Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 51
Filtrar
Más filtros

Banco de datos
País/Región como asunto
Tipo del documento
Intervalo de año de publicación
1.
Proc Natl Acad Sci U S A ; 120(21): e2218478120, 2023 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-37192167

RESUMEN

Aneuploidy syndromes impact multiple organ systems but understanding of tissue-specific aneuploidy effects remains limited-especially for the comparison between peripheral tissues and relatively inaccessible tissues like brain. Here, we address this gap in knowledge by studying the transcriptomic effects of chromosome X, Y, and 21 aneuploidies in lymphoblastoid cell lines, fibroblasts and iPSC-derived neuronal cells (LCLs, FCL, and iNs, respectively). We root our analyses in sex chromosome aneuploidies, which offer a uniquely wide karyotype range for dosage effect analysis. We first harness a large LCL RNA-seq dataset from 197 individuals with one of 6 sex chromosome dosages (SCDs: XX, XXX, XY, XXY, XYY, and XXYY) to i) validate theoretical models of SCD sensitivity and ii) define an expanded set of 41 genes that show obligate dosage sensitivity to SCD and are all in cis (i.e., reside on the X or Y chromosome). We then use multiple complementary analyses to show that cis effects of SCD in LCLs are preserved in both FCLs (n = 32) and iNs (n = 24), whereas trans effects (i.e., those on autosomal gene expression) are mostly not preserved. Analysis of additional datasets confirms that the greater cross-cell type reproducibility of cis vs. trans effects is also seen in trisomy 21 cell lines. These findings i) expand our understanding of X, Y, and 21 chromosome dosage effects on human gene expression and ii) suggest that LCLs may provide a good model system for understanding cis effects of aneuploidy in harder-to-access cell types.


Asunto(s)
Aneuploidia , Síndrome de Down , Humanos , Reproducibilidad de los Resultados , Síndrome de Down/genética , Cromosomas Sexuales , Expresión Génica
2.
Genome Res ; 31(6): 1069-1081, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-34011578

RESUMEN

Single-cell RNA sequencing (scRNA-seq) is a widely used method for identifying cell types and trajectories in biologically heterogeneous samples, but it is limited in its detection and quantification of lowly expressed genes. This results in missing important biological signals, such as the expression of key transcription factors (TFs) driving cellular differentiation. We show that targeted sequencing of ∼1000 TFs (scCapture-seq) in iPSC-derived neuronal cultures greatly improves the biological information garnered from scRNA-seq. Increased TF resolution enhanced cell type identification, developmental trajectories, and gene regulatory networks. This allowed us to resolve differences among neuronal populations, which were generated in two different laboratories using the same differentiation protocol. ScCapture-seq improved TF-gene regulatory network inference and thus identified divergent patterns of neurogenesis into either excitatory cortical neurons or inhibitory interneurons. Furthermore, scCapture-seq revealed a role for of retinoic acid signaling in the developmental divergence between these different neuronal populations. Our results show that TF targeting improves the characterization of human cellular models and allows identification of the essential differences between cellular populations, which would otherwise be missed in traditional scRNA-seq. scCapture-seq TF targeting represents a cost-effective enhancement of scRNA-seq, which could be broadly applied to improve scRNA-seq resolution.


Asunto(s)
Células Madre Pluripotentes Inducidas , Análisis de la Célula Individual , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
3.
Ann Neurol ; 90(2): 193-202, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34184781

RESUMEN

OBJECTIVE: This study was undertaken to identify susceptibility loci for cluster headache and obtain insights into relevant disease pathways. METHODS: We carried out a genome-wide association study, where 852 UK and 591 Swedish cluster headache cases were compared with 5,614 and 1,134 controls, respectively. Following quality control and imputation, single variant association testing was conducted using a logistic mixed model for each cohort. The 2 cohorts were subsequently combined in a merged analysis. Downstream analyses, such as gene-set enrichment, functional variant annotation, prediction and pathway analyses, were performed. RESULTS: Initial independent analysis identified 2 replicable cluster headache susceptibility loci on chromosome 2. A merged analysis identified an additional locus on chromosome 1 and confirmed a locus significant in the UK analysis on chromosome 6, which overlaps with a previously known migraine locus. The lead single nucleotide polymorphisms were rs113658130 (p = 1.92 × 10-17 , odds ratio [OR] = 1.51, 95% confidence interval [CI] = 1.37-1.66) and rs4519530 (p = 6.98 × 10-17 , OR = 1.47, 95% CI = 1.34-1.61) on chromosome 2, rs12121134 on chromosome 1 (p = 1.66 × 10-8 , OR = 1.36, 95% CI = 1.22-1.52), and rs11153082 (p = 1.85 × 10-8 , OR = 1.30, 95% CI = 1.19-1.42) on chromosome 6. Downstream analyses implicated immunological processes in the pathogenesis of cluster headache. INTERPRETATION: We identified and replicated several genome-wide significant associations supporting a genetic predisposition in cluster headache in a genome-wide association study involving 1,443 cases. Replication in larger independent cohorts combined with comprehensive phenotyping, in relation to, for example, treatment response and cluster headache subtypes, could provide unprecedented insights into genotype-phenotype correlations and the pathophysiological pathways underlying cluster headache. ANN NEUROL 2021;90:193-202.


Asunto(s)
Cefalalgia Histamínica/epidemiología , Cefalalgia Histamínica/genética , Sitios Genéticos/genética , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo/métodos , Estudios de Casos y Controles , Cefalalgia Histamínica/diagnóstico , Estudios de Cohortes , Femenino , Humanos , Masculino , Suecia/epidemiología , Reino Unido/epidemiología
4.
Mol Psychiatry ; 26(9): 5252-5265, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-32404948

RESUMEN

Bipolar disorder is a chronic neuropsychiatric condition associated with mood instability, where patients present significant sleep and circadian rhythm abnormalities. Currently, the pathophysiology of bipolar disorder remains elusive, but treatment with lithium continues as the benchmark pharmacotherapy, functioning as a potent mood stabilizer in most, but not all patients. Lithium is well documented to induce period lengthening and amplitude enhancement of the circadian clock. Based on this, we sought to investigate whether lithium differentially impacts circadian rhythms in bipolar patient cell lines and crucially if lithium's effect on the clock is fundamental to its mood-stabilizing effects. We analyzed the circadian rhythms of bipolar patient-derived fibroblasts (n = 39) and their responses to lithium and three further chronomodulators. Here we show, relative to controls (n = 23), patients exhibited a wider distribution of circadian period (p < 0.05), and that patients with longer periods were medicated with a wider range of drugs, suggesting lower effectiveness of lithium. In agreement, patient fibroblasts with longer periods displayed muted circadian responses to lithium as well as to other chronomodulators that phenocopy lithium. These results show that lithium differentially impacts the circadian system in a patient-specific manner and its effect is dependent on the patient's circadian phenotype. We also found that lithium-induced behavioral changes in mice were phenocopied by modulation of the circadian system with drugs that target the clock, and that a dysfunctional clock ablates this response. Thus, chronomodulatory compounds offer a promising route to a novel treatment paradigm. These findings, upon larger-scale validation, could facilitate the implementation of a personalized approach for mood stabilization.


Asunto(s)
Trastorno Bipolar , Litio , Animales , Trastorno Bipolar/tratamiento farmacológico , Ritmo Circadiano , Fibroblastos , Humanos , Compuestos de Litio/farmacología , Ratones
5.
J Biol Chem ; 294(17): 7085-7097, 2019 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-30872401

RESUMEN

The cellular prion protein (PrPC) is a key neuronal receptor for ß-amyloid oligomers (AßO), mediating their neurotoxicity, which contributes to the neurodegeneration in Alzheimer's disease (AD). Similarly to the amyloid precursor protein (APP), PrPC is proteolytically cleaved from the cell surface by a disintegrin and metalloprotease, ADAM10. We hypothesized that ADAM10-modulated PrPC shedding would alter the cellular binding and cytotoxicity of AßO. Here, we found that in human neuroblastoma cells, activation of ADAM10 with the muscarinic agonist carbachol promotes PrPC shedding and reduces the binding of AßO to the cell surface, which could be blocked with an ADAM10 inhibitor. Conversely, siRNA-mediated ADAM10 knockdown reduced PrPC shedding and increased AßO binding, which was blocked by the PrPC-specific antibody 6D11. The retinoic acid receptor analog acitretin, which up-regulates ADAM10, also promoted PrPC shedding and decreased AßO binding in the neuroblastoma cells and in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Pretreatment with acitretin abolished activation of Fyn kinase and prevented an increase in reactive oxygen species caused by AßO binding to PrPC Besides blocking AßO binding and toxicity, acitretin also increased the nonamyloidogenic processing of APP. However, in the iPSC-derived neurons, Aß and other amyloidogenic processing products did not exhibit a reciprocal decrease upon acitretin treatment. These results indicate that by promoting the shedding of PrPC in human neurons, ADAM10 activation prevents the binding and cytotoxicity of AßO, revealing a potential therapeutic benefit of ADAM10 activation in AD.


Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Biopolímeros/metabolismo , Proteínas de la Membrana/metabolismo , Proteína ADAM10/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Línea Celular Tumoral , Activación Enzimática , Técnicas de Silenciamiento del Gen , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/genética , Proteínas Priónicas/metabolismo , Unión Proteica , Proteolisis , Especies Reactivas de Oxígeno/metabolismo
6.
RNA Biol ; 17(12): 1741-1753, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32597303

RESUMEN

RNA-seq is the standard method for profiling gene expression in many biological systems. Due to the wide dynamic range and complex nature of the transcriptome, RNA-seq provides an incomplete characterization, especially of lowly expressed genes and transcripts. Targeted RNA sequencing (RNA CaptureSeq) focuses sequencing on genes of interest, providing exquisite sensitivity for transcript detection and quantification. However, uses of CaptureSeq have focused on bulk samples and its performance on very small populations of cells is unknown. Here we show CaptureSeq greatly enhances transcriptomic profiling of target genes in ultra-low-input samples and provides equivalent performance to that on bulk samples. We validate the performance of CaptureSeq using multiple probe sets on samples of iPSC-derived cortical neurons. We demonstrate up to 275-fold enrichment for target genes, the detection of 10% additional genes and a greater than 5-fold increase in identified gene isoforms. Analysis of spike-in controls demonstrated CaptureSeq improved both detection sensitivity and expression quantification. Comparison to the CORTECON database of cerebral cortex development revealed CaptureSeq enhanced the identification of sample differentiation stage. CaptureSeq provides sensitive, reliable and quantitative expression measurements on hundreds-to-thousands of target genes from ultra-low-input samples and has the potential to greatly enhance transcriptomic profiling when samples are limiting.


Asunto(s)
Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN , Transcriptoma , Diferenciación Celular/genética , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neuronas/citología , Neuronas/metabolismo , Análisis de Secuencia de ARN/métodos , Factores de Transcripción/metabolismo
7.
Brain ; 142(12): 3852-3867, 2019 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-31742594

RESUMEN

The two-pore potassium channel, TRESK has been implicated in nociception and pain disorders. We have for the first time investigated TRESK function in human nociceptive neurons using induced pluripotent stem cell-based models. Nociceptors from migraine patients with the F139WfsX2 mutation show loss of functional TRESK at the membrane, with a corresponding significant increase in neuronal excitability. Furthermore, using CRISPR-Cas9 engineering to correct the F139WfsX2 mutation, we show a reversal of the heightened neuronal excitability, linking the phenotype to the mutation. In contrast we find no change in excitability in induced pluripotent stem cell derived nociceptors with the C110R mutation and preserved TRESK current; thereby confirming that only the frameshift mutation is associated with loss of function and a migraine relevant cellular phenotype. We then demonstrate the importance of TRESK to pain states by showing that the TRESK activator, cloxyquin, can reduce the spontaneous firing of nociceptors in an in vitro human pain model. Using the chronic nitroglycerine rodent migraine model, we demonstrate that mice lacking TRESK develop exaggerated nitroglycerine-induced mechanical and thermal hyperalgesia, and furthermore, show that cloxyquin conversely is able to prevent sensitization. Collectively, our findings provide evidence for a role of TRESK in migraine pathogenesis and its suitability as a therapeutic target.


Asunto(s)
Mutación con Pérdida de Función , Trastornos Migrañosos/genética , Nocicepción/fisiología , Nociceptores/metabolismo , Canales de Potasio/genética , Animales , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Trastornos Migrañosos/inducido químicamente , Trastornos Migrañosos/metabolismo , Nitroglicerina , Dimensión del Dolor , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo
8.
Proc Natl Acad Sci U S A ; 114(16): E3324-E3333, 2017 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-28351971

RESUMEN

Charcot-Marie-Tooth disease type 2D (CMT2D) is a peripheral nerve disorder caused by dominant, toxic, gain-of-function mutations in the widely expressed, housekeeping gene, GARS The mechanisms underlying selective nerve pathology in CMT2D remain unresolved, as does the cause of the mild-to-moderate sensory involvement that distinguishes CMT2D from the allelic disorder distal spinal muscular atrophy type V. To elucidate the mechanism responsible for the underlying afferent nerve pathology, we examined the sensory nervous system of CMT2D mice. We show that the equilibrium between functional subtypes of sensory neuron in dorsal root ganglia is distorted by Gars mutations, leading to sensory defects in peripheral tissues and correlating with overall disease severity. CMT2D mice display changes in sensory behavior concordant with the afferent imbalance, which is present at birth and nonprogressive, indicating that sensory neuron identity is prenatally perturbed and that a critical developmental insult is key to the afferent pathology. Through in vitro experiments, mutant, but not wild-type, GlyRS was shown to aberrantly interact with the Trk receptors and cause misactivation of Trk signaling, which is essential for sensory neuron differentiation and development. Together, this work suggests that both neurodevelopmental and neurodegenerative mechanisms contribute to CMT2D pathogenesis, and thus has profound implications for the timing of future therapeutic treatments.


Asunto(s)
Enfermedad de Charcot-Marie-Tooth/patología , Glicina-ARNt Ligasa/fisiología , Mutación , Receptor trkA/metabolismo , Células Receptoras Sensoriales/patología , Animales , Células Cultivadas , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Receptor trkA/genética , Células Receptoras Sensoriales/metabolismo
9.
Brain ; 141(3): 673-687, 2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29415205

RESUMEN

Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.


Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/terapia , Glicina-ARNt Ligasa/genética , Histona Desacetilasa 6/metabolismo , Mutación/genética , Animales , Transporte Axonal/genética , Células Cultivadas , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/uso terapéutico , Ganglios Espinales/citología , Histona Desacetilasa 6/genética , Ácidos Hidroxámicos/uso terapéutico , Indoles/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Conducción Nerviosa/genética , Unión Neuromuscular/patología , Unión Neuromuscular/fisiopatología , Desempeño Psicomotor/fisiología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Tubulina (Proteína)/metabolismo
10.
Epilepsy Behav ; 101(Pt B): 106581, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31761686

RESUMEN

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder caused by deletions in the TSC1 or TSC2 genes that is associated with epilepsy in up to 90% of patients. Seizures are suggested to start in benign brain tumors, cortical tubers, or in the perituberal tissue making these tubers an interesting target for further research into mechanisms underlying epileptogenesis in TSC. Animal models of TSC insufficiently capture the neurodevelopmental biology of cortical tubers, and hence, human stem cell-based in vitro models of TSC are being increasingly explored in attempts to recapitulate tuber development and epileptogenesis in TSC. However, in vitro culture conditions for stem cell-derived neurons do not necessarily mimic physiological conditions. For example, very high glucose concentrations of up to 25 mM are common in culture media formulations. As TSC is potentially caused by a disruption of the mechanistic target of rapamycin (mTOR) pathway, a main integrator of metabolic information and intracellular signaling, we aimed to examine the impact of different glucose concentrations in the culture media on cellular phenotypes implicated in tuber characteristics. Here, we present preliminary data from a pilot study exploring cortical neuronal differentiation on human embryonic stem cells (hES) harboring a TSC2 knockout mutation (TSC2-/-) and an isogenic control line (TSC2+/+). We show that the commonly used high glucose media profoundly mask cellular phenotypes in TSC2-/- cultures during neuronal differentiation. These phenotypes only become apparent when differentiating TSC2+/+ and TSC2-/- cultures in more physiologically relevant conditions of 5 mM glucose suggesting that the careful consideration of culture conditions is vital to ensuring biological relevance and translatability of stem cell models for neurological disorders such as TSC. This article is part of the Special Issue "Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures".


Asunto(s)
Glucosa/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/ultraestructura , Esclerosis Tuberosa/patología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Células Madre Embrionarias/ultraestructura , Técnicas de Inactivación de Genes , Humanos , Modelos Neurológicos , Mutación/efectos de los fármacos , Neurogénesis , Fenotipo , Proyectos Piloto , Serina-Treonina Quinasas TOR/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética
11.
Hum Mol Genet ; 25(5): 989-1000, 2016 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-26740550

RESUMEN

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.


Asunto(s)
Corteza Cerebral/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Transcriptoma , Adulto , Anciano , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular , Corteza Cerebral/citología , Femenino , Feto , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual
12.
Clin Sci (Lond) ; 132(3): 399-418, 2018 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-29444850

RESUMEN

From the earliest stages of development, when cerebral angiogenesis and neurogenesis are entwined, to the end of life, the interplay between vascular and neural systems of the brain is critical in health and disease. Cerebral microvascular endothelial cells constitute the blood-brain barrier and in concert with pericytes or smooth muscle cells, glia and neurons, integrate into a functional neurovascular unit (NVU). This multicellular NVU maintains homoeostasis of the brain's microenvironment by restricting the entry of systemic pathogens and neurotoxins as well as meeting the metabolic demands of neural activity. Recent evidence of cerebral microvascular pathologies in vascular diseases and dementia, including Alzheimer's disease, has challenged the notion that vascular events are merely the consequence of neuronal pathology. This review focuses on molecular mechanisms of neurovascular dysfunction in dementia and outlines currently employed in vitro models to decode such mechanisms. Deciphering neurovascular crosstalk is likely to be more important in understanding the molecular mechanisms of disease than previously anticipated and may offer novel therapeutic opportunities for dementia and related conditions.


Asunto(s)
Barrera Hematoencefálica/fisiopatología , Encéfalo/irrigación sanguínea , Circulación Cerebrovascular/fisiología , Acoplamiento Neurovascular/fisiología , Animales , Barrera Hematoencefálica/patología , Humanos , Neuronas/citología , Pericitos/citología
13.
Clin Sci (Lond) ; 132(8): 851-868, 2018 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-29712883

RESUMEN

Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.


Asunto(s)
Enfermedades de los Pequeños Vasos Cerebrales/etiología , Investigación Biomédica Traslacional , Animales , Humanos
14.
Biotechnol Lett ; 40(3): 601-607, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29313254

RESUMEN

OBJECTIVES: To test a 3D approach for neural network formation, alignment, and patterning that is reproducible and sufficiently stable to allow for easy manipulation. RESULTS: A novel cell culture system was designed by engineering a method for the directional growth of neurons. This uses NG108-15 neuroblastoma x glioma hybrid cells cultured on suspended and aligned electrospun fibers. These fiber networks improved cellular directionality, with alignment angle standard deviations significantly lower on fibers than on regular culture surfaces. Morphological studies found nuclear aspect ratios and cell projection lengths to be unchanged, indicating that cells maintained neural morphology while growing on fibers and forming a 3D network. Furthermore, fibronectin-coated fibers enhanced neurite extensions for all investigated time points. Differentiated neurons exhibited significant increases in average neurite lengths 96 h post plating, and formed neurite extensions parallel to suspended fibers, as visualized through scanning electron microscopy. CONCLUSIONS: The developed model has the potential to serve as the basis for advanced 3D studies, providing an original approach to neural network patterning and setting the groundwork for further investigations into functionality.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Técnicas Electroquímicas/métodos , Modelos Neurológicos , Fibras Nerviosas/fisiología , Red Nerviosa/citología , Ingeniería de Tejidos/métodos , Animales , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular Tumoral , Diseño de Equipo , Fibronectinas/farmacología , Glioma , Neuritas/fisiología , Neuroblastoma , Poliésteres , Ratas
15.
Hum Mol Genet ; 24(15): 4397-406, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-25972375

RESUMEN

Charcot-Marie-Tooth (CMT) neuropathies are collectively the most common hereditary neurological condition and a major health burden for society. Dominant mutations in the gene GARS, encoding the ubiquitous enzyme, glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and lead to CMT disease type 2D. This genetic disorder exemplifies a recurring motif in neurodegeneration, whereby mutations in essential, widely expressed genes have selective deleterious consequences for the nervous system. Here, using novel Drosophila models, we show a potential solution to this phenomenon. Ubiquitous expression of mutant GlyRS leads to motor deficits, progressive neuromuscular junction (NMJ) denervation and pre-synaptic build-up of mutant GlyRS. Intriguingly, neuronal toxicity is, at least in part, non-cell autonomous, as expression of mutant GlyRS in mesoderm or muscle alone results in similar pathology. This mutant GlyRS toxic gain-of-function, which is WHEP domain-dependent, coincides with abnormal NMJ assembly, leading to synaptic degeneration, and, ultimately, reduced viability. Our findings suggest that mutant GlyRS gains access to ectopic sub-compartments of the motor neuron, providing a possible explanation for the selective neuropathology caused by mutations in a widely expressed gene.


Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Glicina-ARNt Ligasa/genética , Degeneración Nerviosa/genética , Unión Neuromuscular/genética , Animales , Enfermedad de Charcot-Marie-Tooth/patología , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Glicina-ARNt Ligasa/biosíntesis , Humanos , Cuerpos Pedunculados/patología , Mutación , Degeneración Nerviosa/patología , Unión Neuromuscular/crecimiento & desarrollo , Unión Neuromuscular/patología , Bulbo Olfatorio/patología , Nervios Periféricos/patología
16.
Anal Chem ; 89(4): 2440-2448, 2017 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-28192931

RESUMEN

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.


Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Proteoma/análisis , Proteómica , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Espectrometría de Masas/métodos , Proteoma/metabolismo , Piel/citología , Factores de Transcripción/análisis , Factores de Transcripción/metabolismo
17.
Hum Mol Genet ; 23(10): 2639-50, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24368416

RESUMEN

Dominant mutations in GARS, encoding the essential enzyme glycyl-tRNA synthetase (GlyRS), result in a form of Charcot-Marie-Tooth disease, type 2D (CMT2D), predominantly characterized by lower motor nerve degeneration. GlyRS charges the amino acid glycine with its cognate tRNA and is therefore essential for protein translation. However, the underlying mechanisms linking toxic gain-of-function GARS mutations to lower motor neuron degeneration remain unidentified. The neuromuscular junction (NMJ) appears to be an early target for pathology in a number of peripheral nerve diseases and becomes denervated at later stages in two mouse models of CMT2D. We therefore performed a detailed longitudinal examination of NMJs in the distal lumbrical muscles and the proximal transversus abdominis (TVA) muscles of wild-type and Gars mutant mice. We determined that mutant lumbrical NMJs display a persistent defect in maturation that precedes a progressive, age-dependent degeneration. Conversely, the TVA remains relatively unaffected, with only a subtle, short-lived impairment in pre- and post-synaptic development and no reduction in lower motor neuron connectivity to muscle. Together, these observations suggest that mutant Gars is associated with compromised development of the NMJ prior to synaptic degeneration and highlight the neuromuscular synapse as an important site of early, selective pathology in CMT2D mice.


Asunto(s)
Enfermedad de Charcot-Marie-Tooth/patología , Neuronas Motoras/patología , Unión Neuromuscular/patología , Músculos Abdominales/inervación , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Glicina-ARNt Ligasa/genética , Miembro Posterior/inervación , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense
18.
Cephalalgia ; 35(11): 1025-30, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25631169

RESUMEN

BACKGROUND: Visual aura is present in about one-third of migraine patients and triggering by bright or flickering lights is frequently reported. METHOD: Using migraine with visual aura patients, we investigated the neurochemical profile of the visual cortex using magnetic resonance spectroscopy. Specifically, glutamate/creatine and GABA/creatine ratios were quantified in the occipital cortex of female migraine patients. RESULTS: GABA levels in the occipital cortex of migraine patients were lower than that of controls. Glutamate levels in migraine patients, but not controls, correlated with the blood-oxygenation-level-dependent (BOLD) signal in the primary visual cortex during visual stimulation. CONCLUSION: Migraine with visual aura appears to disrupt the excitation-inhibition coupling in the occipital cortex.


Asunto(s)
Ácido Glutámico/análisis , Migraña con Aura/metabolismo , Migraña con Aura/fisiopatología , Lóbulo Occipital/metabolismo , Ácido gamma-Aminobutírico/análisis , Adulto , Química Encefálica , Femenino , Ácido Glutámico/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Lóbulo Occipital/química , Ácido gamma-Aminobutírico/metabolismo
19.
PLoS Genet ; 8(12): e1003074, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23236289

RESUMEN

ß-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding ß-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as "Lincoln ataxia," because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of ß-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that ß-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.


Asunto(s)
Cerebelo , Espectrina/genética , Ataxias Espinocerebelosas , Adulto , Animales , Cerebelo/crecimiento & desarrollo , Cerebelo/patología , Mapeo Cromosómico , Trastornos del Conocimiento/genética , Humanos , Ratones , Ratones Noqueados , Mutación , Neuronas/metabolismo , Neuronas/patología , Células de Purkinje/patología , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/fisiopatología
20.
Nat Genet ; 37(10): 1108-12, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16186814

RESUMEN

Genetic susceptibility to multiple sclerosis is associated with genes of the major histocompatibility complex (MHC), particularly HLA-DRB1 and HLA-DQB1 (ref. 1). Both locus and allelic heterogeneity have been reported in this genomic region. To clarify whether HLA-DRB1 itself, nearby genes in the region encoding the MHC or combinations of these loci underlie susceptibility to multiple sclerosis, we genotyped 1,185 Canadian and Finnish families with multiple sclerosis (n = 4,203 individuals) with a high-density SNP panel spanning the genes encoding the MHC and flanking genomic regions. Strong associations in Canadian and Finnish samples were observed with blocks in the HLA class II genomic region (P < 4.9 x 10(-13) and P < 2.0 x 10(-16), respectively), but the strongest association was with HLA-DRB1 (P < 4.4 x 10(-17)). Conditioning on either HLA-DRB1 or the most significant HLA class II haplotype block found no additional block or SNP association independent of the HLA class II genomic region. This study therefore indicates that MHC-associated susceptibility to multiple sclerosis is determined by HLA class II alleles, their interactions and closely neighboring variants.


Asunto(s)
Antígenos HLA-DR/genética , Antígenos de Histocompatibilidad Clase II/genética , Esclerosis Múltiple/genética , Polimorfismo de Nucleótido Simple , Canadá , Finlandia , Predisposición Genética a la Enfermedad , Cadenas HLA-DRB1 , Humanos , Complejo Mayor de Histocompatibilidad/genética , Esclerosis Múltiple/etnología , Población Blanca
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA