RESUMEN
Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Dipéptidos/metabolismo , Demencia Frontotemporal/metabolismo , Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Proteína C9orf72 , Nucléolo Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , Expansión de las Repeticiones de ADN , Dipéptidos/genética , Drosophila melanogaster/genética , Demencia Frontotemporal/genética , Humanos , Membranas Intracelulares/metabolismo , Poro Nuclear/metabolismo , Péptidos/genética , Péptidos/metabolismo , Proteínas/genéticaRESUMEN
BACKGROUND: Perceiving oneself as obese has been associated with weight loss attempts. However, such a perception may not sufficiently drive significant weight reduction in many individuals. Hence, relying solely on the traditionally emphasized perceived risk of behavioral changes in obesity is challenging. This study used an extended parallel process model and a risk perception attitude framework to explore the influence of perceived risk and perceived efficacy on individual obesity knowledge and obesity prevention behaviors. METHODS: Data were obtained from 1,100 Korean adults aged 40-69 years through an online survey conducted in October 2022. Multinomial logistic regression and analysis of variance were employed to assess the relationships among perceived risk, perceived efficacy, obesity knowledge, and obesity prevention behaviors. RESULTS: Sex was associated with being underweight, overweight, and obese. Moreover, perceived severity was associated with obesity, whereas perceived susceptibility was associated with overweight and obese. Response efficacy was related to being overweight alone, whereas self-efficacy was associated with being underweight, overweight, and obese. The main effects of sex and perceived risk, and their interaction effect were statistically significant for obesity knowledge. Additionally, the main effects of sex, perceived risk, and perceived efficacy on obesity prevention behaviors were statistically significant. CONCLUSIONS: The extended parallel process model and risk perception attitude framework proved effective in classifying obesity based on body mass index, obesity knowledge, and obesity prevention behaviors.
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Sobrepeso , Delgadez , Adulto , Humanos , Obesidad/prevención & control , Índice de Masa Corporal , Pérdida de Peso , Percepción , República de Corea/epidemiología , Peso CorporalRESUMEN
The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Expansión de las Repeticiones de ADN/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Sistemas de Lectura Abierta/genética , Proteínas/genética , Transporte de ARN/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Proteína C9orf72 , Drosophila melanogaster/genética , Ojo/metabolismo , Femenino , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Células HeLa , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Músculos/citología , Músculos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Poro Nuclear/patología , Fenotipo , Biosíntesis de Proteínas , ARN/genética , ARN/metabolismo , Glándulas Salivales/citología , Glándulas Salivales/metabolismo , Glándulas Salivales/patologíaRESUMEN
Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Distrofia Muscular de Cinturas/genética , Proteínas Mutantes/genética , Mutación/genética , Miositis por Cuerpos de Inclusión/genética , Osteítis Deformante/genética , Priones/química , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Masculino , Ratones , Datos de Secuencia Molecular , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/patología , Osteítis Deformante/metabolismo , Osteítis Deformante/patología , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Priones/metabolismo , Estructura Terciaria de Proteína/genética , ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Adult-onset inherited myopathies with similar pathological features, including hereditary inclusion body myopathy (hIBM) and limb-girdle muscular dystrophy (LGMD), are a genetically heterogeneous group of muscle diseases. It is unclear whether these inherited myopathies initiated by mutations in distinct classes of genes are etiologically related. Here, we exploit a genetic model system to establish a mechanistic link between diseases caused by mutations in two distinct genes, hnRNPA2B1 and DNAJB6. Hrb98DE and mrj are the Drosophila melanogaster homologs of human hnRNPA2B1 and DNAJB6, respectively. We introduced disease-homologous mutations to Hrb98DE, thus capturing mutation-dependent phenotypes in a genetically tractable model system. Ectopic expression of the disease-associated mutant form of hnRNPA2B1 or Hrb98DE in fly muscle resulted in progressive, age-dependent cytoplasmic inclusion pathology, as observed in humans with hnRNPA2B1-related myopathy. Cytoplasmic inclusions consisted of hnRNPA2B1 or Hrb98DE protein in association with the stress granule marker ROX8 and additional endogenous RNA-binding proteins (RBPs), suggesting that these pathological inclusions are related to stress granules. Notably, TDP-43 was also recruited to these cytoplasmic inclusions. Remarkably, overexpression of MRJ rescued this phenotype and suppressed the formation of cytoplasmic inclusions, whereas reduction of endogenous MRJ by a classical loss of function allele enhanced it. Moreover, wild-type, but not disease-associated, mutant forms of MRJ interacted with RBPs after heat shock and prevented their accumulation in aggregates. These results indicate both genetic and physical interactions between disease-linked RBPs and DNAJB6/mrj, suggesting etiologic overlap between the pathogenesis of hIBM and LGMD initiated by mutations in hnRNPA2B1 and DNAJB6.
Asunto(s)
Contractura/congénito , Drosophila melanogaster/genética , Proteínas del Choque Térmico HSP40/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Chaperonas Moleculares/genética , Distrofia Muscular de Cinturas/genética , Miositis por Cuerpos de Inclusión/congénito , Proteínas del Tejido Nervioso/genética , Oftalmoplejía/genética , Adulto , Edad de Inicio , Secuencia de Aminoácidos , Animales , Contractura/genética , Contractura/metabolismo , Contractura/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP40/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Músculos/metabolismo , Músculos/patología , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Mutación , Miositis por Cuerpos de Inclusión/genética , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/patología , Proteínas del Tejido Nervioso/metabolismo , Oftalmoplejía/metabolismo , Oftalmoplejía/patología , Fenotipo , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Neuronal function depends on the retrograde relay of growth and survival signals from the synaptic terminal, where the neuron interacts with its targets, to the nucleus, where gene transcription is regulated. Activation of the Bone Morphogenetic Protein (BMP) pathway at the Drosophila larval neuromuscular junction results in nuclear accumulation of the phosphorylated form of the transcription factor Mad in the motoneuron nucleus. This in turn regulates transcription of genes that control synaptic growth. How BMP signaling at the synaptic terminal is relayed to the cell body and nucleus of the motoneuron to regulate transcription is unknown. We show that the BMP receptors are endocytosed at the synaptic terminal and transported retrogradely along the axon. Furthermore, this transport is dependent on BMP pathway activity, as it decreases in the absence of ligand or receptors. We further demonstrate that receptor traffic is severely impaired when Dynein motors are inhibited, a condition that has previously been shown to block BMP pathway activation. In contrast to these results, we find no evidence for transport of phosphorylated Mad along the axons, and axonal traffic of Mad is not affected in mutants defective in BMP signaling or retrograde transport. These data support a model in which complexes of activated BMP receptors are actively transported along the axon towards the cell body to relay the synaptogenic signal, and that phosphorylated Mad at the synaptic terminal and cell body represent two distinct molecular populations.
Asunto(s)
Transporte Axonal/fisiología , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Neuronas Motoras/metabolismo , Terminales Presinápticos/metabolismo , Animales , Dineínas Axonemales/metabolismo , Axones/metabolismo , Receptores de Proteínas Morfogenéticas Óseas/genética , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/genética , Endosomas/genética , Endosomas/metabolismo , Neuronas Motoras/citología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND/AIMS: Autophagy plays critical roles in both cell survival and cell death. Beclin-1, a key modulator of autophagy function, is considered a haploinsufficient tumor suppressor. The role of Beclin-1 expression in cancer is still controversial. Some studies favor the idea that autophagy suppresses tumor development, whereas other researchers suggest that autophagy enhances tumorigenesis. The expression and function of Beclin-1 in gallbladder cancer (GBCA) remain largely unknown. METHODOLOGY: Methodology: We performed immunohistochemical staining for Beclin-1 in 119 GBCA cases, and investigated whether Beclin-1 expression correlated with clinicopathologic characteristics and prognosis of patients. RESULTS: Beclin-1 was expressed in the cytoplasm of cancer cells with occasional nuclear staining in 53 (44.5%) of the 119 cases of GBCA with no expression in adjacent normal epithelial cells. Increased expression of Beclin-1 was significantly associated with longer survival rate of patients with GBCA in univariate (p=0.006) and multivariate analyses (p=0.005). There is no association between Beclin-1 expression and clinicopathologic characteristics. CONCLUSIONS: Beclin-1 was highly expressed in GBCA, and positive expression in cancer cells was significantly related with favorable prognosis in GBCA patients. Our results suggest that the expression of Beclin-1 may be an independent predictive marker of favorable prognosis in GBCA.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/análisis , Biomarcadores de Tumor/análisis , Neoplasias de la Vesícula Biliar/química , Proteínas de la Membrana/análisis , Adulto , Anciano , Anciano de 80 o más Años , Beclina-1 , Distribución de Chi-Cuadrado , Femenino , Neoplasias de la Vesícula Biliar/mortalidad , Neoplasias de la Vesícula Biliar/patología , Neoplasias de la Vesícula Biliar/terapia , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Modelos de Riesgos Proporcionales , Factores de Riesgo , Regulación hacia ArribaRESUMEN
With a rising interest in smart windows and optical displays, the utilization of metal oxides (MOs) has garnered significant attention owing to their high active sites, flexibility, and tunable electronic and optical properties. Despite these advantages, achieving precise tuning of optical properties in MOs-based quantum dots and their mass production remains a challenge. In this study, we present an easily scalable approach to generate WO3 quantum dots with diverse sizes through sequential insertion/exfoliation processes in solvents with suitable surface tension. Additionally, we utilized the prepared WO3 quantum dots in the fabrication of luminescent transparent wood via an impregnation process. These quantum dots manifested three distinct emitting colors: red, green, and blue. Through characterizations of the structural and optical properties of the WO3 quantum dots, we verified that quantum dots with sizes around 30 nm, 50 nm, and 70 nm showcase a monoclinic crystal structure with oxygen-related defect sites. Notably, as the size of the WO3 quantum dots decreased, the maximum emitting peak underwent a blue shift, with peaks observed at 407 nm (blue), 493 nm (green), and 676 nm (red) under excitation by a He-Cd laser (310 nm), respectively. Transparent woods infused with various WO3 quantum dots exhibited luminescence in blue/white emitting colors. These results suggest substantial potential in diverse applications, such as building materials and optoelectronics.
RESUMEN
One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research.
Asunto(s)
Sistemas CRISPR-Cas , Proteínas de Drosophila , Color del Ojo , Edición Génica , Animales , Edición Génica/métodos , Proteínas de Drosophila/genética , Color del Ojo/genética , Animales Modificados Genéticamente , Factores de Transcripción/genética , Drosophila/genética , Estudiantes , Drosophila melanogaster/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Proteínas del Ojo , Transportadoras de Casetes de Unión a ATPRESUMEN
Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10( CHCHD10 ) have been identified as a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia(ALS-FTD). In our previous studies using in vivo Drosophila model expressing C2C10H S81L , and human cell models expressing CHCHD10 S59L , we have identified that the PINK1/Parkin pathway is activated and causes cellular toxicity. Furthermore, we demonstrated that pseudo-substrate inhibitors for PINK1 and mitofusin2 agonists mitigated the cellular toxicity of CHCHD10 S59L . Evidences using in vitro/ in vivo genetic and chemical tools indicate that inhibiting PINK1 would be the most promising treatment for CHCHD10 S59L -induced diseases. Therefore, we have investigated cellular pathways that can modulate the PINK1/Parkin pathway and reduce CHCHD10 S59L -induced cytotoxicity. Here, we report that FDA-approved PDE4 inhibitors reduced CHCHD10 S59L -induced morphological and functional mitochondrial defects in human cells and an in vivo Drosophila model expressing C2C10H S81L . Multiple PDE4 inhibitors decreased PINK1 accumulation and downstream mitophagy induced by CHCHD10 S59L . These findings suggest that PDE4 inhibitors currently available in the market may be repositioned to treat CHCHD10 S59L -induced ALS-FTD and possibly other related diseases.
RESUMEN
Background: Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) have been identified as a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia(ALS-FTD). In our previous studies using in vivo Drosophila model expressing CHCHD10S59L, and human cell models expressing CHCHD10S59L, we have identified that the PINK1/Parkin pathway is activated and causes cellular toxicity. Furthermore, we demonstrated that pseudo-substrate inhibitors for PINK1 and mitofusin2 agonists mitigated the cellular toxicity of CHCHD10S59L. Evidences using in vitro, in vivo genetic, and chemical tools indicate that inhibiting PINK1 would be the most promising treatment for CHCHD10S59L-induced diseases. Methods: An in vivo human cell culture and in vivo Drosophila models expressing CHCHD10S59L mutant were utilized in this study to evaluate the effect of PDE4 inhibitors in PINK-parkin mediated cytotoxicity through immunohistochemical and seahorse assays. Data were analysed using one-way ANOVA and post-hoc Dunnett's test for statistical significance. Results: We investigated cellular pathways that can modulate the PINK1/Parkin pathway and reduce CHCHD10S59L-induced cytotoxicity. Here, we report that FDA-approved PDE4 inhibitors reduced CHCHD10S59L-induced morphological and functional mitochondrial defects in human cells and an in vivo Drosophila model expressing C2C10HS81L. Multiple PDE4 inhibitors decreased PINK1 accumulation and downstream mitophagy induced by CHCHD10S59L. Conclusion: These findings suggest that PDE4 inhibitors currently available in the market may be repositioned to treat CHCHD10S59L-induced ALS-FTD and possibly other related diseases, and that disease treatment with PDE4 inhibitors should include careful consideration of the PINK1/Parkin pathway, as it is generally recognized as a protective pathway.
RESUMEN
The Drosophila melanogaster compound eye is a well-structured and comprehensive array of around 800 ommatidia, exhibiting a symmetrical and hexagonal pattern. This regularity and ease of observation make the Drosophila eye system a powerful tool to model various human neurodegenerative diseases. However, ways of quantifying abnormal phenotypes, such as manual ranking of eye severity scores, have limitations, especially when ranking weak alterations in eye morphology. To overcome these limitations, computational approaches have been developed such as Flynotyper. The use of a ring light allows for better qualitative images accessing the intactness of individual ommatidia. However, these images cannot be analyzed by Flynotyper directly due to shadows on ommatidia introduced by the ring light. Here, we describe an unbiased way to quantify rough eye phenotypes observed in Drosophila disease models by combining two software, ilastik and Flynotyper. By preprocessing the images with ilastik, successful quantification of the rough eye phenotype can be achieved with Flynotyper.
Asunto(s)
Drosophila melanogaster , Fenotipo , Animales , Programas Informáticos , Ojo/anatomía & histología , Ojo/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
OBJECTIVES: In 2012, Liu et al. reported that miR-34 is an age-related miRNA regulating age-associated events and long-term brain integrity in Drosophila. They demonstrated that modulating miR-34 and its downstream target, Eip74EF, showed beneficial effects on an age-related disease using a Drosophila model of Spinocerebellar ataxia type 3 expressing SCA3trQ78. These results imply that miR-34 could be a general genetic modifier and therapeutic candidate for age-related diseases. Thus, the goal of this study was to examine the effect of miR-34 and Eip47EF on another age-related Drosophila disease model. RESULTS: Using a Drosophila eye model expressing mutant Drosophila VCP (dVCP) that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), or multisystem proteinopathy (MSP), we demonstrated that abnormal eye phenotypes generated by dVCPR152H were rescued by Eip74EF siRNA expression. Contrary to our expectations, miR-34 overexpression alone in the eyes with GMR-GAL4 resulted in complete lethality due to the leaky expression of GMR-GAL4 in other tissues. Interestingly, when miR-34 was co-expressed with dVCPR152H, a few survivors were produced; however, their eye degeneration was greatly exacerbated. Our data indicate that, while confirming that the downregulation of Eip74EF is beneficial to the dVCPR152HDrosophila eye model, the high expression level of miR-34 is actually toxic to the developing flies and the role of miR-34 in dVCPR152H-mediated pathogenesis is inconclusive in the GMR-GAL4 eye model. Identifying the transcriptional targets of Eip74EF might provide valuable insights into diseases caused by mutations in VCP such as ALS, FTD, and MSP.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteínas de Drosophila , Demencia Frontotemporal , MicroARNs , Animales , Esclerosis Amiotrófica Lateral/genética , Drosophila/genética , Proteínas de Drosophila/genética , Demencia Frontotemporal/genética , MicroARNs/genética , Fenotipo , Factores de TranscripciónRESUMEN
Transition metal dichalcogenide-based quantum dots are promising materials for applications in diverse fields, such as sensors, electronics, catalysis, and biomedicine, because of their outstanding physicochemical properties. In this study, we propose bio-imaging characteristics through utilizing water-soluble MoS2 quantum dots (MoS2-QDs) with two different sizes (i.e., ~5 and ~10 nm). The structural and optical properties of the fabricated metallic phase MoS2-QDs (m-MoS2-QDs) were characterized by transmission electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, UV-vis absorption spectroscopy, and photoluminescence. The synthesized m-MoS2-QDs showed clear photophysical characteristic peaks derived from the quantum confinement effect and defect sites, such as oxygen functional groups. When the diameter of the synthesized m-MoS2-QD was decreased, the emission peak was blue-shifted from 436 to 486 nm under excitation by a He-Cd laser (325 nm). Density functional theory calculations confirmed that the size decrease of m-MoS2-QDs led to an increase in the bandgap because of quantum confinement effects. In addition, when incorporated into the bio-imaging of HeLa cells, m-MoS2-QDs were quite biocompatible with bright luminescence and exhibited low toxicity. Our results are commercially applicable for achieving high-performance bio-imaging probes.
RESUMEN
Bone Morphogenetic Protein (BMP) signaling mediated by the receptor Wishful thinking (Wit) is essential for nervous system development in Drosophila. Mutants lacking wit function show defects in neuromuscular junction development and function, specification of neurosecretory phenotypes, and eclosion behavior that result in lethality. The ligand is Glass bottom boat, the Drosophila ortholog of mammalian BMP-7, which acts as a retrograde signal through the Wit receptor. In order to identify transcriptional targets of the BMP pathway in the Drosophila nervous system, we have analyzed the gene expression profile of wit mutant larval central nervous system. Genes differentially expressed identified by microarray analysis have been verified by quantitative PCR and studied by in situ hybridization. Among the genes thus identified, we find solute transporters, neuropeptides, mitochondrial proteins, and novel genes. In addition, several genes are regulated by wit in an isoform-specific manner that suggest regulation of alternative splicing by BMP signaling.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Sistema Nervioso Central , Drosophila melanogaster , Transducción de Señal/fisiología , Animales , Proteínas Morfogenéticas Óseas/genética , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/fisiología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Análisis por Micromatrices , Datos de Secuencia Molecular , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reproducibilidad de los Resultados , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. Here, we generate CHCH10S59L-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10S59L mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10S59L independently affects the TDP-43 and PINK1 pathways. CHCHD10S59L expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10S59L-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. Our findings suggest that CHCHD10S59L-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Demencia Frontotemporal/genética , Proteínas Mitocondriales/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Demencia Frontotemporal/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopía Confocal , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/genética , Homología de Secuencia de AminoácidoRESUMEN
Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by toxic gain of function of the androgen receptor (AR). Previously, we found that co-regulator binding through the activation function-2 (AF2) domain of AR is essential for pathogenesis, suggesting that AF2 may be a potential drug target for selective modulation of toxic AR activity. We screened previously identified AF2 modulators for their ability to rescue toxicity in a Drosophila model of SBMA. We identified two compounds, tolfenamic acid (TA) and 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole (MEPB), as top candidates for rescuing lethality, locomotor function and neuromuscular junction defects in SBMA flies. Pharmacokinetic analyses in mice revealed a more favorable bioavailability and tissue retention of MEPB compared with TA in muscle, brain and spinal cord. In a preclinical trial in a new mouse model of SBMA, MEPB treatment yielded a dose-dependent rescue from loss of body weight, rotarod activity and grip strength. In addition, MEPB ameliorated neuronal loss, neurogenic atrophy and testicular atrophy, validating AF2 modulation as a potent androgen-sparing strategy for SBMA therapy.
Asunto(s)
Atrofia Muscular Espinal/patología , Degeneración Nerviosa/patología , Receptores Androgénicos/química , Receptores Androgénicos/metabolismo , Animales , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Proteínas Co-Represoras/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster , Células HEK293 , Humanos , Masculino , Ratones Transgénicos , Atrofia Muscular Espinal/tratamiento farmacológico , Degeneración Nerviosa/tratamiento farmacológico , Fenotipo , Proyectos Piloto , Dominios Proteicos , Expansión de Repetición de Trinucleótido/genética , ortoaminobenzoatos/farmacología , ortoaminobenzoatos/uso terapéuticoRESUMEN
Nutrigenomics is the study of gene-nutrient interactions and how they affect the health and metabolism of an organism. Combining nutrigenomics with longevity studies is a natural extension and promises to help identify mechanisms whereby nutrients affect the aging process, life span, and, with the incorporation of age-dependent functional measures, health span. The topics we discuss in this chapter are genetic techniques, dietary manipulations, metabolic studies, and microarray analysis methods to investigate how nutrition affects gene expression, life span, triglyceride levels, total protein levels, and live weight in Drosophila. To better illustrate nutrigenomic techniques, we analyzed Drosophila larvae or adults fed control diets (high sucrose) and compared these with larvae or adults fed diets high in the saturated fat palmitic acid, soy, or 95% lean ground beef. The main results of these studies are, surprisingly, that triglyceride and total protein levels are significantly decreased by the beef diet in all adults, and total protein levels are significantly increased in male flies fed the soy diet. Furthermore, and less surprisingly, we found that all three experimental diets significantly decreased longevity and increased the length of time to develop from egg to adult. We also describe preliminary microarray results with adult flies fed the different diets, which suggest that only about 2-3% of the approx 18,000 genes have significantly altered mRNA expression levels compared with flies fed a control sucrose diet. The significance of these results and other types of nutrigenomics and longevity analyses is discussed.