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The epigenetic modification of chromatin structure and its effect on complex neuronal processes like learning and memory is an emerging field in neuroscience. However, little is known about the \"writers\" of the neuronal epigenome and how they lay down the basis for proper cognition. Here, we have dissected the neuronal function of the Drosophila euchromatin histone methyltransferase (EHMT), a member of a conserved protein family that methylates histone 3 at lysine 9 (H3K9). EHMT is widely expressed in the nervous system and other tissues, yet EHMT mutant flies are viable. Neurodevelopmental and behavioral analyses identified EHMT as a regulator of peripheral dendrite development, larval locomotor behavior, non-associative learning, and courtship memory. The requirement for EHMT in memory was mapped to 7B-Gal4 positive cells, which are, in adult brains, predominantly mushroom body neurons. Moreover, memory was restored by EHMT re-expression during adulthood, indicating that cognitive defects are reversible in EHMT mutants. To uncover the underlying molecular mechanisms, we generated genome-wide H3K9 dimethylation profiles by ChIP-seq. Loss of H3K9 dimethylation in EHMT mutants occurs at 5% of the euchromatic genome and is enriched at the 5' and 3' ends of distinct classes of genes that control neuronal and behavioral processes that are corrupted in EHMT mutants. Our study identifies Drosophila EHMT as a key regulator of cognition that orchestrates an epigenetic program featuring classic learning and memory genes. Our findings are relevant to the pathophysiological mechanisms underlying Kleefstra Syndrome, a severe form of intellectual disability caused by mutations in human EHMT1, and have potential therapeutic implications. Our work thus provides novel insights into the epigenetic control of cognition in health and disease.

Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features.

One of the major functions of programmed cell death (apoptosis) is the removal of cells that suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes that, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS promoters at both ends, which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, 7 genes were identified, which suppressed the p53-induced apoptosis. In 4 mutants, the suppression effect resulted from single genes activated by 1 UAS promoter (Pka-R2, Rga, crol, and Spt5). In the other 3 (Orct2, Polr2M, and stg), deleting either UAS promoter eliminated the suppression effect. In qPCR experiments, we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eukaryotic genomes, there are coexpressed gene clusters. Three of the DEP insertion mutants are included, and 2 are in close vicinity of separate coexpressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.

Background GATA zinc finger domain containing 2B (GATAD2B) encodes a subunit of the MeCP1-Mi-2/nucleosome remodelling and deacetylase complex involved in chromatin modification and regulation of transcription. We recently identified two de novo loss-of-function mutations in GATAD2B by whole exome sequencing in two unrelated individuals with severe intellectual disability. Methods To identify additional individuals with GATAD2B aberrations, we searched for microdeletions overlapping with GATAD2B in inhouse and international databases, and performed targeted Sanger sequencing of the GATAD2B locus in a selected cohort of 80 individuals based on an overlap with the clinical features in the two index cases. To address whether GATAD2B is required directly in neurones for cognition and neuronal development, we investigated the role of Drosophila GATAD2B orthologue simjang (simj) in learning and synaptic connectivity. Results We identified a third individual with a 240 kb microdeletion encompassing GATAD2B and a fourth unrelated individual with GATAD2B loss-of-function mutation. Detailed clinical description showed that all four individuals with a GATAD2B aberration had a distinctive phenotype with childhood hypotonia, severe intellectual disability, limited speech, tubular shaped nose with broad nasal tip, short philtrum, sparse hair and strabismus. Neuronal knockdown of Drosophila GATAD2B orthologue, simj, resulted in impaired learning and altered synapse morphology. Conclusions We hereby define a novel clinically recognisable intellectual disability syndrome caused by loss-of-function of GATAD2B. Our results in Drosophila suggest that GATAD2B is required directly in neurones for normal cognitive performance and synapse development.

Most insertional mutagenesis screens of Drosophila performed to date have not used target chromosomes that have been checked for their suitability for phenotypic screens for viable phenotypes. To address this, we have generated a selection of stocks carrying either isogenized second chromosomes or isogenized third chromosomes, in a genetic background derived from a Canton-S wild-type strain. We have tested these stocks for a range of behavioral and other viable phenotypes. As expected, most lines are statistically indistinguishable from Canton-S in most phenotypes tested. The lines generated are now being used as target chromosomes in mutagenesis screens, and the characterization reported here will facilitate their use in screens of these lines for behavioral and other viable phenotypes.

Background: Although habituation is one of the most ancient and fundamental forms of learning, its regulators and relevance for human disease are poorly understood. Methods: We manipulated the orthologs of 286 genes implicated in intellectual disability (ID) with or without comorbid autism spectrum disorder (ASD) specifically in Drosophila neurons, and tested these models in light-off jump habituation. We dissected neuronal substrates underlying the identified habituation deficits and integrated genotype-phenotype annotations, gene ontologies and interaction networks to determine the clinical features and molecular processes that are associated with habituation deficits. Results: We identified more than 100 genes required for habituation learning. For the vast majority of these, 93 genes, a role in habituation learning was previously unknown. These genes characterize ID disorders with macrocephaly/overgrowth and comorbid ASD. Moreover, ASD individuals from the Simons Simplex Collection (SSC) carrying damaging de novo mutations in these genes exhibit increased aberrant behaviors associated with inappropriate, stereotypic speech. At the molecular level, ID genes required for normal habituation are enriched in synaptic function and converge on Ras-MAPK signaling. Both increased Ras-MAPK signaling in GABAergic and decreased Ras-MAPK signaling in cholinergic neurons specifically inhibit the adaptive habituation response. Conclusions: Our work supports the relevance of habituation learning to autism, identifies an unprecedented number of novel habituation players, supports an emerging role for inhibitory neurons in habituation and reveals an opposing, circuit-level-based mechanism for Ras-MAPK signaling. This establishes habituation as a possible, widely applicable functional readout and target for pharmacologic intervention in ID/ASD.

Apoptosis, the programmed cell death, is responsible for the removal of cells seriously damaged or unwanted in development. A major function of apoptosis is the removal of cells which suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of the DEP transposon, a Pelement derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes which, when overexpressed, suppress the p53-activated apoptosis. TheDEP element has Gal4-activatable, outward-directed UAS-promoters at both ends which can be deleted separately in vivo. In theDEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS-promoters, and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, seven genes were identified which suppressed the p53-induced apoptosis. In four mutants, the suppression effect was resulted by single genes activated by one UAS-promoter (Pka-R2, Rga, crol, Spt5). In the other three (Orct2, Polr2M, stg), deleting either UAS-promoter eliminated the suppression effect. In qPCR experiments we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eucaryotic genomes there are co-expressed gene clusters. Three of the DEP insertion mutants are included and two are in close vicinity of separate co-expressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.

Background Consultation-Liaison Psychiatry (CLP) provides services for patients with medical-psychiatric comorbidity at the general hospital. Referral satisfaction is considered as one of the most important outcome measures of CLP interventions. Our aim was to assess the levels of satisfaction with the CLP service amongst medical staff at a university hospital in Denmark. Methods Medical staff answered an online survey regarding their experience with different aspects of inpatient and outpatient CLP services. Results There were 152 responses from 16 medical units, with a survey return rate above 85%. Measured on a 5-point Likert scale, there was a median rating of 4 in response to questions regarding communication and organizational aspects, a median rating of 5 in response to questions regarding overall evaluation of the CLP service on both inpatient and outpatient questionnaire. The questions regarding treatment quality were rated with a median of 4 on the inpatient questionnaire and 2 of the outpatient questionnaire items, and with a median of 5 on 2 outpatient items. Physicians´ evaluations were statistically more positive than nurses´. As a group, respondents already employed before the CLP unit was established and those who used the CLP services more were statistically significantly more satisfied then respondents employed after the establishment of the CLP unit and those who used the CLP service less. Conclusion The CLP services were positively appreciated and considered to be valuable among medical hospital staff. We believe that Consultation-Liaison Psychiatry deserves further help to implement and expand its services in general hospital settings. In addition, our results underline the feasibility of surveys as quality measures of clinical care.

Consultation-Liaison Psychiatry (CLP) provides services for patients with medical-psychiatric comorbidity at the general hospital. Referral satisfaction is considered as one of the most important outcome measures of CLP interventions. Our aim was to assess the levels of satisfaction with the CLP service amongst medical staff at a university hospital in Denmark. Medical staff answered an online survey regarding their experience with different aspects of inpatient and outpatient CLP services. There were 152 responses from 16 medical units, with a survey return rate above 85%. Measured on a 5-point Likert scale, there was a median rating of 4 in response to questions regarding communication and organizational aspects, a median rating of 5 in response to questions regarding overall evaluation of the CLP service on both inpatient and outpatient questionnaire. The questions regarding treatment quality were rated with a median of 4 on the inpatient questionnaire and 2 of the outpatient questionnaire items, and with a median of 5 on 2 outpatient items. Physicians´ evaluations were statistically more positive than nurses´. As a group, respondents already employed before the CLP unit was established and those who used the CLP services more were statistically significantly more satisfied then respondents employed after the establishment of the CLP unit and those who used the CLP service less. The CLP services were positively appreciated and considered to be valuable among medical hospital staff. We believe that Consultation-Liaison Psychiatry deserves further help to implement and expand its services in general hospital settings. In addition, our results underline the feasibility of surveys as quality measures of clinical care.