RESUMEN
BACKGROUND: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. METHODS: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. RESULTS: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. CONCLUSIONS: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.
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Cardiomiopatía Dilatada , Femenino , Embarazo , Animales , Humanos , Cardiomiopatía Dilatada/genética , Pez Cebra , Volumen Sistólico , Función Ventricular Izquierda , Centrosoma/metabolismo , Miocitos CardíacosRESUMEN
MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at â¼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors.
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Carcinogénesis/metabolismo , Cromatina/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Anisotropía , Sitios de Unión/genética , Carcinogénesis/genética , Cromatina/química , Cromatina/genética , Polarización de Fluorescencia , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genética , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
In December 2020, the Food and Drug Administration (FDA) issued Emergency Use Authorizations (EUAs) for Pfizer-BioNTech and Moderna COVID-19 vaccines, and in February 2021, FDA issued an EUA for the Janssen (Johnson & Johnson) COVID-19 vaccine. After each EUA, the Advisory Committee on Immunization Practices (ACIP) issued interim recommendations for vaccine use; currently Pfizer-BioNTech is authorized and recommended for persons aged ≥12 years and Moderna and Janssen for persons aged ≥18 years (1-3). Both Pfizer-BioNTech and Moderna vaccines, administered as 2-dose series, are mRNA-based COVID-19 vaccines, whereas the Janssen COVID-19 vaccine, administered as a single dose, is a recombinant replication-incompetent adenovirus-vector vaccine. As of July 22, 2021, 187 million persons in the United States had received at least 1 dose of COVID-19 vaccine (4); close monitoring of safety surveillance has demonstrated that serious adverse events after COVID-19 vaccination are rare (5,6). Three medical conditions have been reported in temporal association with receipt of COVID-19 vaccines. Two of these (thrombosis with thrombocytopenia syndrome [TTS], a rare syndrome characterized by venous or arterial thrombosis and thrombocytopenia, and Guillain-Barré syndrome [GBS], a rare autoimmune neurologic disorder characterized by ascending weakness and paralysis) have been reported after Janssen COVID-19 vaccination. One (myocarditis, cardiac inflammation) has been reported after Pfizer-BioNTech COVID-19 vaccination or Moderna COVID-19 vaccination, particularly after the second dose; these were reviewed together and will hereafter be referred to as mRNA COVID-19 vaccination. ACIP has met three times to review the data associated with these reports of serious adverse events and has comprehensively assessed the benefits and risks associated with receipt of these vaccines. During the most recent meeting in July 2021, ACIP determined that, overall, the benefits of COVID-19 vaccination in preventing COVID-19 morbidity and mortality outweigh the risks for these rare serious adverse events in adults aged ≥18 years; this balance of benefits and risks varied by age and sex. ACIP continues to recommend COVID-19 vaccination in all persons aged ≥12 years. CDC and FDA continue to closely monitor reports of serious adverse events and will present any additional data to ACIP for consideration. Information regarding risks and how they vary by age and sex and type of vaccine should be disseminated to providers, vaccine recipients, and the public.
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Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/efectos adversos , COVID-19/prevención & control , Inmunización/normas , Guías de Práctica Clínica como Asunto , Adulto , Sistemas de Registro de Reacción Adversa a Medicamentos , Comités Consultivos , COVID-19/epidemiología , Aprobación de Drogas , Humanos , Estados Unidos/epidemiología , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
There is a need for valves and pumps that operate at the microscale with precision and accuracy, are versatile in their application, and are easily fabricated. To that end, we developed a new rotary planar multiport valve to faithfully select solutions (contamination = 5.22 ± 0.06 ppb) and a rotary planar peristaltic pump to precisely control fluid delivery (flow rate = 2.4 ± 1.7 to 890 ± 77 µL/min). Both the valve and pump were implemented in a planar format amenable to single-layer soft lithographic fabrication. These planar microfluidics were actuated by a rotary motor controlled remotely by custom software. Together, these two devices constitute an innovative microformulator that was used to prepare precise, high-fidelity mixtures of up to five solutions (deviation from prescribed mixture = ±|0.02 ± 0.02| %). This system weighed less than a kilogram, occupied around 500 cm3, and generated pressures of 255 ± 47 kPa. This microformulator was then combined with an electrochemical sensor creating a microclinical analyzer (µCA) for detecting glutamate in real time. Using the chamber of the µCA as an in-line bioreactor, we compared glutamate homeostasis in human astrocytes differentiated from human-induced pluripotent stem cells (hiPSCs) from a control subject (CC-3) and a Tuberous Sclerosis Complex (TSC) patient carrying a pathogenic TSC2 mutation. When challenged with glutamate, TSC astrocytes took up less glutamate than control cells. These data validate the analytical power of the µCA and the utility of the microformulator by leveraging it to assess disease-related alterations in cellular homeostasis.
RESUMEN
Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disease caused by heterozygous de novo missense mutations in the ATP1A3 gene that encodes the neuronal specific α3 subunit of the Na,K-ATPase (NKA) pump. Mechanisms underlying patient episodes including environmental triggers remain poorly understood, and there are no empirically proven treatments for AHC. In this study, we generated patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls for the E815K ATP1A3 mutation that causes the most phenotypically severe form of AHC. Using an in vitro iPSC-derived cortical neuron disease model, we found elevated levels of ATP1A3 mRNA in AHC lines compared to controls, without significant perturbations in protein expression. Microelectrode array analyses demonstrated that in cortical neuronal cultures, ATP1A3+/E815K iPSC-derived neurons displayed less overall activity than neurons differentiated from isogenic mutation-corrected and unrelated control cell lines. However, induction of cellular stress by elevated temperature revealed a hyperactivity phenotype following heat stress in ATP1A3+/E815K neurons compared to control lines. Treatment with flunarizine, a drug commonly used to prevent AHC episodes, did not impact this stress-triggered phenotype. These findings support the use of iPSC-derived neuronal cultures for studying complex neurodevelopmental conditions such as AHC and provide a platform for mechanistic discovery in a human disease model.
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Hemiplejía/metabolismo , Neuronas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Diferenciación Celular , Células Cultivadas , Corteza Cerebral/metabolismo , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Mutación Missense , Fenotipo , ARN Mensajero/metabolismoRESUMEN
Over 1250 mutations in SCN1A, the Nav1.1 voltage-gated sodium channel gene, are associated with seizure disorders including GEFS+. To evaluate how a specific mutation, independent of genetic background, causes seizure activity we generated two pairs of isogenic human iPSC lines by CRISPR/Cas9 gene editing. One pair is a control line from an unaffected sibling, and the mutated control carrying the GEFS+ K1270T SCN1A mutation. The second pair is a GEFS+ patient line with the K1270T mutation, and the corrected patient line. By comparing the electrophysiological properties in inhibitory and excitatory iPSC-derived neurons from these pairs, we found the K1270T mutation causes cell type-specific alterations in sodium current density and evoked firing, resulting in hyperactive neural networks. We also identified differences associated with genetic background and interaction between the mutation and genetic background. Comparisons within and between dual pairs of isogenic iPSC-derived neuronal cultures provide a novel platform for evaluating cellular mechanisms underlying a disease phenotype and for developing patient-specific anti-seizure therapies.
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Epilepsia/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Neuronas , Genotipo , Humanos , Células Madre Pluripotentes Inducidas , Mutación , Fenotipo , Convulsiones Febriles/genéticaRESUMEN
Mutations in the DEPDC5 gene can cause epilepsy, including forms with and without brain malformations. The goal of this study was to investigate the contribution of DEPDC5 gene dosage to the underlying neuropathology of DEPDC5-related epilepsies. We generated induced pluripotent stem cells (iPSCs) from epilepsy patients harboring heterozygous loss of function mutations in DEPDC5. Patient iPSCs displayed increases in both phosphorylation of ribosomal protein S6 and proliferation rate, consistent with elevated mTORC1 activation. In line with these findings, we observed increased soma size in patient iPSC-derived cortical neurons that was rescued with rapamycin treatment. These data indicate that human cells heterozygous for DEPDC5 loss-of-function mutations are haploinsufficient for control of mTORC1 signaling. Our findings suggest that human pathology differs from mouse models of DEPDC5-related epilepsies, which do not show consistent phenotypic differences in heterozygous neurons, and support the need for human-based models to affirm and augment the findings from animal models of DEPDC5-related epilepsy.
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Epilepsia Refractaria/genética , Proteínas Activadoras de GTPasa/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neuronas/metabolismo , Neuronas/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Epilepsia Refractaria/metabolismo , Haploinsuficiencia , Humanos , Células Madre Pluripotentes Inducidas , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Tuberous sclerosis complex 2 (TSC2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin signaling pathway that controls cell survival, proliferation, growth, and migration. Loss of Tsc2 function manifests in organ-specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of the kidney has identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice as well as a in a novel cell population. The impact in adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2, lacking exons 36-37 with an intact 3' region and polycystin 1, is driven by Abcg2. Here, we demonstrate that selective expression of Tsc2fl36-37 in the Abcg2pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2-expressing proximal tubule epithelium and mesenchyme during the development of cystic lesions and remodeling of kidney parenchyma.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Fibrosis/patología , Enfermedades Renales Poliquísticas/patología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Animales , Linaje de la Célula , Femenino , Fibrosis/genética , Túbulos Renales Proximales/patología , Masculino , Ratones , Miofibroblastos/fisiología , Enfermedades Renales Poliquísticas/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
Astrocytes serve many functions in the human brain, many of which focus on maintenance of homeostasis. Astrocyte dysfunction in Tuberous Sclerosis Complex (TSC) has long been appreciated with activation of the mTORC1 signaling pathway resulting in gliosis and possibly contributing to the very frequent phenotype of epilepsy. We hypothesized that aberrant expression of the astrocyte protein aquaporin-4 (AQP4) may be present in TSC and contribute to disease pathology. Characterization of AQP4 expression in epileptic cortex from TSC patients demonstrated a diffuse increase in AQP4. To determine if this was due to exposure to seizures, we examined Aqp4 expression in mouse models of TSC in which Tsc1 or Tsc2 inactivation was targeted to astrocytes or glial progenitors, respectively. Loss of either Tsc1 or Tsc2 from astrocytes resulted in a marked increase in Aqp4 expression which was sensitive to mTORC1 inhibition with rapamycin. Our findings in both TSC epileptogenic cortex and in a variety of astrocyte culture models demonstrate for the first time that AQP4 expression is dysregulated in TSC. The extent to which AQP4 contributes to epilepsy in TSC is not known, though the similarities in AQP4 expression between TSC and temporal lobe epilepsy supports further studies targeting AQP4 in TSC.
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Acuaporina 4/biosíntesis , Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Convulsiones/metabolismo , Esclerosis Tuberosa/metabolismo , Adolescente , Animales , Niño , Preescolar , Femenino , Humanos , Recién Nacido , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Convulsiones/etiología , Esclerosis Tuberosa/complicacionesRESUMEN
Tuberous sclerosis complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprogramming plasmid containing a shRNA against TP53. We also found that loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. These results support important contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming.
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Reprogramación Celular/genética , Células Madre Pluripotentes Inducidas/fisiología , Pérdida de Heterocigocidad , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Adolescente , Adulto , Alelos , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Genes p53 , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Masculino , Mutación , ARN Interferente Pequeño/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
MRI is a valuable tool to assess myelin during development and demyelinating disease processes. While multiexponential T2 and quantitative magnetization transfer measures correlate with myelin content, neither provides the total myelin volume fraction. In many cases correlative measures are adequate; but to assess microstructure of myelin, (e.g. calculate the g-ratio using MRI), an accurate measure of myelin volume fraction is imperative. Using a volumetric model of white matter, we relate MRI measures of myelin to absolute measures of myelin volume fraction and compare them to quantitative histology. We assess our approach in control mice along with two models of hypomyelination and one model of hypermyelination and find strong agreement between MRI and histology amongst models. This work investigates the sensitivities of MRI myelin measures to changes in axon geometry and displays promise for estimating g-ratio from MRI.
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Enfermedades Desmielinizantes/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Modelos Teóricos , Vaina de Mielina , Neuroimagen/métodos , Sustancia Blanca/anatomía & histología , Sustancia Blanca/diagnóstico por imagen , Animales , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Imagen por Resonancia Magnética/normas , Ratones , Ratones Noqueados , Vaina de Mielina/metabolismo , Neuroimagen/normas , Sensibilidad y Especificidad , Sustancia Blanca/patologíaRESUMEN
Mutations in ATP1A3 encoding the catalytic subunit of the Na/K-ATPase expressed in mammalian neurons cause alternating hemiplegia of childhood (AHC) as well as an expanding spectrum of other neurodevelopmental syndromes and neurological phenotypes. Most AHC cases are explained by de novo heterozygous ATP1A3 mutations, but the fundamental molecular and cellular consequences of these mutations in human neurons are not known. In this study, we investigated the electrophysiological properties of neurons generated from AHC patient-specific induced pluripotent stem cells (iPSCs) to ascertain functional disturbances underlying this neurological disease. Fibroblasts derived from two subjects with AHC, a male and a female, both heterozygous for the common ATP1A3 mutation G947R, were reprogrammed to iPSCs. Neuronal differentiation of iPSCs was initiated by neurogenin-2 (NGN2) induction followed by co-culture with mouse glial cells to promote maturation of cortical excitatory neurons. Whole-cell current clamp recording demonstrated that, compared with control iPSC-derived neurons, neurons differentiated from AHC iPSCs exhibited a significantly lower level of ouabain-sensitive outward current ('pump current'). This finding correlated with significantly depolarized potassium equilibrium potential and depolarized resting membrane potential in AHC neurons compared with control neurons. In this cellular model, we also observed a lower evoked action potential firing frequency when neurons were held at their resting potential. However, evoked action potential firing frequencies were not different between AHC and control neurons when the membrane potential was clamped to -80â¯mV. Impaired neuronal excitability could be explained by lower voltage-gated sodium channel availability at the depolarized membrane potential observed in AHC neurons. Our findings provide direct evidence of impaired neuronal Na/K-ATPase ion transport activity in human AHC neurons and demonstrate the potential impact of this genetic defect on cellular excitability.
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Hemiplejía/diagnóstico , Hemiplejía/fisiopatología , Potenciales de la Membrana/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Adulto , Animales , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Técnicas de Cocultivo , Femenino , Hemiplejía/genética , Humanos , Lactante , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Ratones , Adulto JovenRESUMEN
The essential micronutrient manganese is enriched in brain, especially in the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested that alterations in striatal manganese handling occur in Huntington's disease (HD) models. We found that p53 phosphorylation at serine 15 is the most responsive cell signaling event to manganese exposure (of 18 tested) in human neuroprogenitors and a mouse striatal cell line. Manganese-dependent activation of p53 was severely diminished in HD cells. Inhibitors of ataxia telangiectasia mutated (ATM) kinase decreased manganese-dependent phosphorylation of p53. Likewise, analysis of ATM autophosphorylation and additional ATM kinase targets, H2AX and CHK2, support a role for ATM in the activation of p53 by manganese and that a defect in this process occurs in HD. Furthermore, the deficit in Mn-dependent activation of ATM kinase in HD neuroprogenitors was highly selective, as DNA damage and oxidative injury, canonical activators of ATM, did not show similar deficits. We assessed cellular manganese handling to test for correlations with the ATM-p53 pathway, and we observed reduced Mn accumulation in HD human neuroprogenitors and HD mouse striatal cells at manganese exposures associated with altered p53 activation. To determine if this phenotype contributes to the deficit in manganese-dependent ATM activation, we used pharmacological manipulation to equalize manganese levels between HD and control mouse striatal cells and rescued the ATM-p53 signaling deficit. Collectively, our data demonstrate selective alterations in manganese biology in cellular models of HD manifest in ATM-p53 signaling.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Cuerpo Estriado/metabolismo , Enfermedad de Huntington/metabolismo , Manganeso/metabolismo , Células-Madre Neurales/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular , Cuerpo Estriado/enzimología , Daño del ADN , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/genética , Masculino , Ratones , Células-Madre Neurales/enzimología , Fosforilación , Transducción de Señal , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), and DKI-derived white matter tract integrity metrics (WMTI) were experimentally evaluated ex vivo through comparisons to histological measurements and established magnetic resonance imaging (MRI) measures of myelin in two knockout mouse models with varying degrees of hypomyelination. DKI metrics of mean and radial kurtosis were found to be better indicators of myelin content than conventional DTI metrics. The biophysical WMTI model based on the DKI framework reported on axon water fraction with good accuracy in cases with near normal axon density, but did not provide additional specificity to myelination. Overall, DKI provided additional information regarding white matter microstructure compared with DTI, making it an attractive method for future assessments of white matter development and pathology.
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Encéfalo/ultraestructura , Imagen de Difusión Tensora/métodos , Imagen por Resonancia Magnética/métodos , Vaina de Mielina/ultraestructura , Esclerosis Tuberosa/patología , Sustancia Blanca/ultraestructura , Animales , Axones/ultraestructura , Proteínas Portadoras/genética , Difusión , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Proteína Asociada al mTOR Insensible a la Rapamicina , Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa(vu463) ) that has an inactivating mutation in the etfa gene. dxa(vu463) recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa(vu463) zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa(vu463) zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.
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Flavoproteínas Transportadoras de Electrones/genética , Enfermedades Mitocondriales/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Complejos Multiproteicos/genética , Serina-Treonina Quinasas TOR/genética , Animales , Modelos Animales de Enfermedad , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Mitocondrias/genética , Mitocondrias/patología , Enfermedades Mitocondriales/fisiopatología , Terapia Molecular Dirigida , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/fisiopatología , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/terapia , Complejos Multiproteicos/antagonistas & inhibidores , Transducción de Señal , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.
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Autofagia/genética , Proteínas de Ciclo Celular , Ribosomas , Serina-Treonina Quinasas TOR , Proteína p53 Supresora de Tumor , Proteínas de Pez Cebra , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular , Genes Letales/genética , Mutación , Biosíntesis de Proteínas/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Poorly-defined interactions between environmental and genetic risk factors underlie Parkinson's disease (PD) etiology. Here we tested the hypothesis that human stem cell derived forebrain neuroprogenitors from patients with known familial risk for early onset PD will exhibit enhanced sensitivity to PD environmental risk factors compared to healthy control subjects without a family history of PD. Two male siblings (SM and PM) with biallelic loss-of-function mutations in PARK2 were identified. Human induced pluripotent stem cells (hiPSCs) from SM, PM, and four control subjects with no known family histories of PD or related neurodegenerative diseases were utilized. We tested the hypothesis that hiPSC-derived neuroprogenitors from patients with PARK2 mutations would show heightened cell death, mitochondrial dysfunction, and reactive oxygen species generation compared to control cells as a result of exposure to heavy metals (PD environmental risk factors). We report that PARK2 mutant neuroprogenitors showed increased cytotoxicity with copper (Cu) and cadmium (Cd) exposure but not manganese (Mn) or methyl mercury (MeHg) relative to control neuroprogenitors. PARK2 mutant neuroprogenitors also showed a substantial increase in mitochondrial fragmentation, initial ROS generation, and loss of mitochondrial membrane potential following Cu exposure. Our data substantiate Cu exposure as an environmental risk factor for PD. Furthermore, we report a shift in the lowest observable effect level (LOEL) for greater sensitivity to Cu-dependent mitochondrial dysfunction in patients SM and PM relative to controls, correlating with their increased genetic risk for PD.
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Cadmio/metabolismo , Cobre/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Células-Madre Neurales/metabolismo , Enfermedad de Parkinson/metabolismo , Ubiquitina-Proteína Ligasas , Adulto , Línea Celular , Predisposición Genética a la Enfermedad , Humanos , Masculino , Manganeso/metabolismo , Potencial de la Membrana Mitocondrial , Compuestos de Metilmercurio/metabolismo , Mutación , Enfermedad de Parkinson/genética , Factores de RiesgoRESUMEN
Tuberous sclerosis complex (TSC) is a multisystem genetic disorder with severe neurologic manifestations, including epilepsy, autism, anxiety and attention deficit hyperactivity disorder. TSC is caused by the loss of either the TSC1 or TSC2 genes that normally regulate the mammalian target of rapamycin (mTOR) kinase. mTOR exists within two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Loss of either TSC gene leads to increased mTORC1 but decreased mTORC2 signaling. As the contribution of decreased mTORC2 signaling to neural development and homeostasis has not been well studied, we generated a conditional knockout (CKO) of Rictor, a key component of mTORC2. mTORC2 signaling is impaired in the brain, whereas mTORC1 signaling is unchanged. Rictor CKO mice have small brains and bodies, normal lifespan and are fertile. Cortical layering is normal, but neurons are smaller than those in control brains. Seizures were not observed, although excessive slow activity was seen on electroencephalography. Rictor CKO mice are hyperactive and have reduced anxiety-like behavior. Finally, there is decreased white matter and increased levels of monoamine neurotransmitters in the cerebral cortex. Loss of mTORC2 signaling in the cortex independent of mTORC1 can disrupt normal brain development and function and may contribute to some of the neurologic manifestations seen in TSC.
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Proteínas Portadoras/genética , Eliminación de Gen , Complejos Multiproteicos/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Esclerosis Tuberosa/metabolismo , Animales , Ansiedad/genética , Conducta Animal , Western Blotting , Electroencefalografía , Técnica del Anticuerpo Fluorescente , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Noqueados , Proteína Asociada al mTOR Insensible a la Rapamicina , Convulsiones/genética , Convulsiones/fisiopatología , SueñoRESUMEN
Understanding differences in gene expression that increase risk for pulmonary arterial hypertension (PAH) is essential to understanding the molecular basis for disease. Previous studies on patient samples were limited by end-stage disease effects or by use of nonadherent cells, which are not ideal to model vascular cells in vivo. These studies addressed the hypothesis that pathological processes associated with PAH may be identified via a genetic signature common across multiple cell types. Expression array experiments were initially conducted to analyze cell types at different stages of vascular differentiation (mesenchymal stromal and endothelial) derived from PAH patient-specific induced pluripotent stem (iPS) cells. Molecular pathways that were altered in the PAH cell lines were then compared with those in fibroblasts from 21 patients, including those with idiopathic and heritable PAH. Wnt was identified as a target pathway and was validated in vitro using primary patient mesenchymal and endothelial cells. Taken together, our data suggest that the molecular lesions that cause PAH are present in all cell types evaluated, regardless of origin, and that stimulation of the Wnt signaling pathway was a common molecular defect in both heritable and idiopathic PAH.
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Diferenciación Celular/genética , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Células Madre Pluripotentes/patología , Vía de Señalización Wnt/genética , Línea Celular , Células Cultivadas , Células Endoteliales/patología , Células Endoteliales/fisiología , Hipertensión Pulmonar Primaria Familiar , Humanos , Células Madre Pluripotentes/fisiología , Mucosa Respiratoria/patología , Mucosa Respiratoria/fisiologíaRESUMEN
Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, the most severe symptoms of TSC are often neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes, we observed aberrant early neurodevelopment in vitro, including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions, including loci associated with key genes in neurodevelopment. Collectively, these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated, with implications for whether and how prenatal treatment should be pursued.