RESUMEN
Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.
Asunto(s)
Agenesia del Cuerpo Calloso/genética , Cerebelo/anomalías , Regulación del Desarrollo de la Expresión Génica/genética , Malformaciones del Desarrollo Cortical/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación/genética , Malformaciones del Sistema Nervioso/genética , Agenesia del Cuerpo Calloso/complicaciones , Agenesia del Cuerpo Calloso/diagnóstico por imagen , Agenesia del Cuerpo Calloso/patología , Animales , Animales Recién Nacidos , Apoptosis/genética , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Cerebelo/diagnóstico por imagen , Niño , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/genética , Modelos Animales de Enfermedad , Embrión de Mamíferos , Femenino , Humanos , Masculino , Malformaciones del Desarrollo Cortical/complicaciones , Malformaciones del Desarrollo Cortical/diagnóstico por imagen , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Malformaciones del Sistema Nervioso/complicaciones , Malformaciones del Sistema Nervioso/diagnóstico por imagen , Factor de Transcripción PAX6/metabolismoRESUMEN
In the supplementary information PDF originally posted, there were discrepancies from the integrated supplementary information that appeared in the HTML; the former has been corrected as follows. In the legend to Supplementary Fig. 2c, "major organs of the mouse" has been changed to "major organs of the adult mouse." In the legend to Supplementary Fig. 6d,h, "At E14.5 Mbe/Mbe mutants have a smaller percentage of Brdu positive cells in bin 3" has been changed to "At E14.5 Mbe/Mbe mutants have a higher percentage of Brdu positive cells in bin 3."
RESUMEN
The formation of the vertebrate brain requires the generation, migration, differentiation and survival of neurons. Genetic mutations that perturb these critical cellular events can result in malformations of the telencephalon, providing a molecular window into brain development. Here we report the identification of an N-ethyl-N-nitrosourea-induced mouse mutant characterized by a fractured hippocampal pyramidal cell layer, attributable to defects in neuronal migration. We show that this is caused by a hypomorphic mutation in Vps15 that perturbs endosomal-lysosomal trafficking and autophagy, resulting in an upregulation of Nischarin, which inhibits Pak1 signaling. The complete ablation of Vps15 results in the accumulation of autophagic substrates, the induction of apoptosis and severe cortical atrophy. Finally, we report that mutations in VPS15 are associated with cortical atrophy and epilepsy in humans. These data highlight the importance of the Vps15-Vps34 complex and the Nischarin-Pak1 signaling hub in the development of the telencephalon.
Asunto(s)
Movimiento Celular/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Mutación/efectos de los fármacos , Trastornos del Neurodesarrollo , Neuronas/patología , ATPasas de Translocación de Protón Vacuolares/genética , Alquilantes/toxicidad , Animales , Animales Recién Nacidos , Atrofia/inducido químicamente , Atrofia/genética , Atrofia/patología , Autofagia/efectos de los fármacos , Autofagia/genética , Encéfalo/efectos de los fármacos , Encéfalo/patología , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Embrión de Mamíferos , Etilnitrosourea/toxicidad , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trastornos del Neurodesarrollo/inducido químicamente , Trastornos del Neurodesarrollo/diagnóstico por imagen , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , ATPasas de Translocación de Protón Vacuolares/efectos de los fármacosRESUMEN
The development of the vertebrate central nervous system is reliant on a complex cascade of biological processes that include mitotic division, relocation of migrating neurons, and the extension of dendritic and axonal processes. Each of these cellular events requires the diverse functional repertoire of the microtubule cytoskeleton for the generation of forces, assembly of macromolecular complexes and transport of molecules and organelles. The tubulins are a multi-gene family that encode for the constituents of microtubules, and have been implicated in a spectrum of neurological disorders. Evidence is building that different tubulins tune the functional properties of the microtubule cytoskeleton dependent on the cell type, developmental profile and subcellular localisation. Here we review of the origins of the functional specification of the tubulin gene family in the developing brain at a transcriptional, translational, and post-transcriptional level. We remind the reader that tubulins are not just loading controls for your average Western blot.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Humanos , Procesamiento Proteico-Postraduccional/fisiología , ProteómicaRESUMEN
Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death.
Asunto(s)
Apoptosis/genética , Ciclo Celular/genética , Microcefalia/genética , Microtúbulos/genética , Tubulina (Proteína)/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Encéfalo/anomalías , Encéfalo/embriología , Diferenciación Celular , Modelos Animales de Enfermedad , Embrión de Mamíferos/embriología , Técnicas de Sustitución del Gen , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microtúbulos/metabolismo , Células-Madre Neurales/citología , Factores de Transcripción SOXB1/metabolismo , Huso Acromático/genética , Células Madre/citología , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
The development of the mammalian brain requires the generation, migration, and differentiation of neurons, cellular processes that are dependent on a dynamic microtubule cytoskeleton. Mutations in tubulin genes, which encode for the structural subunits of microtubules, cause detrimental neurological disorders known as the tubulinopathies. The disease spectra associated with different tubulin genes are overlapping but distinct, an observation believed to reflect functional specification of this multigene family. Perturbation of the ß-tubulin TUBB2B is known to cause polymicrogyria, pachygyria, microcephaly, and axon guidance defects. Here we provide a detailed analysis of the expression pattern of its murine homolog Tubb2b. The generation and characterization of BAC-transgenic eGFP reporter mouse lines has revealed that it is highly expressed in progenitors and postmitotic neurons during cortical development. This contrasts with the 8-week-old cortex, in which Tubb2b expression is restricted to macroglia, and expression is almost completely absent in mature neurons. This developmental transition in neurons is mirrored in the adult hippocampus and the cerebellum but is not a universal feature of Tubb2b; its expression persists in a population of postmitotic neurons in the 8-week-old retina. We propose that the dynamic spatial and temporal expression of Tubb2b reflects specific functional requirements of the microtubule cytoskeleton.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Neuronas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Imagenología Tridimensional , Immunoblotting , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Células-Madre Neurales/metabolismo , Neuroglía/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/crecimiento & desarrollo , Retina/metabolismo , Tubulina (Proteína)/genéticaRESUMEN
The formation of the mammalian cortex requires the generation, migration, and differentiation of neurons. The vital role that the microtubule cytoskeleton plays in these cellular processes is reflected by the discovery that mutations in various tubulin isotypes cause different neurodevelopmental diseases, including lissencephaly (TUBA1A), polymicrogyria (TUBA1A, TUBB2B, TUBB3), and an ocular motility disorder (TUBB3). Here, we show that Tubb5 is expressed in neurogenic progenitors in the mouse and that its depletion in vivo perturbs the cell cycle of progenitors and alters the position of migrating neurons. We report the occurrence of three microcephalic patients with structural brain abnormalities harboring de novo mutations in TUBB5 (M299V, V353I, and E401K). These mutant proteins, which affect the chaperone-dependent assembly of tubulin heterodimers in different ways, disrupt neurogenic division and/or migration in vivo. Our results provide insight into the functional repertoire of the tubulin gene family, specifically implicating TUBB5 in embryonic neurogenesis and microcephaly.
Asunto(s)
Encéfalo/anomalías , Encéfalo/metabolismo , Microcefalia/metabolismo , Mutación Missense , Células-Madre Neurales/metabolismo , Tubulina (Proteína)/metabolismo , Sustitución de Aminoácidos , Animales , Encéfalo/embriología , Encéfalo/patología , Femenino , Humanos , Masculino , Ratones , Ratones Mutantes , Microcefalia/embriología , Microcefalia/genética , Microcefalia/patología , Células-Madre Neurales/patología , Neurogénesis/genética , Tubulina (Proteína)/genéticaRESUMEN
The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing--both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal.
Asunto(s)
ARN/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Aminoácidos Básicos/fisiología , Cationes/química , Entropía , Mutagénesis , Conformación de Ácido Nucleico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
In bacteria, transcription, translation and gene regulation are highly coupled processes. The achievement of a certain functional structure at a distinct temporal and spatial position is therefore essential for RNA molecules. Proteins that facilitate this proper folding of RNA molecules are called RNA chaperones. Here a prominent example from E. coli is reviewed: the nucleoid associated protein StpA. Based on its various RNA remodeling functions, we propose a mechanistic model that explains how StpA promotes RNA folding. Through transient interactions via the RNA backbone, thereby shielding repelling charges in RNA, it pre-positions the RNA molecules for the successful formation of transition states from encounter complexes.