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1.
Mol Psychiatry ; 27(2): 819-830, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34112971

RESUMEN

Copy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca2+ channel activity. These findings provide biological insight into the neuropathological mechanism underlying 1q21.1 associated brain disorder and indicate a potential target for therapeutic interventions.


Asunto(s)
Variaciones en el Número de Copia de ADN , Células Madre Pluripotentes Inducidas , Anomalías Múltiples , Animales , Deleción Cromosómica , Cromosomas Humanos Par 1 , Variaciones en el Número de Copia de ADN/genética , Humanos , Megalencefalia , Ratones , Neuronas , Fenotipo
2.
BMC Med ; 20(1): 123, 2022 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-35440050

RESUMEN

BACKGROUND: Tuberous sclerosis complex (TSC) is a rare multi-system genetic disorder characterised by the presence of benign tumours throughout multiple organs including the brain, kidneys, heart, liver, eyes, lungs and skin, in addition to neurological and neuropsychiatric complications. Intracardiac tumour (rhabdomyoma), neurodevelopmental disorders (NDDs) and kidney disorders (KD) are common manifestations of TSC and have been linked with TSC1 and TSC2 loss-of-function mutations independently, but the dynamic relationship between these organ manifestations remains unexplored. Therefore, this study aims to characterise the nature of the relationship specifically between these three organs' manifestations in TSC1 and TSC2 mutation patients. METHODS: Clinical data gathered from TSC patients across South Wales registered with Cardiff and Vale University Health Board (CAV UHB) between 1990 and 2020 were analysed retrospectively to evaluate abnormalities in the heart, brain and kidney development. TSC-related abnormalities such as tumour prevalence, location and size were analysed for each organ in addition to neuropsychiatric involvement and were compared between TSC1 and TSC2 mutant genotypes. Lastly, statistical co-occurrence between organ manifestations co-morbidity was quantified, and trajectories of disease progression throughout organs were modelled. RESULTS: This study found a significantly greater mutational frequency at the TSC2 locus in the cohort in comparison to TSC1. An equal proportion of male and female patients were observed in this group and by meta-analysis of previous studies. No significant difference in characterisation of heart involvement was observed between TSC1 and TSC2 patients. Brain involvement was seen with increased severity in TSC2 patients, characterised by a greater prevalence of cortical tubers and communication disorders. Renal pathology was further enhanced in TSC2 patients, marked by increased bilateral angiomyolipoma prevalence. Furthermore, co-occurrence of NDDs and KDs was the most positively correlated out of investigated manifestations, regardless of genotype. Analysis of disease trajectories revealed a more diverse clinical outcome for TSC2 patients: however, a chronological association of rhabdomyoma, NDD and KD was most frequently observed for TSC1 patients. CONCLUSIONS: This study marks the first empirical investigation of the co-morbidity between congenital heart defects (CHD), NDDs, and KDs in TSC1 and TSC2 patients. This remains a unique first step towards the characterisation of the dynamic role between genetics, heart function, brain function and kidney function during the early development in the context of TSC.


Asunto(s)
Rabdomioma , Esclerosis Tuberosa , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Mutación , Estudios Retrospectivos , Esclerosis Tuberosa/complicaciones , Esclerosis Tuberosa/epidemiología , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteínas Supresoras de Tumor/genética
3.
BMC Biol ; 19(1): 172, 2021 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-34429112

RESUMEN

BACKGROUND: Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. RESULTS: We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of 'barcoded' mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. CONCLUSIONS: We present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.


Asunto(s)
Dictyostelium , Dictyostelium/genética , Genoma , Genómica , Tecnología
4.
Ergonomics ; 65(1): 39-59, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-34006206

RESUMEN

The fashion industry cannot use 3 D Body Scanning to create custom garment patterns because its measurements fail to meet ISO 20685:2010's tolerances. To advance 3 D Body Scanning's precision, we present Gryphon: an algorithm that removes the two most extreme measurements from five body scans; removing potentially erroneous data. We assess Gryphon's precision against current industry practice, determine if consecutive and non-consecutive data capture influences precision, and determine 3 D Body Scanning's inherent imprecision inherent. We analyse 97 participants over 121 industry-standard measurements for consecutive and non-consecutive data-capture through MANOVA statistical analysis. Under current industry practice, only one measurement meets ISO 20685. However, under Gryphon and consecutive scanning, 97.5% of measurements meet ISO 20685. We also prove that the body's in-scan movement does not affect reliability. Ultimately, we offer the fashion industry, ergonomists, and practitioners an accessible method to increase 3 D Body Scanning's precision at a level unavailable under previous methods. Practitioner Summary: Ergonomists need precise data, yet we prove 0% of 3 D Body Scanning's measurements meet ISO 20685's tolerances. Our analysis of 97 participants scans, shows consecutive scanning is necessary to achieve data suitable for anthropometric applications. We develop the Gryphon process with consecutive scanning, making 97.3% of measurements meet ISO 20685. Abbreviations: ISO: International Organisation for Standards; Three Dimensional: 3D; MANOVA: Multivariate analysis of variance.


Asunto(s)
Imagenología Tridimensional , Proyectos de Investigación , Antropometría , Recolección de Datos , Humanos , Reproducibilidad de los Resultados
5.
EMBO Rep ; 20(6)2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31036712

RESUMEN

Nucleosome positioning is important for neurodevelopment, and genes mediating chromatin remodelling are strongly associated with human neurodevelopmental disorders. To investigate changes in nucleosome positioning during neural differentiation, we generate genome-wide nucleosome maps from an undifferentiated human-induced pluripotent stem cell (hiPSC) line and after its differentiation to the neural progenitor cell (NPC) stage. We find that nearly 3% of nucleosomes are highly positioned in NPC, but significantly, there are eightfold fewer positioned nucleosomes in pluripotent cells, indicating increased positioning during cell differentiation. Positioned nucleosomes do not strongly correlate with active chromatin marks or gene transcription. Unexpectedly, we find a small population of nucleosomes that occupy similar positions in pluripotent and neural progenitor cells and are found at binding sites of the key gene regulators NRSF/REST and CTCF Remarkably, the presence of these nucleosomes appears to be independent of the associated regulatory complexes. Together, these results present a scenario in human cells, where positioned nucleosomes are sparse and dynamic, but may act to alter gene expression at a distance via the structural conformation at sites of chromatin regulation.


Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Neurogénesis , Nucleosomas/metabolismo , Sitios de Unión , Biomarcadores , Regulación del Desarrollo de la Expresión Génica , Humanos , Unión Proteica , Factores de Transcripción
6.
Genome Res ; 27(4): 591-600, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28330902

RESUMEN

Nucleosome placement and repositioning can direct transcription of individual genes; however, the precise interactions of these events are complex and largely unresolved at the whole-genome level. The Chromodomain-Helicase-DNA binding (CHD) Type III proteins are a subfamily of SWI2/SNF2 proteins that control nucleosome positioning and are associated with several complex human disorders, including CHARGE syndrome and autism. Type III CHDs are required for multicellular development of animals and Dictyostelium but are absent in plants and yeast. These CHDs can mediate nucleosome translocation in vitro, but their in vivo mechanism is unknown. Here, we use genome-wide analysis of nucleosome positioning and transcription profiling to investigate the in vivo relationship between nucleosome positioning and gene expression during development of wild-type (WT) Dictyostelium and mutant cells lacking ChdC, a Type III CHD protein ortholog. We demonstrate major nucleosome positional changes associated with developmental gene regulation in WT. Loss of chdC caused an increase of intragenic nucleosome spacing and misregulation of gene expression, affecting ∼50% of the genes that are repositioned during WT development. These analyses demonstrate active nucleosome repositioning during Dictyostelium multicellular development, establish an in vivo function of CHD Type III chromatin remodeling proteins in this process, and reveal the detailed relationship between nucleosome positioning and gene regulation, as cells transition between developmental states.


Asunto(s)
ADN Helicasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Nucleosomas/genética , Proteínas Protozoarias/metabolismo , Ensamble y Desensamble de Cromatina , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Nucleosomas/metabolismo
7.
Development ; 140(24): 4926-36, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24301467

RESUMEN

Control of chromatin structure is crucial for multicellular development and regulation of cell differentiation. The CHD (chromodomain-helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are three mammalian CHD subfamilies and their impaired functions are associated with several human diseases. Here, we identify three CHD orthologs (ChdA, ChdB and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, using genome-wide (RNA-seq) transcriptome profiling for each null strain, we show that the different CHDs regulate distinct gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC nulls developed in pure populations or with low levels of wild-type cells. This study provides novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression.


Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Dictyostelium/crecimiento & desarrollo , Diferenciación Celular , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Expresión Génica , Perfilación de la Expresión Génica , Transducción de Señal/genética , Transcriptoma
8.
Res Sq ; 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-39070657

RESUMEN

Background: Aberrant activation of mTORC1 is clearly defined in TSC, causing uncontrolled cell growth. While mTORC1 inhibitors show efficacy to stabilise tumour growth in TSC, they are not fully curative. Disease facets of TSC that are not restored with mTOR inhibitors might involve NF-κB. The study aimed to characterise NF-κB in the context of TSC. Results: Enrichment of NF-κB-regulated genes was observed in TSC patient tumours, SEN/SEGAs, cortical tubers and a TSC tumour-derived cell line (621 - 101). Highlighting an inflammatory component of TSC, TSC cell models showed an elevated level of NF-κB and STAT3 activation. Herein, we report a dysregulated inflammatory phenotype of TSC2-deficient cells where NF-κB promotes autocrine signalling involving IL-6. Of importance, mTORC1 inhibition does not block this inflammatory signal to promote STAT3, while NF-κB inhibition was much more effective. Combined mTORC1 and NF-κB inhibition was potent at preventing anchorage-independent growth of TSC2-deficient cells, and unlike mTORC1 inhibition alone was sufficient to prevent colony regrowth after cessation of treatment. Conclusion: This study reveals autocrine signalling crosstalk between NF-κB and STAT3 in TSC cell models. Furthermore, the data presented indicate that NF-κB pathway inhibitors could be a viable adjunct therapy with the current mTOR inhibitors to treat TSC.

9.
Front Cell Dev Biol ; 11: 1166808, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37255597

RESUMEN

Introduction: There is a growing interest in the possibility of dietary supplementation with polyunsaturated fatty acids (PUFAs) for treatment and prevention of neurodevelopmental and neuropsychiatric disorders. Studies have suggested that of the two important classes of polyunsaturated fatty acids, omega-6 (n-6) and omega-3 (n-3), n-3 polyunsaturated fatty acids support brain development and function, and when used as a dietary supplement may have beneficial effects for maintenance of a healthy brain. However, to date epidemiological studies and clinical trials on children and adults have been inconclusive regarding treatment length, dosage and use of specific n-3 polyunsaturated fatty acids. The aim of this study is to generate a simplified in vitro cell-based model system to test how different n-6 to n-3 polyunsaturated fatty acids ratios affect human-derived neurons activity as a cellular correlate for brain function and to probe the mechanism of their action. Methods: All experiments were performed by use of human induced pluripotent stem cells (iPSCs). In this study, we examined the effect of different ratios of linoleic acid (n-6) to alpha-linolenic acid in cell growth medium on induced pluripotent stem cell proliferation, generation of neuronal precursors and electrophysiology of cortical glutamatergic neurons by multielectrode array (MEA) analysis. Results: This study shows that at a n-6:n-3 ratio of 5:1 polyunsaturated fatty acids induce stem cell proliferation, generating a large increase in number of cells after 72 h treatment; suppress generation of neuronal progenitor cells, as measured by decreased expression of FOXG1 and Nestin in neuronal precursor cells (NPC) after 20 days of development; and disrupt neuronal activity in vitro, increasing spontaneous neuronal firing, reducing synchronized bursting receptor subunits. We observed no significant differences for neuronal precursor cells treated with ratios 1:3 and 3:1, in comparison to 1:1 control ratio, but higher ratios of n-6 to n-3 polyunsaturated fatty acids adversely affect early stages of neuronal differentiation. Moreover, a 5:1 ratio in cortical glutamatergic neurons induce expression of GABA receptors which may explain the observed abnormal electrophysiological activity.

10.
Sci Rep ; 13(1): 3060, 2023 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-36810617

RESUMEN

Hay fever affects people differently and can change over a lifetime, but data is lacking on how environmental factors may influence this. This study is the first to combine atmospheric sensor data with real-time, geo-positioned hay fever symptom reports to examine the relationship between symptom severity and air quality, weather and land use. We study 36145 symptom reports submitted over 5 years by over 700 UK residents using a mobile application. Scores were recorded for nose, eyes and breathing. Symptom reports are labelled as urban or rural using land-use data from the UK's Office for National Statistics. Reports are compared with AURN network pollution measurements and pollen and meteorological data taken from the UK Met Office. Our analysis suggests urban areas record significantly higher symptom severity for all years except 2017. Rural areas do not record significantly higher symptom severity in any year. Additionally, symptom severity correlates with more air quality markers in urban areas than rural areas, indicating that differences in allergy symptoms may be due to variations in the levels of pollutants, pollen counts and seasonality across land-use types. The results suggest that a relationship exists between urban surroundings and hay fever symptoms.


Asunto(s)
Contaminación del Aire , Rinitis Alérgica Estacional , Humanos , Rinitis Alérgica Estacional/diagnóstico , Polen , Nariz , Reino Unido
11.
Nat Commun ; 13(1): 319, 2022 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-35031602

RESUMEN

Natural selection should favour generalist predators that outperform specialists across all prey types. Two genetic solutions could explain why intraspecific variation in predatory performance is, nonetheless, widespread: mutations beneficial on one prey type are costly on another (antagonistic pleiotropy), or mutational effects are prey-specific, which weakens selection, allowing variation to persist (relaxed selection). To understand the relative importance of these alternatives, we characterised natural variation in predatory performance in the microbial predator Dictyostelium discoideum. We found widespread nontransitive differences among strains in predatory success across different bacterial prey, which can facilitate stain coexistence in multi-prey environments. To understand the genetic basis, we developed methods for high throughput experimental evolution on different prey (REMI-seq). Most mutations (~77%) had prey-specific effects, with very few (~4%) showing antagonistic pleiotropy. This highlights the potential for prey-specific effects to dilute selection, which would inhibit the purging of variation and prevent the emergence of an optimal generalist predator.


Asunto(s)
Dictyostelium/genética , Conducta Alimentaria , Bacterias/metabolismo , Evolución Biológica , Dictyostelium/crecimiento & desarrollo , Dictyostelium/fisiología , Cadena Alimentaria , Mutación
12.
Transl Psychiatry ; 12(1): 438, 2022 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-36216811

RESUMEN

Genetic evidence indicates disrupted epigenetic regulation as a major risk factor for psychiatric disorders, but the molecular mechanisms that drive this association remain to be determined. EHMT1 is an epigenetic repressor that is causal for Kleefstra Syndrome (KS), a genetic disorder linked with neurodevelopmental disorders and associated with schizophrenia. Here, we show that reduced EHMT1 activity decreases NRSF/REST protein leading to abnormal neuronal gene expression and progression of neurodevelopment in human iPSC. We further show that EHMT1 regulates NRSF/REST indirectly via repression of miRNA and leads to aberrant neuronal gene regulation and neurodevelopment timing. Expression of a NRSF/REST mRNA that lacks the miRNA-binding sites restores neuronal gene regulation to EHMT1 deficient cells. Significantly, the EHMT1-regulated miRNA gene set not only controls NRSF/REST but is enriched for association for Intellectual Disability (ID) and schizophrenia. This reveals a broad molecular interaction between H3K9 demethylation, NSRF/REST regulation and risk for ID and Schizophrenia.


Asunto(s)
Discapacidad Intelectual , MicroARNs , Proteínas Represoras , Esquizofrenia , Epigénesis Genética , Humanos , Discapacidad Intelectual/genética , MicroARNs/genética , ARN Mensajero/genética , Proteínas Represoras/genética , Esquizofrenia/genética
13.
Nat Commun ; 13(1): 27, 2022 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-35031607

RESUMEN

Coordinated programs of gene expression drive brain development. It is unclear which transcriptional programs, in which cell-types, are affected in neuropsychiatric disorders such as schizophrenia. Here we integrate human genetics with transcriptomic data from differentiation of human embryonic stem cells into cortical excitatory neurons. We identify transcriptional programs expressed during early neurogenesis in vitro and in human foetal cortex that are down-regulated in DLG2-/- lines. Down-regulation impacted neuronal differentiation and maturation, impairing migration, morphology and action potential generation. Genetic variation in these programs is associated with neuropsychiatric disorders and cognitive function, with associated variants predominantly concentrated in loss-of-function intolerant genes. Neurogenic programs also overlap schizophrenia GWAS enrichment previously identified in mature excitatory neurons, suggesting that pathways active during prenatal cortical development may also be associated with mature neuronal dysfunction. Our data from human embryonic stem cells, when combined with analysis of available foetal cortical gene expression data, de novo rare variants and GWAS statistics for neuropsychiatric disorders and cognition, reveal a convergence on transcriptional programs regulating excitatory cortical neurogenesis.


Asunto(s)
Corteza Cerebral/embriología , Regulación del Desarrollo de la Expresión Génica , Guanilato-Quinasas/genética , Neurogénesis , Proteínas Supresoras de Tumor/genética , Animales , Diferenciación Celular , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Predisposición Genética a la Enfermedad , Guanilato-Quinasas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Trastornos Mentales/genética , Neurogénesis/genética , Neurogénesis/fisiología , Neuronas , Embarazo , Esquizofrenia/genética , Transcriptoma , Proteínas Supresoras de Tumor/metabolismo
14.
Anal Chem ; 83(23): 8900-5, 2011 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-22029261

RESUMEN

Interactions between biomolecules are an important feature of biological systems and understanding these interactions is a key goal in biochemical studies. Using conventional techniques, such as surface plasmon resonance and isothermal titration calorimetry, the determination of the binding constants requires a significant amount of time and resources to produce and purify sufficient quantities of biomolecules in order to measure the affinity of biological interactions. Using DNA hybridization, we have demonstrated a new technique based on the use of nanotethers and time-resolved Forster resonance energy transfer (FRET) that significantly reduces the amount of material required to carry out quantitative binding assays. Test biomolecules were colocalized and attached to a surface using DNA tethers constructed from overlapping oligonucleotides. The length of the tethers defines the concentration of the tethered biomolecule. Effective end concentrations ranging from 56 nM to 3.8 µM were demonstrated. The use of variable length tethers may have wider applications in the quantitative measurement of affinity binding parameters.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Nanoestructuras/química , ADN de Cadena Simple/química , Colorantes Fluorescentes/química , Método de Montecarlo , Hibridación de Ácido Nucleico , Oligonucleótidos/química
15.
Curr Opin Cell Biol ; 16(5): 470-6, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15363795

RESUMEN

Cell adhesion is a basic property of animal cells, but is also present in many other eukaryotes. Did cell adhesion systems arise independently in different eukaryotic groups, or do they share common origins? Recent results show that cell adhesion proteins related to cadherin, IgG-like CAM and C-type lectin are present both in sponges, the most distant animal branch, and in eukaryote groups outside the metazoan lineage, indicating that these forms of adhesion arose prior to animal evolution. Furthermore, proteins containing features of animal adhesion systems, such as Fas-1 and thrombospondin domains, are distributed throughout the eukaryotes and function in cell adhesion.


Asunto(s)
Moléculas de Adhesión Celular/genética , Células Eucariotas/metabolismo , Evolución Molecular , Filogenia , Animales , Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Eucariotas/citología
16.
Proc Natl Acad Sci U S A ; 105(16): 5998-6003, 2008 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-18413611

RESUMEN

The Rapoport-Luebering glycolytic bypass comprises evolutionarily conserved reactions that generate and dephosphorylate 2,3-bisphosphoglycerate (2,3-BPG). For >30 years, these reactions have been considered the responsibility of a single enzyme, the 2,3-BPG synthase/2-phosphatase (BPGM). Here, we show that Dictyostelium, birds, and mammals contain an additional 2,3-BPG phosphatase that, unlike BPGM, removes the 3-phosphate. This discovery reveals that the glycolytic pathway can bypass the formation of 3-phosphoglycerate, which is a precursor for serine biosynthesis and an activator of AMP-activated protein kinase. Our 2,3-BPG phosphatase activity is encoded by the previously identified gene for multiple inositol polyphosphate phosphatase (MIPP1), which we now show to have dual substrate specificity. By genetically manipulating Mipp1 expression in Dictyostelium, we demonstrated that this enzyme provides physiologically relevant regulation of cellular 2,3-BPG content. Mammalian erythrocytes possess the highest content of 2,3-BPG, which controls oxygen binding to hemoglobin. We determined that total MIPP1 activity in erythrocytes at 37 degrees C is 0.6 mmol 2,3-BPG hydrolyzed per liter of cells per h, matching previously published estimates of the phosphatase activity of BPGM. MIPP1 is active at 4 degrees C, revealing a clinically significant contribution to 2,3-BPG loss during the storage of erythrocytes for transfusion. Hydrolysis of 2,3-BPG by human MIPP1 is sensitive to physiologic alkalosis; activity decreases 50% when pH rises from 7.0 to 7.4. This phenomenon provides a homeostatic mechanism for elevating 2,3-BPG levels, thereby enhancing oxygen release to tissues. Our data indicate greater biological significance of the Rapoport-Luebering shunt than previously considered.


Asunto(s)
2,3-Difosfoglicerato/metabolismo , Evolución Molecular , Glucólisis , Monoéster Fosfórico Hidrolasas/metabolismo , 2,3-Difosfoglicerato/análisis , 2,3-Difosfoglicerato/química , Alcalosis , Secuencia de Aminoácidos , Animales , Aves , Dictyostelium/enzimología , Eritrocitos/química , Eritrocitos/enzimología , Eritrocitos/metabolismo , Hemoglobinas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Oxígeno/metabolismo , Monoéster Fosfórico Hidrolasas/química , Fosforilación , Proteínas Protozoarias/química , Ratas
17.
Mol Biol Cell ; 18(12): 4772-9, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17898079

RESUMEN

Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] gradient within the plasma membrane is important for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P(3) on the membrane nearest the polarizing signal and PI(3,4,5)P(3) dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is not only the substrate of PI3K and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C (PLC) activity, which hydrolyzes PI(4,5)P(2), could have important consequences for PI(3,4,5)P(3) localization. We investigate the role of PLC in PI(3,4,5)P(3)-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor LY294002 and produce little PI(3,4,5)P(3) after cAMP stimulation, as monitored by the PI(3,4,5)P(3)-specific pleckstrin homology (PH)-domain of CRAC (PH(CRAC)GFP). In contrast, PLC overexpression elevates PI(3,4,5)P(3) and impairs chemotaxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal PI(3,4,5)P(3) gradient.


Asunto(s)
Quimiotaxis , Dictyostelium/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Línea Celular , Quimiotaxis/efectos de los fármacos , Cromonas/farmacología , AMP Cíclico/metabolismo , Dictyostelium/citología , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Regulación Enzimológica de la Expresión Génica , Morfolinas/farmacología , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Fosfolipasas de Tipo C/deficiencia , Fosfolipasas de Tipo C/genética
18.
Mol Autism ; 11(1): 80, 2020 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-33076974

RESUMEN

BACKGROUND: Tuberous sclerosis complex (TSC) is a rare genetic multisystemic disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes. It is characterised by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and has severe neurodevelopmental and neurological components including autism, intellectual disability and epilepsy. In human and rodent models, loss of the TSC proteins causes neuronal hyperexcitability and synaptic dysfunction, although the consequences of these changes for the developing central nervous system are currently unclear. METHODS: Here we apply multi-electrode array-based assays to study the effects of TSC2 loss on neuronal network activity using autism spectrum disorder (ASD) patient-derived iPSCs. We examine both temporal synchronisation of neuronal bursting and spatial connectivity between electrodes across the network. RESULTS: We find that ASD patient-derived neurons with a functional loss of TSC2, in addition to possessing neuronal hyperactivity, develop a dysfunctional neuronal network with reduced synchronisation of neuronal bursting and lower spatial connectivity. These deficits of network function are associated with elevated expression of genes for inhibitory GABA signalling and glutamate signalling, indicating a potential abnormality of synaptic inhibitory-excitatory signalling. mTORC1 activity functions within a homeostatic triad of protein kinases, mTOR, AMP-dependent protein Kinase 1 (AMPK) and Unc-51 like Autophagy Activating Kinase 1 (ULK1) that orchestrate the interplay of anabolic cell growth and catabolic autophagy while balancing energy and nutrient homeostasis. The mTOR inhibitor rapamycin suppresses neuronal hyperactivity, but does not increase synchronised network activity, whereas activation of AMPK restores some aspects of network activity. In contrast, the ULK1 activator, LYN-1604, increases the network behaviour, shortens the network burst lengths and reduces the number of uncorrelated spikes. LIMITATIONS: Although a robust and consistent phenotype is observed across multiple independent iPSC cultures, the results are based on one patient. There may be more subtle differences between patients with different TSC2 mutations or differences of polygenic background within their genomes. This may affect the severity of the network deficit or the pharmacological response between TSC2 patients. CONCLUSIONS: Our observations suggest that there is a reduction in the network connectivity of the in vitro neuronal network associated with ASD patients with TSC2 mutation, which may arise via an excitatory/inhibitory imbalance due to increased GABA-signalling at inhibitory synapses. This abnormality can be effectively suppressed via activation of ULK1.


Asunto(s)
Trastorno del Espectro Autista/tratamiento farmacológico , Trastorno del Espectro Autista/genética , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Red Nerviosa/patología , Neuronas/patología , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Adolescente , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Biomarcadores/metabolismo , Electrodos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lactante , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Red Nerviosa/efectos de los fármacos , Red Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ribonucleótidos/farmacología , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo
19.
Brain Neurosci Adv ; 4: 2398212820928647, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32954001

RESUMEN

Regulators of chromatin dynamics and transcription are increasingly implicated in the aetiology of neurodevelopmental disorders. Haploinsufficiency of EHMT1, encoding a histone methyltransferase, is associated with several neurodevelopmental disorders, including Kleefstra syndrome, developmental delay and autism spectrum disorder. Using a mouse model of Ehmt1 haploinsufficiency (Ehmt1 D6Cre/+), we examined a number of brain and behavioural endophenotypes of relevance to neurodevelopmental disorders. Specifically, we show that Ehmt1 D6Cre/+ mice have deficits in information processing, evidenced by abnormal sensory-motor gating, a complete absence of object recognition memory, and a reduced magnitude of auditory evoked potentials in both paired-pulse inhibition and mismatch negativity. The electrophysiological experiments show that differences in magnitude response to auditory stimulus were associated with marked reductions in total and evoked beta- and gamma-band oscillatory activity, as well as significant reductions in phase synchronisation. The pattern of electrophysiological deficits in Ehmt1 D6Cre/+ matches those seen in control mice following administration of the selective NMDA-R antagonist, ketamine. This, coupled with reduction of Grin1 mRNA expression in Ehmt1 D6Cre/+ hippocampus, suggests that Ehmt1 haploinsufficiency may lead to disruption in NMDA-R. Taken together, these data indicate that reduced Ehmt1 dosage during forebrain development leads to abnormal circuitry formation, which in turn results in profound information processing deficits. Such information processing deficits are likely paramount to our understanding of the cognitive and neurological dysfunctions shared across the neurodevelopmental disorders associated with EHMT1 haploinsufficiency.

20.
Artículo en Inglés | MEDLINE | ID: mdl-33321999

RESUMEN

Copy number variants (CNVs) play an important role in the genetic underpinnings of neuropsychiatric/neurodevelopmental disorders. The chromosomal region 16p11.2 (BP4-BP5) harbours both deletions and duplications that are associated in carriers with neurodevelopmental and neuropsychiatric conditions as well as several rare disorders including congenital malformation syndromes. The aim of this article is to provide a review of the current knowledge of the diverse neurodevelopmental disorders (NDD) associated with 16p11.2 deletions and duplications reported in published cohorts. A literature review was conducted using the PubMed/MEDLINE electronic database limited to papers published in English between 1 January 2010 and 31 July 2020, describing 16p11.2 deletions and duplications carriers' cohorts. Twelve articles meeting inclusion criteria were reviewed from the 75 articles identified by the search. Of these twelve papers, eight described both deletions and duplications, three described deletions only and one described duplications only. This study highlights the heterogeneity of NDD descriptions of the selected cohorts and inconsistencies concerning accuracy of data reporting.


Asunto(s)
Variaciones en el Número de Copia de ADN , Trastornos Mentales/genética , Trastornos del Neurodesarrollo/genética , Deleción Cromosómica , Trastornos de los Cromosomas/genética , Duplicación Cromosómica , Cromosomas Humanos Par 16/genética , Heterocigoto , Humanos
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