Publications by authors named "Deepak Srivastava"

241 Publications

Transcriptome-wide association study reveals two genes that influence mismatch negativity.

Cell Rep 2021 Mar;34(11):108868

Division of Psychiatry, University College London, London, UK; Institute of Cognitive Neuroscience, University College London, London, UK; Institute of Psychiatry, Psychology, and Neuroscience, King's College London, London, UK; Camden and Islington NHS Foundation Trust, London, UK. Electronic address:

Mismatch negativity (MMN) is a differential electrophysiological response measuring cortical adaptability to unpredictable stimuli. MMN is consistently attenuated in patients with psychosis. However, the genetics of MMN are uncharted, limiting the validation of MMN as a psychosis endophenotype. Here, we perform a transcriptome-wide association study of 728 individuals, which reveals 2 genes (FAM89A and ENGASE) whose expression in cortical tissues is associated with MMN. Enrichment analyses of neurodevelopmental expression signatures show that genes associated with MMN tend to be overexpressed in the frontal cortex during prenatal development but are significantly downregulated in adulthood. Endophenotype ranking value calculations comparing MMN and three other candidate psychosis endophenotypes (lateral ventricular volume and two auditory-verbal learning measures) find MMN to be considerably superior. These results yield promising insights into sensory processing in the cortex and endorse the notion of MMN as a psychosis endophenotype.
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http://dx.doi.org/10.1016/j.celrep.2021.108868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7972991PMC
March 2021

Effects of chronic exposure to haloperidol, olanzapine or lithium on SV2A and NLGN synaptic puncta in the rat frontal cortex.

Behav Brain Res 2021 May 23;405:113203. Epub 2021 Feb 23.

Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Maurice Wohl Clinical Neuroscience Institute, King's College London, 5 Cutcombe Road, London SE5 9RT, UK; MRC Centre for Neurodevelopmental Disorders, King's College London, London SE1 1UL, UK. Electronic address:

Positron emission tomography studies using the synaptic vesicle glycoprotein 2A (SV2A) radioligand [C]-UCB-J provide in vivo evidence for synaptic dysfunction and/or loss in the cingulate and frontal cortex of patients with schizophrenia. In exploring potential confounding effects of antipsychotic medication, we previously demonstrated that chronic (28-day) exposure to clinically relevant doses of haloperidol does not affect [H]-UCB-J radioligand binding in the cingulate and frontal cortex of male rats. Furthermore, neither chronic haloperidol nor olanzapine exposure had any effect on SV2A protein levels in these brain regions. These data do not exclude the possibility, however, that more subtle changes in SV2A may occur at pre-synaptic terminals, or the post-synaptic density, following chronic antipsychotic drug exposure. Moreover, relatively little is known about the potential effects of psychotropic drugs other than antipsychotics on SV2A. To address these questions directly, we herein used immunostaining and confocal microscopy to explore the effect of chronic (28-day) exposure to clinically relevant doses of haloperidol, olanzapine or the mood stabilizer lithium on presynaptic SV2A, postsynaptic Neuroligin (NLGN) puncta and their overlap as a measure of total synaptic density in the rat prefrontal and anterior cingulate cortex. We found that, under the conditions tested here, exposure to antipsychotics had no effect on SV2A, NLGN, or overall synaptic puncta count. In contrast, chronic lithium exposure significantly increased NLGN puncta density relative to vehicle, with no effect on either SV2A or total synaptic puncta. Future studies are required to understand the functional consequences of these changes.
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http://dx.doi.org/10.1016/j.bbr.2021.113203DOI Listing
May 2021

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids.

Mol Autism 2021 01 22;12(1). Epub 2021 Jan 22.

MRC Centre for Neurodevelopmental Disorders, King's College London, London, UK.

Background: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.

Results: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes.

Conclusion: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.
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http://dx.doi.org/10.1186/s13229-021-00413-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821651PMC
January 2021

Mouse gastruloids take heart.

Nat Rev Cardiol 2021 Apr;18(4):233-234

Gladstone Institutes, San Francisco, CA, USA.

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http://dx.doi.org/10.1038/s41569-020-00501-4DOI Listing
April 2021

Network-based screen in iPSC-derived cells reveals therapeutic candidate for heart valve disease.

Science 2021 02 10;371(6530). Epub 2020 Dec 10.

Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA.

Mapping the gene-regulatory networks dysregulated in human disease would allow the design of network-correcting therapies that treat the core disease mechanism. However, small molecules are traditionally screened for their effects on one to several outputs at most, biasing discovery and limiting the likelihood of true disease-modifying drug candidates. Here, we developed a machine-learning approach to identify small molecules that broadly correct gene networks dysregulated in a human induced pluripotent stem cell (iPSC) disease model of a common form of heart disease involving the aortic valve (AV). Gene network correction by the most efficacious therapeutic candidate, XCT790, generalized to patient-derived primary AV cells and was sufficient to prevent and treat AV disease in vivo in a mouse model. This strategy, made feasible by human iPSC technology, network analysis, and machine learning, may represent an effective path for drug discovery.
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http://dx.doi.org/10.1126/science.abd0724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880903PMC
February 2021

BRD4 (Bromodomain-Containing Protein 4) Interacts with GATA4 (GATA Binding Protein 4) to Govern Mitochondrial Homeostasis in Adult Cardiomyocytes.

Circulation 2020 Dec 23;142(24):2338-2355. Epub 2020 Oct 23.

Gladstone Institute of Cardiovascular Disease, San Francisco, CA (A.P., M.A., B.G.-T., Y.H., A.H., Q.D., S.A.B.W., F.F., J.A.P.-B., S.M.H., D.S.).

Background: Gene regulatory networks control tissue homeostasis and disease progression in a cell type-specific manner. Ubiquitously expressed chromatin regulators modulate these networks, yet the mechanisms governing how tissue specificity of their function is achieved are poorly understood. BRD4 (bromodomain-containing protein 4), a member of the BET (bromo- and extraterminal domain) family of ubiquitously expressed acetyl-lysine reader proteins, plays a pivotal role as a coactivator of enhancer signaling across diverse tissue types in both health and disease and has been implicated as a pharmacological target in heart failure. However, the cell-specific role of BRD4 in adult cardiomyocytes remains unknown.

Methods: We combined conditional mouse genetics, unbiased transcriptomic and epigenomic analyses, and classic molecular biology and biochemical approaches to understand the mechanism by which BRD4 in adult cardiomyocyte homeostasis.

Results: Here, we show that cardiomyocyte-specific deletion of in adult mice leads to acute deterioration of cardiac contractile function with mutant animals demonstrating a transcriptomic signature characterized by decreased expression of genes critical for mitochondrial energy production. Genome-wide occupancy data show that BRD4 enriches at many downregulated genes (including the master coactivators , and their downstream targets) and preferentially colocalizes with GATA4 (GATA binding protein 4), a lineage-determining cardiac transcription factor not previously implicated in regulation of adult cardiac metabolism. BRD4 and GATA4 form an endogenous complex in cardiomyocytes and interact in a bromodomain-independent manner, revealing a new functional interaction partner for BRD4 that can direct its locus and tissue specificity.

Conclusions: These results highlight a novel role for a BRD4-GATA4 module in cooperative regulation of a cardiomyocyte-specific gene program governing bioenergetic homeostasis in the adult heart.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.047753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736290PMC
December 2020

Association of Damaging Variants in Genes With Increased Cancer Risk Among Patients With Congenital Heart Disease.

JAMA Cardiol 2021 Apr;6(4):457-462

Cardiovascular Division, Brigham and Women's Hospital, Boston, Massachusetts.

Importance: Patients with congenital heart disease (CHD), the most common birth defect, have increased risks for cancer. Identification of the variables that contribute to cancer risk is essential for recognizing patients with CHD who warrant longitudinal surveillance and early interventions.

Objective: To compare the frequency of damaging variants in cancer risk genes among patients with CHD and control participants and identify associated clinical variables in patients with CHD who have cancer risk variants.

Design, Setting, And Participants: This multicenter case-control study included participants with CHD who had previously been recruited to the Pediatric Cardiac Genomics Consortium based on presence of structural cardiac anomaly without genetic diagnosis at the time of enrollment. Permission to use published sequencing data from unaffected adult participants was obtained from 2 parent studies. Data were collected for this study from December 2010 to April 2019.

Exposures: Presence of rare (allele frequency, <1 × 10-5) loss-of-function (LoF) variants in cancer risk genes.

Main Outcomes And Measures: Frequency of LoF variants in cancer risk genes (defined in the Catalogue of Somatic Mutations in Cancer-Cancer Gene Consensus database), were statistically assessed by binomial tests in patients with CHD and control participants.

Results: A total of 4443 individuals with CHD (mean [range] age, 13.0 [0-84] years; 2225 of 3771 with reported sex [59.0%] male) and 9808 control participants (mean [range] age, 52.1 [1-92] years; 4967 of 9808 [50.6%] male) were included. The frequency of LoF variants in regulatory cancer risk genes was significantly higher in patients with CHD than control participants (143 of 4443 [3.2%] vs 166 of 9808 [1.7%]; odds ratio [OR], 1.93 [95% CI, 1.54-2.42]; P = 1.38 × 10-12), and among CHD genes previously associated with cancer risk (58 of 4443 [1.3%] vs 18 of 9808 [0.18%]; OR, 7.2 [95% CI, 4.2-12.2]; P < 2.2 × 10-16). The LoF variants were also nominally increased in 14 constrained cancer risk genes with high expression in the developing heart. Seven of these genes (ARHGEF12, CTNNB1, LPP, MLLT4, PTEN, TCF12, and TFRC) harbored LoF variants in multiple patients with unexplained CHD. The highest rates for LoF variants in cancer risk genes occurred in patients with CHD and extracardiac anomalies (248 of 1482 individuals [16.7%]; control: 1099 of 9808 individuals [11.2%]; OR, 1.59 [95% CI, 1.37-1.85]; P = 1.3 × 10-10) and/or neurodevelopmental delay (209 of 1393 individuals [15.0%]; control: 1099 of 9808 individuals [11.2%]; OR, 1.40 [95% CI, 1.19-1.64]; P = 9.6 × 10-6).

Conclusions And Relevance: Genotypes of CHD may account for increased cancer risks. In this cohort, damaging variants were prominent in the 216 genes that predominantly encode regulatory proteins. Consistent with their fundamental developmental functions, patients with CHD and damaging variants in these genes often had extracardiac manifestations. These data may also implicate cancer risk genes that are repeatedly varied in patients with unexplained CHD as CHD genes.
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http://dx.doi.org/10.1001/jamacardio.2020.4947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578917PMC
April 2021

mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm.

Elife 2020 10 15;9. Epub 2020 Oct 15.

Department of Genetics, Harvard Medical School, Boston, United States.

Damaging variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating that activates and that with orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct expression and pancreatic development. Remarkably, an exon 4 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing , and expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct variants.
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http://dx.doi.org/10.7554/eLife.53278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593088PMC
October 2020

Planar Airy beam light-sheet for two-photon microscopy.

Biomed Opt Express 2020 Jul 22;11(7):3927-3935. Epub 2020 Jun 22.

School of Physics and Astronomy, University of Exeter, EX4 4QL, Exeter, UK.

We demonstrate the first planar Airy light-sheet microscope. Fluorescence light-sheet microscopy has become the method of choice to study large biological samples with cellular or sub-cellular resolution. The propagation-invariant Airy beam enables a ten-fold increase in field-of-view with single-photon excitation; however, the characteristic asymmetry of the light-sheet limits its potential for multi-photon excitation. Here we show how a planar light-sheet can be formed from the curved propagation-invariant Airy beam. The resulting symmetric light sheet excites two-photon fluorescence uniformly across an extended field-of-view without the need for deconvolution. We demonstrate the method for rapid two-photon imaging of large volumes of neuronal tissue.
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http://dx.doi.org/10.1364/BOE.395547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510906PMC
July 2020

Dopamine-induced interactions of female mouse hypothalamic proteins with progestin receptor-A in the absence of hormone.

J Neuroendocrinol 2020 10 30;32(10):e12904. Epub 2020 Sep 30.

Neuroscience Department, Wellesley College, Wellesley, MA, USA.

Neural progestin receptors (PR) function in reproduction, neural development, neuroprotection, learning, memory and the anxiety response. In the absence of progestins, PR can be activated by dopamine (DA) in the rodent hypothalamus to elicit female sexual behaviour. The present study investigated mechanisms of DA activation of PR by testing the hypothesis that proteins from DA-treated hypothalami interact with PR in the absence of progestins. Ovariectomised, oestradiol-primed mice were infused with a D1-receptor agonist, SKF38393 (SKF), into the third ventricle 30 minutes prior to death. Proteins from SKF-treated hypothalami were pulled-down with glutathione S-transferase-tagged mouse PR-A or PR-B and the interactomes were analysed by mass spectrometry. The largest functional group to interact with PR-A in a DA-dependent manner was synaptic proteins. To test the hypothesis that DA activation of PR regulates synaptic proteins, we developed oestradiol-induced PR-expressing hypothalamic-like neurones derived from human-induced pluripotent stem cells (hiPSCs). Similar to progesterone (P4), SKF treatment of hiPSCs increased synapsin1/2 expression. This SKF-dependent effect was blocked by the PR antagonist RU486, suggesting that PR are necessary for this DA-induced increase. The second largest DA-dependent PR-A protein interactome comprised metabolic regulators involved in glucose metabolism, lipid synthesis and mitochondrial energy production. Interestingly, hypothalamic proteins interacted with PR-A, but not PR-B, in an SKF-dependent manner, suggesting that DA promotes the interaction of multiple hypothalamic proteins with PR-A. These in vivo and in vitro results indicate novel mechanisms by which DA can differentially activate PR isoforms in the absence of P4 and provide a better understanding of ligand-independent PR activation in reproductive, metabolic and mental health disorders in women.
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http://dx.doi.org/10.1111/jne.12904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591852PMC
October 2020

Interferon-γ signaling in human iPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes.

Sci Adv 2020 Aug 19;6(34):eaay9506. Epub 2020 Aug 19.

Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK.

Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-γ (IFN-γ), in offspring's brains play a central role. IFN-γ activates an antiviral cellular state, limiting viral entry and replication. Moreover, IFN-γ is implicated in brain development. We tested the hypothesis that IFN-γ signaling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. Transient IFN-γ treatment of neural progenitors derived from human induced pluripotent stem cells increased neurite outgrowth. RNA sequencing analysis revealed that major histocompatibility complex class I (MHCI) genes were persistently up-regulated through neuronal differentiation-an effect that was mediated by IFN-γ-induced promyelocytic leukemia protein (PML) nuclear bodies. Critically, IFN-γ-induced neurite outgrowth required both PML and MHCI. We also found evidence that IFN-γ disproportionately altered the expression of genes associated with schizophrenia and autism, suggesting convergence between genetic and environmental risk factors. Together, these data implicate IFN-γ signaling in neurodevelopmental disorder etiology.
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http://dx.doi.org/10.1126/sciadv.aay9506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438100PMC
August 2020

Apolipoprotein E expression pattern in human induced pluripotent stem cells during neural induction.

F1000Res 2020 12;9:353. Epub 2020 May 12.

Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology, and Neuroscience, King's College London, London, SE5 9NU, UK.

Apolipoprotein E (APOE) is a multifunctional protein that plays significant roles in important cellular mechanisms in peripheral tissues and is as well expressed in the central nervous system, notably by adult neural stem cells (NSCs) in the hippocampus. Evidence from animal studies suggest that APOE is critical for adult NSC maintenance. However, whether APOE has the potential to play a similar role in human NSCs has not been directly investigated. To address this question, we conducted a focused study characterising gene and protein expression in an model of neural differentiation utilising human induced pluripotent stem cells. We found that gene expression was dramatically decreased as the cells became more differentiated, indicating that expression levels reflect the degree of cellular differentiation during neural induction. Furthermore, qualitative analysis results of immunocytochemistry showed that intracellular localisation of APOE protein becomes more pronounced as neural differentiation progresses. Taken together, our findings suggest a potential role for APOE in human NSC maintenance and justify further investigations being carried out to understand whether changes in APOE levels can directly impact the neurogenic capacity of human stem cells.
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http://dx.doi.org/10.12688/f1000research.23580.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355222PMC
March 2021

Emerging Developments in Human Induced Pluripotent Stem Cell-Derived Microglia: Implications for Modelling Psychiatric Disorders With a Neurodevelopmental Origin.

Front Psychiatry 2020 11;11:789. Epub 2020 Aug 11.

Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Maurice Wohl Clinical Neuroscience Institute, King's College London, London, United Kingdom.

Microglia, the resident tissue macrophages of the brain, are increasingly implicated in the pathophysiology of psychiatric disorders with a neurodevelopmental origin, including schizophrenia. To date, however, our understanding of the potential role for these cells in schizophrenia has been informed by studies of aged samples, low resolution neuroimaging and rodent models. Whilst these have provided important insights, including signs of the heterogeneous nature of microglia, we currently lack a validated human system to characterize microglia in the context of brain health and disease during neurodevelopment. Primarily, this reflects a lack of access to human primary tissue during developmental stages. In this review, we first describe microglia, including their ontogeny and heterogeneity and consider their role in brain development. We then provide an evaluation of the potential for differentiating microglia from human induced pluripotent stem cells (hiPSCs) as a robust human model system to study these cells. We find the majority of protocols for hiPSC-derived microglia generate cells characteristically similar to foetal stage microglia when exposed to neuronal environment-like cues. This may represent a robust and relevant model for the study of cellular and molecular mechanisms in schizophrenia. Each protocol however, provides unique benefits as well as shortcomings, highlighting the need for context-dependent protocol choice and cross-lab collaboration and communication to identify the most robust and translatable microglia model.
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http://dx.doi.org/10.3389/fpsyt.2020.00789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7433763PMC
August 2020

Atypical Neurogenesis in Induced Pluripotent Stem Cells From Autistic Individuals.

Biol Psychiatry 2021 03 23;89(5):486-496. Epub 2020 Jun 23.

Autism Research Centre, Department of Psychiatry, University of Cambridge, Cambridge, United Kingdom.

Background: Autism is a heterogeneous collection of disorders with a complex molecular underpinning. Evidence from postmortem brain studies have indicated that early prenatal development may be altered in autism. Induced pluripotent stem cells (iPSCs) generated from individuals with autism with macrocephaly also indicate prenatal development as a critical period for this condition. But little is known about early altered cellular events during prenatal stages in autism.

Methods: iPSCs were generated from 9 unrelated individuals with autism without macrocephaly and with heterogeneous genetic backgrounds, and 6 typically developing control individuals. iPSCs were differentiated toward either cortical or midbrain fates. Gene expression and high throughput cellular phenotyping was used to characterize iPSCs at different stages of differentiation.

Results: A subset of autism-iPSC cortical neurons were RNA-sequenced to reveal autism-specific signatures similar to postmortem brain studies, indicating a potential common biological mechanism. Autism-iPSCs differentiated toward a cortical fate displayed impairments in the ability to self-form into neural rosettes. In addition, autism-iPSCs demonstrated significant differences in rate of cell type assignment of cortical precursors and dorsal and ventral forebrain precursors. These cellular phenotypes occurred in the absence of alterations in cell proliferation during cortical differentiation, differing from previous studies. Acquisition of cell fate during midbrain differentiation was not different between control- and autism-iPSCs.

Conclusions: Taken together, our data indicate that autism-iPSCs diverge from control-iPSCs at a cellular level during early stage of neurodevelopment. This suggests that unique developmental differences associated with autism may be established at early prenatal stages.
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http://dx.doi.org/10.1016/j.biopsych.2020.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843956PMC
March 2021

De Novo Damaging Variants, Clinical Phenotypes, and Post-Operative Outcomes in Congenital Heart Disease.

Circ Genom Precis Med 2020 08 30;13(4):e002836. Epub 2020 Jun 30.

Department of Genetics (R.L., M.B.), Yale University School of Medicine, New Haven, CT.

Background: De novo genic and copy number variants are enriched in patients with congenital heart disease, particularly those with extra-cardiac anomalies. The impact of de novo damaging variants on outcomes following cardiac repair is unknown.

Methods: We studied 2517 patients with congenital heart disease who had undergone whole-exome sequencing as part of the CHD GENES study (Congenital Heart Disease Genetic Network).

Results: Two hundred ninety-four patients (11.7%) had clinically significant de novo variants. Patients with de novo damaging variants were 2.4 times more likely to have extra-cardiac anomalies (=5.63×10). In 1268 patients (50.4%) who had surgical data available and underwent open-heart surgery exclusive of heart transplantation as their first operation, we analyzed transplant-free survival following the first operation. Median follow-up was 2.65 years. De novo variants were associated with worse transplant-free survival (hazard ratio, 3.51; =5.33×10) and longer times to final extubation (hazard ratio, 0.74; =0.005). As de novo variants had a significant interaction with extra-cardiac anomalies for transplant-free survival (=0.003), de novo variants conveyed no additional risk for transplant-free survival for patients with these anomalies (adjusted hazard ratio, 1.96; =0.06). By contrast, de novo variants in patients without extra-cardiac anomalies were associated with worse transplant-free survival during follow-up (hazard ratio, 11.21; =1.61×10) than that of patients with no de novo variants. Using agnostic machine-learning algorithms, we identified de novo copy number variants at 15q25.2 and 15q11.2 as being associated with worse transplant-free survival and 15q25.2, 22q11.21, and 3p25.2 as being associated with prolonged time to final extubation.

Conclusions: In patients with congenital heart disease undergoing open-heart surgery, de novo variants were associated with worse transplant-free survival and longer times on the ventilator. De novo variants were most strongly associated with adverse outcomes among patients without extra-cardiac anomalies, suggesting a benefit for preoperative genetic testing even when genetic abnormalities are not suspected during routine clinical practice. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01196182.
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http://dx.doi.org/10.1161/CIRCGEN.119.002836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439931PMC
August 2020

Genomic analyses implicate noncoding de novo variants in congenital heart disease.

Nat Genet 2020 08 29;52(8):769-777. Epub 2020 Jun 29.

Flatiron Institute, Simons Foundation, New York, NY, USA.

A genetic etiology is identified for one-third of patients with congenital heart disease (CHD), with 8% of cases attributable to coding de novo variants (DNVs). To assess the contribution of noncoding DNVs to CHD, we compared genome sequences from 749 CHD probands and their parents with those from 1,611 unaffected trios. Neural network prediction of noncoding DNV transcriptional impact identified a burden of DNVs in individuals with CHD (n = 2,238 DNVs) compared to controls (n = 4,177; P = 8.7 × 10). Independent analyses of enhancers showed an excess of DNVs in associated genes (27 genes versus 3.7 expected, P = 1 × 10). We observed significant overlap between these transcription-based approaches (odds ratio (OR) = 2.5, 95% confidence interval (CI) 1.1-5.0, P = 5.4 × 10). CHD DNVs altered transcription levels in 5 of 31 enhancers assayed. Finally, we observed a DNV burden in RNA-binding-protein regulatory sites (OR = 1.13, 95% CI 1.1-1.2, P = 8.8 × 10). Our findings demonstrate an enrichment of potentially disruptive regulatory noncoding DNVs in a fraction of CHD at least as high as that observed for damaging coding DNVs.
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http://dx.doi.org/10.1038/s41588-020-0652-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415662PMC
August 2020

Systems Analysis Implicates WAVE2 Complex in the Pathogenesis of Developmental Left-Sided Obstructive Heart Defects.

JACC Basic Transl Sci 2020 Apr 8;5(4):376-386. Epub 2020 Apr 8.

Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, New York.

Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to identify causative variants is limited. To identify causal CHD genes that are associated with specific molecular functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe CHD. This approach enabled the authors to identify an association between left ventricular outflow tract obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins , , and and the regulators of small GTPase signaling and are critical to cardiac development.
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http://dx.doi.org/10.1016/j.jacbts.2020.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188873PMC
April 2020

EM-mosaic detects mosaic point mutations that contribute to congenital heart disease.

Genome Med 2020 04 29;12(1):42. Epub 2020 Apr 29.

Columbia University Medical Center, 1130 St Nicholas Ave, New York, NY, 10032, USA.

Background: The contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined.

Methods: We developed a new computational method, EM-mosaic (Expectation-Maximization-based detection of mosaicism), to analyze mosaicism in exome sequences derived primarily from blood DNA of 2530 CHD proband-parent trios. To optimize this method, we measured mosaic detection power as a function of sequencing depth. In parallel, we analyzed our cohort using MosaicHunter, a Bayesian genotyping algorithm-based mosaic detection tool, and compared the two methods. The accuracy of these mosaic variant detection algorithms was assessed using an independent resequencing method. We then applied both methods to detect mosaicism in cardiac tissue-derived exome sequences of 66 participants for which matched blood and heart tissue was available.

Results: EM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The estimated true frequency of mosaic variants above 10% mosaicism was 0.14/person in blood and 0.21/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction.

Conclusions: We estimate that ~ 1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants with relatively higher allele fraction. Although blood is a readily available DNA source, cardiac tissues analyzed contributed ~ 5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses.
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http://dx.doi.org/10.1186/s13073-020-00738-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189690PMC
April 2020

Brain-synthesized oestrogens regulate cortical migration in a sexually divergent manner.

Eur J Neurosci 2020 07 11;52(1):2646-2663. Epub 2020 Jun 11.

Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK.

Oestrogens play an important role in brain development where they have been implicated in controlling various cellular processes. Several lines of evidence have been presented showing that oestrogens can be synthesized locally within the brain. Studies have demonstrated that aromatase, the enzyme responsible for the conversion of androgens to oestrogens, is expressed during early development in both male and female cortices. Furthermore, 17β-oestradiol has been measured in foetal brain tissue from multiple species. 17β-oestradiol regulates neural progenitor proliferation as well as the development of early neuronal morphology. However, what role locally derived oestrogens play in regulating cortical migration and, moreover, whether these effects are the same in males and females are unknown. Here, we investigated the impact of knockdown expression of Cyp19a1, which encodes aromatase, between embryonic day (E) 14.5 and postnatal day 0 (P0) had on neural migration within the cortex. Aromatase was expressed in the developing cortex of both sexes, but at significantly higher levels in male than female mice. Under basal conditions, no obvious differences in cortical migration between male and female mice were observed. However, knockdown of Cyp19a1 resulted in an increase in cells within the cortical plate, and a concurrent decrease in the subventricular zone/ventricular zone in P0 male mice. Interestingly, the opposite effect was observed in females, who displayed a significant reduction in cells migrating to the cortical plate. Together, these findings indicate that brain-derived oestrogens regulate radial migration through distinct mechanisms in males and females.
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http://dx.doi.org/10.1111/ejn.14755DOI Listing
July 2020

Estradiol reverses excitatory synapse loss in a cellular model of neuropsychiatric disorders.

Transl Psychiatry 2020 01 21;10(1):16. Epub 2020 Jan 21.

Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, SE5 9RT, UK.

Loss of glutamatergic synapses is thought to be a key cellular pathology associated with neuropsychiatric disorders including schizophrenia (SCZ) and major depressive disorder (MDD). Genetic and cellular studies of SCZ and MDD using in vivo and in vitro systems have supported a key role for dysfunction of excitatory synapses in the pathophysiology of these disorders. Recent clinical studies have demonstrated that the estrogen, 17β-estradiol can ameliorate many of the symptoms experienced by patients. Yet, to date, our understanding of how 17β-estradiol exerted these beneficial effects is limited. In this study, we have tested the hypothesis that 17β-estradiol can restore dendritic spine number in a cellular model that recapitulates the loss of synapses associated with SCZ and MDD. Ectopic expression of wildtype, mutant or shRNA-mediated knockdown of Disrupted in Schizophrenia 1 (DISC1) reduced dendritic spine density in primary cortical neurons. Acute or chronic treatment with 17β-estradiol increased spine density to control levels in neurons with altered DISC1 levels. In addition, 17β-estradiol reduced the extent to which ectopic wildtype and mutant DISC1 aggregated. Furthermore, 17β-estradiol also caused the enrichment of synaptic proteins at synapses and increased the number of dendritic spines containing PSD-95 or that overlapped with the pre-synaptic marker bassoon. Taken together, our data indicates that estrogens can restore lost excitatory synapses caused by altered DISC1 expression, potentially through the trafficking of DISC1 and its interacting partners. These data highlight the possibility that estrogens exert their beneficial effects in SCZ and MDD in part by modulating dendritic spine number.
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http://dx.doi.org/10.1038/s41398-020-0682-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026123PMC
January 2020

Δ-tetrahydrocannabinol and 2-AG decreases neurite outgrowth and differentially affects ERK1/2 and Akt signaling in hiPSC-derived cortical neurons.

Mol Cell Neurosci 2020 03 7;103:103463. Epub 2020 Jan 7.

Department of Basic and Clinical Neuroscience, The Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry Psychology and Neuroscience, King's College London, London SE5 8AF, UK; MRC Centre for Neurodevelopmental Disorders, King's College London, London, UK. Electronic address:

Endocannabinoids regulate different aspects of neurodevelopment. In utero exposure to the exogenous psychoactive cannabinoid Δ-tetrahydrocannabinol (Δ-THC), has been linked with abnormal cortical development in animal models. However, much less is known about the actions of endocannabinoids in human neurons. Here we investigated the effect of the endocannabinoid 2-arachidonoyl glycerol (2AG) and Δ-THC on the development of neuronal morphology and activation of signaling kinases, in cortical neurons derived from human induced pluripotent stem cells (hiPSCs). Our data indicate that the cannabinoid type 1 receptor (CB1R), but not the cannabinoid 2 receptor (CB2R), GPR55 or TRPV1 receptors, is expressed in young, immature hiPSC-derived cortical neurons. Consistent with previous reports, 2AG and Δ-THC negatively regulated neurite outgrowth. Interestingly, acute exposure to both 2AG and Δ-THC inhibited phosphorylation of serine/threonine kinase extracellular signal-regulated protein kinases (ERK1/2), whereas Δ-THC also reduced phosphorylation of Akt (aka PKB). Moreover, the CB1R inverse agonist SR 141716A attenuated the decrease in neurite outgrowth and ERK1/2 phosphorylation induced by 2AG and Δ-THC. Taken together, our data suggest that hiPSC-derived cortical neurons express CB1Rs and are responsive to exogenous cannabinoids. Thus, hiPSC-neurons may represent a good cellular model for investigating the role of the endocannabinoid system in regulating cellular processes in developing human neurons.
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http://dx.doi.org/10.1016/j.mcn.2019.103463DOI Listing
March 2020

Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation.

Circ Res 2019 09 14;125(7):662-677. Epub 2019 Aug 14.

From the Department of Medicine, Division of Cardiology (M.S.S., R.A.B., M.B.F., A.S.R., B.Y.E., K.A.K., M.A.C., K.S., M.P.Y.L., T.A.M.), University of Colorado Anschutz Medical Campus, Aurora.

Rationale: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown.

Objective: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart.

Methods And Results: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-β (transforming growth factor-β) signaling to stimulate conversion of quiescent cardiac fibroblasts into ()-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for in TGF-β-mediated cardiac fibroblast activation.

Conclusions: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for . The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310347PMC
September 2019

Single-cell analysis of cardiogenesis reveals basis for organ-level developmental defects.

Nature 2019 08 24;572(7767):120-124. Epub 2019 Jul 24.

Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA.

Organogenesis involves integration of diverse cell types; dysregulation of cell-type-specific gene networks results in birth defects, which affect 5% of live births. Congenital heart defects are the most common malformations, and result from disruption of discrete subsets of cardiac progenitor cells, but the transcriptional changes in individual progenitors that lead to organ-level defects remain unknown. Here we used single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular subpopulations has catastrophic consequences. A network-based computational method for single-cell RNA-sequencing analysis that predicts lineage-specifying transcription factors identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite the failure of right ventricular formation in Hand2-null mice. Temporal single-cell-transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium was specified but failed to properly differentiate and migrate. Loss of Hand2 also led to dysregulation of retinoic acid signalling and disruption of anterior-posterior patterning of cardiac progenitors. This work reveals transcriptional determinants that specify fate and differentiation in individual cardiac progenitor cells, and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework for investigating congenital heart defects.
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http://dx.doi.org/10.1038/s41586-019-1414-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719697PMC
August 2019

Context-Specific Transcription Factor Functions Regulate Epigenomic and Transcriptional Dynamics during Cardiac Reprogramming.

Cell Stem Cell 2019 07;25(1):87-102.e9

Gladstone Institutes, San Francisco, CA 94158, USA; Departments of Pediatrics and Biochemistry & Biophysics, University of California, San Francisco, CA 94143, USA; Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA 94158, USA. Electronic address:

Ectopic expression of combinations of transcription factors (TFs) can drive direct lineage conversion, thereby reprogramming a somatic cell's identity. To determine the molecular mechanisms by which Gata4, Mef2c, and Tbx5 (GMT) induce conversion from a cardiac fibroblast toward an induced cardiomyocyte, we performed comprehensive transcriptomic, DNA-occupancy, and epigenomic interrogation throughout the reprogramming process. Integration of these datasets identified new TFs involved in cardiac reprogramming and revealed context-specific roles for GMT, including the ability of Mef2c and Tbx5 to independently promote chromatin remodeling at previously inaccessible sites. We also find evidence for cooperative facilitation and refinement of each TF's binding profile in a combinatorial setting. A reporter assay employing newly defined regulatory elements confirmed that binding of a single TF can be sufficient for gene activation, suggesting that co-binding events do not necessarily reflect synergy. These results shed light on fundamental mechanisms by which combinations of TFs direct lineage conversion.
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http://dx.doi.org/10.1016/j.stem.2019.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632093PMC
July 2019

Exchange protein directly activated by cAMP 2 is required for corticotropin-releasing hormone-mediated spine loss.

Eur J Neurosci 2019 10 9;50(7):3108-3114. Epub 2019 Jul 9.

Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.

Corticotropin-releasing hormone is produced in response to acute and chronic stress. Previous studies have shown that activation of the corticotropin-releasing hormone receptor 1 (CRHR1) by corticotropin-releasing hormone results in the rapid loss of dendritic spines which correlates with cognitive dysfunction associated with stress. Exchange protein directly activated by cAMP (EPAC2), a guanine nucleotide exchange factor for the small GTPase Rap, plays a critical role in regulating dendritic spine morphology and has been linked with CRHR1 signalling. In this study, we have tested whether EPAC2 links corticotropin-releasing hormone with dendritic spine remodelling. In primary rat cortical neurons, we show that CRHR1 is highly enriched in the dendritic spines. Furthermore, we find that EPAC2 and CRHR1 co-localize in cortical neurons and that acute exposure to corticotropin-releasing hormone induces spine loss. To establish whether EPAC2 was required for corticotropin-releasing hormone-mediated spine loss, we knocked-down EPAC2 in cortical neurons using a short hairpin RNA-mediated approach. In the presence of Epac2 knocked-down, corticotropin-releasing hormone was no longer able to induce spine loss. Taken together, our data indicate that EPAC2 is required for the rapid loss of dendritic spines induced by corticotropin-releasing hormone and may ultimately contribute to responses to acute stress.
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http://dx.doi.org/10.1111/ejn.14487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821562PMC
October 2019

Oligogenic inheritance of a human heart disease involving a genetic modifier.

Science 2019 05 30;364(6443):865-870. Epub 2019 May 30.

Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA.

Complex genetic mechanisms are thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human cardiac anomaly can be caused by a combination of rare, inherited heterozygous mutations. Whole-exome sequencing of a nuclear family revealed that three offspring with childhood-onset cardiomyopathy had inherited three missense single-nucleotide variants in the , , and genes. The and variants were inherited from the affected, asymptomatic father and the rare variant (minor allele frequency, 0.0012) from the unaffected mother. We used CRISPR-Cas9 to generate mice encoding the orthologous variants and found that compound heterozygosity for all three variants recapitulated the human disease phenotype. Analysis of murine hearts and human induced pluripotent stem cell-derived cardiomyocytes provided histologic and molecular evidence for the variant's contribution as a genetic modifier.
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http://dx.doi.org/10.1126/science.aat5056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557373PMC
May 2019

The Psychiatric Risk Gene NT5C2 Regulates Adenosine Monophosphate-Activated Protein Kinase Signaling and Protein Translation in Human Neural Progenitor Cells.

Biol Psychiatry 2019 07 30;86(2):120-130. Epub 2019 Mar 30.

Department of Basic & Clinical Neuroscience, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, United Kingdom; Medical Research Council Centre for Neurodevelopmental Disorders, King's College London, London, United Kingdom. Electronic address:

Background: The 5'-nucleotidase, cytosolic II gene (NT5C2, cN-II) is associated with disorders characterized by psychiatric and psychomotor disturbances. Common psychiatric risk alleles at the NT5C2 locus reduce expression of this gene in the fetal and adult brain, but downstream biological risk mechanisms remain elusive.

Methods: Distribution of the NT5C2 protein in the human dorsolateral prefrontal cortex and cortical human neural progenitor cells (hNPCs) was determined using immunostaining, publicly available expression data, and reverse transcriptase quantitative polymerase chain reaction. Phosphorylation quantification of adenosine monophosphate-activated protein kinase (AMPK) alpha (Thr172) and ribosomal protein S6 (Ser235/Ser236) was performed using Western blotting to infer the degree of activation of AMPK signaling and the rate of protein translation. Knockdowns were induced in hNPCs and Drosophila melanogaster using RNA interference. Transcriptomic profiling of hNPCs was performed using microarrays, and motility behavior was assessed in flies using the climbing assay.

Results: Expression of NT5C2 was higher during neurodevelopment and was neuronally enriched in the adult human cortex. Knockdown in hNPCs affected AMPK signaling, a major nutrient-sensing mechanism involved in energy homeostasis, and protein translation. Transcriptional changes implicated in protein translation were observed in knockdown hNPCs, and expression changes to genes related to AMPK signaling and protein translation were confirmed using reverse transcriptase quantitative polymerase chain reaction. The knockdown in Drosophila was associated with drastic climbing impairment.

Conclusions: We provide an extensive neurobiological characterization of the psychiatric risk gene NT5C2, describing its previously unknown role in the regulation of AMPK signaling and protein translation in neural stem cells and its association with Drosophila melanogaster motility behavior.
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http://dx.doi.org/10.1016/j.biopsych.2019.03.977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614717PMC
July 2019

Loss of EPAC2 alters dendritic spine morphology and inhibitory synapse density.

Mol Cell Neurosci 2019 07 4;98:19-31. Epub 2019 May 4.

Department of Physiology, Institute of Psychiatry, Psychology and Neuroscience, King's College London, 125 Coldharbour Lane, London SE5 8NU, UK; Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine, 303 E. Chicago Avenue, Chicago, IL 60611, USA. Electronic address:

EPAC2 is a guanine nucleotide exchange factor that regulates GTPase activity of the small GTPase Rap and Ras and is highly enriched at synapses. Activation of EPAC2 has been shown to induce dendritic spine shrinkage and increase spine motility, effects that are necessary for synaptic plasticity. These morphological effects are dysregulated by rare mutations of Epac2 associated with autism spectrum disorders. In addition, EPAC2 destabilizes synapses through the removal of synaptic GluA2/3-containing AMPA receptors. Previous work has shown that Epac2 knockout mice (Epac2) display abnormal social interactions, as well as gross disorganization of the frontal cortex and abnormal spine motility in vivo. In this study we sought to further understand the cellular consequences of knocking out Epac2 on the development of neuronal and synaptic structure and organization of cortical neurons. Using primary cortical neurons generated from Epac2 or Epac2 mice, we confirm that EPAC2 is required for cAMP-dependent spine shrinkage. Neurons from Epac2 mice also displayed increased synaptic expression of GluA2/3-containing AMPA receptors, as well as of the adhesion protein N-cadherin. Intriguingly, analysis of excitatory and inhibitory synaptic proteins revealed that loss of EPAC2 resulted in altered expression of vesicular GABA transporter (VGAT) but not vesicular glutamate transporter 1 (VGluT1), indicating an altered ratio of excitatory and inhibitory synapses onto neurons. Finally, examination of cortical neurons located within the anterior cingulate cortex further revealed subtle deficits in the establishment of dendritic arborization in vivo. These data provide evidence that loss of EPAC2 enhances the stability of excitatory synapses and increases the number of inhibitory inputs.
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http://dx.doi.org/10.1016/j.mcn.2019.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639166PMC
July 2019