassium channels. RNA editing has a profound impact on brain development and behavior. Accumulating data reveal that RNA editing plays a role in developmental disorders, mood disorders and inflammation. Methods: Male mice exposed to prenatal stress (PRS) were tested for social interaction behavior and locomotor activity in adulthood (PND75). The effects of haloperidol and clozapine on behavior in PRS and control mice were also measured. The hippocampus was tested for altered RNA editing by QPCR of RNA editing enzymes (ADARs1-3), and targeted next-generation sequencing analysis of the RNA editing levels of 24 genes. We have also performed these tests in the hippocampus of subjects with major depression and subjects without a history of psychiatric illness. Results: We report that mice exposed to PRS had reduced social interaction behavior and reduced hippocampal RNA editing of the AMPAR subunits GluA2-4, the potassium channel Kv1.1, and 5-HT2CR. Social interaction behavior was increased by clozapine, and this behavioral improvement was correlated with the RNA editing of GluA2 in the hippocampus but not in the frontal cortex. No other mRNAs tested had altered RNA editing in the clozapine-treated PRS mice. Analyses of human postmortem subjects are still in progress. Conclusions: Our current data indicate that PRS is associated with many RNA editing alterations in the hippocampal epitranscriptome. Only clozapine alleviated the behavioral deficits induced by PRS. These effects of clozapine, although correlated with ADAR expression, were associated with increased hippocampal RNA editing of GluA2 but no other mRNA targets. Supported By: Chicago Biomedical Consortium Award Keywords: Antipsychotics, Glutamate, Next Generation Sequencing, Hippocampus, Animal Behavior
7. Altered MAP2 Phosphorylation and Dendritic Spine Density in Schizophrenia Megan Garver1, Matthew MacDonald1, Harris Bell Temin1, Ying Ding1, Jason Newman1, David Lewis2, Nathan Yates1, and Robert Sweet1 1 University of Pittsburgh, 2University of Pittsburgh School of Medicine
Background: Lower immunoreactivity for the dendritic microtubule associated protein, MAP2, in the cerebral cortex has been reported in 60% of individuals with schizophrenia. We recently demonstrated that in auditory cortex of subjects with schizophrenia lower MAP2: 1) was associated with dendritic spine
Biological Psychiatry May 15, 2017; 81:S1–S139 www.sobp.org/journal
S3
Biological Psychiatry
Thursday Abstracts
reductions and 2) was not due to reduction in MAP2 protein levels. Hyperphosphorylation of the highly homologous microtubule associated protein, Tau, is known to alter its immunoreactivity and induce dendritic pathology. We therefore hypothesized that MAP2 is abnormally phosphorylated in schizophrenia, and phosphoMAP2 levels correlate with dendritic spine loss. Methods: Whole gray matter was dissected from primary auditory cortex of individuals with schizophrenia and normal comparison subjects matched for age, sex, and postmortem interval, and total protein extracted. Proteins were digested with trypsin and phosphorylated peptides captured with TiO2 and analyzed by nano-flow liquid chromatography tandem mass spectrometry. Extracted ion chromatograms for all peptides were manually inspected and quantified. Temporal and parietal cortex from a cohort of monkeys chronically exposed to haloperidol, olanzapine, or sham were similarly processed and analyzed. Results: Four MAP2 phosphopeptides (of 19 identified) were increased and one decreased in schizophrenia subjects (all p,0.05, Bonferroni corrected). Levels of these phosphopeptides were not altered by antipsychotic exposure in monkeys. Phosphorylations occurred at sites homologous to pathologic phosphorylations in Tau and were significantly correlated with dendritic spine reductions. Conclusions: These findings demonstrate altered MAP2 phosphorylation in schizophrenia. The homology of identified phosphosites with Tau protein provide links to possible mechanisms of dendritic spine loss conserved in schizophrenia and tauopathies. Supported By: MH 071533 and K01 MH 107756 from NIMH; Young Investigator Award from The Brain & Behavior Research Foundation Keywords: Schizophrenia, Human Postmortem Brain, Dendritic spines, cytoskeleton, mass spectrometry
8. CRISPR/Cas9 Genome-Editing of the RERE SuperEnhancer Alters Expression of Genes in Independent Schizophrenia GWAS Regions Cathy Barr, Yu Feng, Aidan Dineen, Karen Wigg, and Ambalika Sarkar Krembil Research Institute Background: The majority of associated markers for psychiatric disorders reside in gene regulatory regions, particularly enhancers and super-enhancers. Enhancers can reside megabases from the gene they regulate (target gene) and their targets are often not the nearest gene. Thus, the assumption that the gene nearest a GWAS-significant marker will be the risk gene will in many cases be incorrect. Methods: To identify the target genes of enhancers with GWAS significant markers, we analyzed Capture-HiC data selecting enhancers for functional studies using CRISPR/Cas9 in human neural precursor cells (hNPCs). The impact on expression was measured by RT-qPCR and RNA-seq in the edited versus the mock-transfected cells. Results: We selected the super-enhancer spanning the 3’ end of the RERE gene for study. Capture-HiC data indicate interactions with RERE (retinoic acid signaling co-repressor/co-activator),
S4
PARK7 (protects neurons from oxidative stress) and PER3. Using CRISPR/Cas9, we deleted a 2kb region in hNPCs. RTqPCR showed RERE and PER3 were reduced in expression however PARK7 was upregulated. RERE, PER3 and PARK7 regulate gene expression thus we used RNA-seq to examine transcriptome changes. 107 genes were differentially expressed, including 14 regulated by retinoic acid. Importantly, 3 of these are located in independent GWAS regions for schizophrenia. Conclusions: Capture-HiC provides important new leads in pinpointing the target genes of enhancer-mediated regulation emanating from the GWAS findings and functional studies confirm altered expression of interacting genes. The finding of altered expression of genes in independent GWAS regions is an important new lead in understanding the regulation of schizophrenia risk genes. Supported By: Krembil Foundation, Ontario Mental Health Foundation Keywords: CRISPR, transcriptome, chromosomal conformation, Schizophrenia, genome-wide association study
9. Polygenic Risk Profile Scores Should Predict Cognition in Schizophrenia but, for Individuals with a Large Premorbid/current IQ Difference, They Don’t Dwight Dickinson1, Sofia Zaidman2, Evan Giangrande2, Daniel Weinberger3, and Karen F. Berman4 National Institutes of Health, 2CTNB, NIMH, NIH, 3Leiber Institute for Brain Development, 4Section on Integrative Neuroimaging, Clinical & Translational Neuroscience Branch, National Institute of Mental Health, Bethesda, MD
1
Background: Prior work indicates that cognitive and schizophrenia genetics overlap in affected individuals and families. We tested whether “cognitive developmental trajectory” subgroups, defined by different patterns of premorbid versus current IQ, showed different associations of schizophrenia polygenic risk profile scores (RPS) with cognitive performance. Methods: Cluster analyses in 550 schizophrenia cases, using premorbid (WRAT) and current (WAIS) IQ as indicators, yielded three groups: 42% with high WRAT and IQ (SzHH), 36% with high WRAT and low IQ (SzHL), and 22% with two low scores (SzLL). Subgroup assignments were applied to 239 unaffected siblings (one per family). Schizophrenia RPS were calculated at 10 p-value thresholds using variants identified by the Psychiatric Genetics Consortium. We tested the association of RPS with general cognitive ability (“g”) by subgroup, controlling for age, sex, and population stratification. Results: Cognitive performance was impaired and RPS scores were elevated in schizophrenia groups relative to controls. Siblings were intermediate. The SzHH showed significant negative associations between RPS and “g” across the more inclusive RPS thresholds (e.g., at RPS_0.05 n5173; p5.002; R250.051). Their siblings showed the same pattern (SibHH at RPS_0.05 n5118; p5.028; R250.042), as did the smaller SzLL subgroup, though non-significantly. The SzHL showed no evidence of RPS association with “g” at any threshold. Conclusions: For many schizophrenia cases and unaffected siblings, RPS are inversely associated with cognition. For
Biological Psychiatry May 15, 2017; 81:S1–S139 www.sobp.org/journal