RESUMEN
Mutations in the Aristaless-related homeobox (ARX) gene are found in a spectrum of epilepsy and X-linked intellectual disability disorders. During development Arx is expressed in pallial ventricular zone (VZ) progenitor cells where the excitatory projection neurons of the cortex are born. Arx(-/Y) mice were shown to have decreased proliferation in the cortical VZ resulting in smaller brains; however, the basis for this reduced proliferation was not established. To determine the role of ARX on cell cycle dynamics in cortical progenitor cells, we generated cerebral cortex-specific Arx mouse mutants (cKO). The loss of pallial Arx resulted in the reduction of cortical progenitor cells, particularly the proliferation of intermediate progenitor cells (IPCs) was affected. Later in development and postnatally cKO brains showed a reduction of upper layer but not deeper layer neurons consistent with the IPC defect. Transcriptional profile analysis of E14.5 Arx-ablated cortices compared with control revealed that CDKN1C, an inhibitor of cell cycle progression, is overexpressed in the cortical VZ and SVZ of Arx KOs throughout corticogenesis. We also identified ARX as a direct regulator of Cdkn1c transcription. Together these data support a model where ARX regulates the expansion of cortical progenitor cells through repression of Cdkn1c.
Asunto(s)
Ciclo Celular/fisiología , Corteza Cerebral/crecimiento & desarrollo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Homeodominio/metabolismo , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Factores de Transcripción/metabolismo , Animales , Recuento de Células , Proliferación Celular/fisiología , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Proteínas de Homeodominio/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitosis/fisiología , Células-Madre Neurales/patología , Neuroglía/patología , Neuroglía/fisiología , Neuronas/patología , Neuronas/fisiología , Bulbo Olfatorio/crecimiento & desarrollo , Bulbo Olfatorio/patología , Bulbo Olfatorio/fisiopatología , Tamaño de los Órganos , Factores de Transcripción/genética , TranscriptomaRESUMEN
Mutations in the Aristaless-Related Homeobox (ARX) gene cause structural anomalies of the brain, epilepsy, and neurocognitive deficits in children. During forebrain development, Arx is expressed in both pallial and subpallial progenitor cells. We previously demonstrated that elimination of Arx from subpallial-derived cortical interneurons generates an epilepsy phenotype with features overlapping those seen in patients with ARX mutations. In this report, we have selectively removed Arx from pallial progenitor cells that give rise to the cerebral cortical projection neurons. While no discernable seizure activity was recorded, these mice exhibited a peculiar constellation of behaviors. They are less anxious, less social, and more active when compared with their wild-type littermates. The overall cortical thickness was reduced, and the corpus callosum and anterior commissure were hypoplastic, consistent with a perturbation in cortical connectivity. Taken together, these data suggest that some of the structural and behavioral anomalies, common in patients with ARX mutations, are specifically due to alterations in pallial progenitor function. Furthermore, our data demonstrate that some of the neurobehavioral features found in patients with ARX mutations may not be due to on-going seizures, as is often postulated, given that epilepsy was eliminated as a confounding variable in these behavior analyses.
Asunto(s)
Ondas Encefálicas/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mutación/genética , Telencéfalo/crecimiento & desarrollo , Telencéfalo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Conducta de Elección/fisiología , Condicionamiento Psicológico/fisiología , Adaptación a la Oscuridad/genética , Discapacidades del Desarrollo/genética , Modelos Animales de Enfermedad , Epilepsia/genética , Conducta Exploratoria/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/genética , Fuerza Muscular/genética , Fenotipo , Olfato/genéticaRESUMEN
Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.
Asunto(s)
Proteínas Hedgehog/metabolismo , Factor Nuclear 3-beta del Hepatocito/biosíntesis , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/metabolismo , Médula Espinal/embriología , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Animales , Embrión de Pollo , Epilepsia/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/biosíntesis , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Tubo Neural/embriología , Tubo Neural/crecimiento & desarrollo , Proteínas Nucleares , Factores de Transcripción/genética , Proteínas de Pez CebraRESUMEN
Polyalanine (poly-A) tracts exist in 494 annotated proteins; to date, expansions in these tracts have been associated with nine human diseases. The pathogenetic mechanism by which a poly-A tract results in these various human disorders remains uncertain. To understand the role of this mutation type, we investigated the change in functional properties of the transcription factor Arx when it has an expanded poly-A tract (Arx(E)), a mutation associated with infantile spasms and intellectual disabilities in humans. We found that although Arx(E) functions normally in the dorsal brain, its function in subpallial-derived populations of neurons is compromised. These contrasting functions are associated with the misregulation of Arx targets through the loss of the ability of Arx(E) to interact with the Arx cofactor Tle1. Our data demonstrate a novel mechanism for poly-A expansion diseases: the misregulation of a subset of target genes normally regulated by a transcription factor.
Asunto(s)
Encéfalo/embriología , Expansión de las Repeticiones de ADN , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Neuronas/fisiología , Poli A/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Encéfalo/citología , Encéfalo/metabolismo , Movimiento Celular , Corteza Cerebral/citología , Corteza Cerebral/embriología , Proteínas Co-Represoras , ADN/metabolismo , Proteínas de Homeodominio/química , Interneuronas/fisiología , Ratones , Mutación , Neurogénesis , Prosencéfalo/embriología , Prosencéfalo/metabolismo , Unión Proteica , Proteínas Represoras/metabolismo , Telencéfalo/citología , Telencéfalo/embriología , Factores de Transcripción/químicaRESUMEN
Citizen science is a productive approach to include non-scientists in research efforts that impact particular issues or communities. In most cases, scientists at advanced career stages design high-quality, exciting projects that enable citizen contribution, a crowdsourcing process that drives discovery forward and engages communities. The challenges of having citizens design their own research with no or limited training and providing access to laboratory tools, reagents, and supplies have limited citizen science efforts. This leaves the incredible life experiences and immersion of citizens in communities that experience health disparities out of the research equation, thus hampering efforts to address community health needs with a full picture of the challenges that must be addressed. Here, we present a robust and reproducible approach that engages participants from Grade 5 through adult in research focused on defining how diet impacts disease signaling. We leverage the powerful genetics, cell biology, and biochemistry of Drosophila oogenesis to define how nutrients impact phenotypes associated with genetic mutants that are implicated in cancer and diabetes. Participants lead the project design and execution, flipping the top-down hierarchy of the prevailing scientific culture to co-create research projects and infuse the research with cultural and community relevance.
Asunto(s)
Drosophila , Salud Pública , Animales , InvestigaciónRESUMEN
Mesothelin is a cell-surface molecule over-expressed on a large fraction of carcinomas, and thus is an attractive target of immunotherapy. A molecularly targeted therapy for these cancers was created by engineering T cells to express a chimeric receptor with high affinity for human mesothelin. Lentiviral vectors were used to express a single-chain variable fragment that binds mesothelin and that is fused to signaling domains derived from T-cell receptor zeta, CD28, and CD137 (4-1BB). When stimulated by mesothelin, lentivirally transduced T cells were induced to proliferate, express the antiapoptotic gene Bcl-X(L), and secrete multiple cytokines, all features characteristic of central memory T cells. When transferred intratumorally or intravenously into NOD/scid/IL2rgamma(-/-) mice engrafted with large pre-established tumors, the engineered T cells reduced the tumor burden, and in some cases resulted in complete eradication of the tumors at low effector-to-target ratios. Incorporation of the CD137 signaling domain specifically reprogrammed cells for multifunctional cytokine secretion and enhanced persistence of T cells. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of poorly immunogenic tumors. Genetically redirected T cells have promise of targeting T lymphocytes to tumor antigens, confer resistance to the tumor microenvironment, and providing immunosurveillance.
Asunto(s)
Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Animales , Antígenos CD28/genética , Línea Celular Tumoral , Humanos , Mesotelina , Ratones , Transducción de Señal/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.
Asunto(s)
IMP Deshidrogenasa/genética , Retina/enzimología , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Sitios de Unión , Dominio Catalítico , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , IMP Deshidrogenasa/química , IMP Deshidrogenasa/ultraestructura , Modelos Moleculares , NAD/metabolismo , Enfermedades de la Retina/genéticaRESUMEN
Mammalian forebrain development requires extensive migration, yet the mechanisms through which migrating neurons sense and respond to guidance cues are not well understood. Similar to the axon growth cone, the leading process and branches of neurons may guide migration, but the cytoskeletal events that regulate branching are unknown. We have previously shown that loss of microtubule-associated protein Lis1 reduces branching during migration compared with wild-type neurons. Using time-lapse imaging of Lis1(+/-) and Lis1(+/+) cells migrating from medial ganglionic eminence explant cultures, we show that the branching defect is not due to a failure to initiate branches but a defect in the stabilization of new branches. The leading processes of Lis1(+/-) neurons have reduced expression of stabilized, acetylated microtubules compared with Lis1(+/+) neurons. To determine whether Lis1 modulates branch stability through its role as the noncatalytic beta regulatory subunit of platelet-activating factor (PAF) acetylhydrolase 1b, exogenous PAF was applied to wild-type cells. Excess PAF added to wild-type neurons phenocopies the branch instability observed in Lis1(+/-) neurons, and a PAF antagonist rescues leading process branching in Lis1(+/-) neurons. These data highlight a role for Lis1, acting through the PAF pathway, in leading process branching and microtubule stabilization.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Movimiento Celular/fisiología , Interneuronas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa/química , 1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa/farmacología , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Interneuronas/citología , Interneuronas/efectos de los fármacos , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/farmacología , Microtúbulos/metabolismo , Microtúbulos/fisiología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Técnicas de Cultivo de Órganos , Prosencéfalo/citología , Prosencéfalo/embriología , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Many different enzymes in intermediate metabolism dynamically assemble filamentous polymers in cells, often in response to changes in physiological conditions. Most of the enzyme filaments known to date have only been observed in cells, but in a handful of cases structural and biochemical studies have revealed the mechanisms and consequences of assembly. In general, enzyme polymerization functions as a mechanism to allosterically tune enzyme kinetics, and it may play a physiological role in integrating metabolic signaling. Here, we highlight some principles of metabolic filaments by focusing on two well-studied examples in nucleotide biosynthesis pathways-inosine-5'-monophosphate (IMP) dehydrogenase and cytosine triphosphate (CTP) synthase.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , IMP Deshidrogenasa/metabolismo , Ligasas de Carbono-Nitrógeno/fisiología , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Células HeLa , Humanos , IMP Deshidrogenasa/fisiología , Polimerizacion , Multimerización de Proteína/fisiologíaRESUMEN
Polymerization of metabolic enzymes into micron-scale assemblies is an emerging mechanism for regulating their activity. CTP synthase (CTPS) is an essential enzyme in the biosynthesis of the nucleotide CTP and undergoes regulated and reversible assembly into large filamentous structures in organisms from bacteria to humans. The purpose of these assemblies is unclear. A major challenge to addressing this question has been the inability to abolish assembly without eliminating CTPS protein. Here we demonstrate that a recently reported point mutant in CTPS, Histidine 355A (H355A), prevents CTPS filament assembly in vivo and dominantly inhibits the assembly of endogenous wild-type CTPS in the Drosophila ovary. Expressing this mutant in ovarian germline cells, we show that disruption of CTPS assembly in early stage egg chambers reduces egg production. This effect is exacerbated in flies fed the glutamine antagonist 6-diazo-5-oxo-L-norleucine, which inhibits de novo CTP synthesis. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Drosophila/fisiología , Células Germinativas/metabolismo , Multimerización de Proteína , Reproducción , Animales , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/genética , Metabolismo Energético , Técnica del Anticuerpo Fluorescente , Expresión Génica , Glutamina/metabolismo , MutaciónRESUMEN
Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas GTP promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and non-assembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.
RESUMEN
Mutations in the aristaless-related homeobox (ARX) gene result in a spectrum of structural and functional nervous system disorders including lissencephaly, movement disorders, intellectual disabilities, and epilepsy. Some patients also have symptoms indicating hypothalamic dysfunction, but little is known about the role of ARX in diencephalic development. To begin evaluating diencephalic defects, we examined the expression of a panel of known genes and gene products that label specific diencephalic nuclei in 2 different Arx mutant mouse lines at E18.5. Male mice engineered to have a polyalanine expansion mutation (Arx) revealed no expression differences in any diencephalic nucleus when compared with wild-type littermates. In contrast, mice null for Arx (Arx) lost expression of specific markers of the thalamic reticular nucleus and zona incerta (ZI) while retaining expression in other thalamic nuclei and in the hypothalamus. Tyrosine hydroxylase, a marker of the dopaminergic A13 subnucleus of ZI, was among those lost, suggesting a requirement for Arx in normal thalamic reticular nucleus and ZI development and, specifically, for A13 dopaminergic fate. Because the ZI and A13 regions make connections to several hypothalamic nuclei, such misspecification may contribute to the "hypothalamic dysfunction" observed in some patients.