RESUMEN
The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development.
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Encéfalo/embriología , Cromosomas Humanos Par 3/genética , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Discapacidad Intelectual/genética , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Proteínas de Xenopus/genética , Animales , Apoptosis/genética , Encéfalo/patología , Ciclo Celular/genética , Deleción Cromosómica , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Humanos , Discapacidad Intelectual/patología , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
The Drosophila ovary serves as a model for pioneering studies of stem cell niches, with defined cell types and signaling pathways supporting both germline and somatic stem cells. The establishment of the niche units begins during larval stages with the formation of terminal filament-cap structures; however, the genetics underlying their development remains largely unknown. Here, we show that the transcription factor Lmx1a is required for ovary morphogenesis. We found that Lmx1a is expressed in early ovarian somatic lineages and becomes progressively restricted to terminal filaments and cap cells. We show that Lmx1a is required for the formation of terminal filaments, during the larval-pupal transition. Finally, our data demonstrate that Lmx1a functions genetically downstream of Bric-à-Brac, and is crucial for the expression of key components of several conserved pathways essential to ovarian stem cell niche development. Importantly, expression of chicken Lmx1b is sufficient to rescue the null Lmx1a phenotype, indicating functional conservation across the animal kingdom. These results significantly expand our understanding of the mechanisms controlling stem cell niche development in the fly ovary.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Proteínas con Homeodominio LIM/metabolismo , Ovario/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Linaje de la Célula , Pollos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Genes de Insecto , Proteínas con Homeodominio LIM/genética , Mutación , Ovario/citología , Ovario/metabolismo , Transducción de Señal , Nicho de Células Madre/genética , Nicho de Células Madre/fisiología , Factores de Transcripción/genéticaRESUMEN
Alzheimer's disease (AD) is the most prevalent of a large group of related proteinopathies for which there is currently no cure. Here, we used Drosophila to explore a strategy to block Aß42 neurotoxicity through engineering of the Heat shock protein 70 (Hsp70), a chaperone that has demonstrated neuroprotective activity against several intracellular amyloids. To target its protective activity against extracellular Aß42, we added a signal peptide to Hsp70. This secreted form of Hsp70 (secHsp70) suppresses Aß42 neurotoxicity in adult eyes, reduces cell death, protects the structural integrity of adult neurons, alleviates locomotor dysfunction, and extends lifespan. SecHsp70 binding to Aß42 through its holdase domain is neuroprotective, but its ATPase activity is not required in the extracellular space. Thus, the holdase activity of secHsp70 masks Aß42 neurotoxicity by promoting the accumulation of nontoxic aggregates. Combined with other approaches, this strategy may contribute to reduce the burden of AD and other extracellular proteinopathies.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Drosophila melanogaster/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/genética , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Ojo/metabolismo , Femenino , Ingeniería Genética/métodos , Células HEK293 , Proteínas HSP70 de Choque Térmico/genética , Humanos , Longevidad/genética , Masculino , Trastornos Motores/genética , Trastornos Motores/metabolismo , Trastornos Motores/prevención & control , Neuronas/metabolismo , Neuroprotección/genética , Fragmentos de Péptidos/genética , Unión ProteicaRESUMEN
Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. We show here that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, we show for the first time that co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, we demonstrate the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides/inmunología , Modelos Animales de Enfermedad , Drosophila , Fragmentos de Péptidos/inmunología , Anticuerpos de Cadena Única/inmunología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/prevención & control , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/toxicidad , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Encéfalo/patología , Ojo Compuesto de los Artrópodos/inmunología , Evaluación Preclínica de Medicamentos/métodos , Sinergismo Farmacológico , Femenino , Masculino , Actividad Motora , Neuronas/metabolismo , Proteínas RecombinantesRESUMEN
Prion diseases encompass a diverse group of neurodegenerative conditions characterized by the accumulation of misfolded prion protein (PrP) isoforms. Other conformational variants of PrP have also been proposed to contribute to neurotoxicity in prion diseases, including misfolded intermediates as well as cytosolic and transmembrane isoforms. To better understand PrP neurotoxicity, we analyzed the role of two highly conserved methionines in helix 3 on PrP biogenesis, folding and pathogenesis. Expression of the PrP-M205S and -M205,212S mutants in Drosophila led to hyperglycosylation, intracellular accumulation and widespread conformational changes due to failure of oxidative folding. Surprisingly, PrP-M205S and -M205,212S acquired a transmembrane topology (Ctm) previously linked to mutations in the signal peptide (SP) and the transmembrane domain (TMD). PrP-M205,212S also disrupted the accumulation of key neurodevelopmental proteins in lipid rafts, resulting in shortened axonal projections. These results uncover a new role for the hydrophobic domain in promoting oxidative folding and preventing the formation of neurotoxic Ctm PrP, mechanisms that may be relevant in the pathogenesis of both inherited and sporadic prion diseases.
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Vesículas Citoplasmáticas/metabolismo , Enfermedades por Prión/fisiopatología , Priones/química , Transporte de Proteínas , Animales , Animales Modificados Genéticamente , Drosophila/genética , Drosophila/metabolismo , Femenino , Genotipo , Humanos , Masculino , Microdominios de Membrana/fisiología , Ratones , Mutación , Oxidación-Reducción , Enfermedades por Prión/patología , Priones/metabolismo , Pliegue de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Spinocerebellar ataxia 13 (SCA13) is an autosomal dominant disease resulting from mutations in KCNC3 (Kv3.3), a voltage-gated potassium channel. The KCNC3(R420H) mutation was first identified as causative for SCA13 in a four-generation Filipino kindred with over 20 affected individuals. Electrophysiological analyses in oocytes previously showed that this mutation did not lead to a functional channel and displayed a dominant negative phenotype. In an effort to identify the molecular basis of this allelic form of SCA13, we first determined that human KCNC3(WT) and KCNC3(R420H) display disparate post-translational modifications, and the mutant protein has reduced complex glycan adducts. Immunohistochemical analyses demonstrated that KCNC3(R420H) was not properly trafficking to the plasma membrane and surface biotinylation demonstrated that KCNC3(R420H) exhibited only 24% as much surface expression as KCNC3(WT). KCNC3(R420H) trafficked through the ER but was retained in the Golgi. KCNC3(R420H) expression results in altered Golgi and cellular morphology. Electron microscopy of KCNC3(R420H) localization further supports retention in the Golgi. These results are specific to the KCNC3(R420H) allele and provide new insight into the molecular basis of disease manifestation in SCA13.
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Arginina/genética , Histidina/genética , Líquido Intracelular/metabolismo , Mutación/genética , Canales de Potasio Shaw/genética , Degeneraciones Espinocerebelosas/genética , Animales , Animales Modificados Genéticamente , Biotinilación , Células COS , Cadherinas/metabolismo , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/metabolismo , Drosophila , Proteínas de Drosophila/genética , Retículo Endoplásmico/metabolismo , Femenino , Humanos , Masculino , Oocitos , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Ataxias Espinocerebelosas/congénito , Degeneraciones Espinocerebelosas/metabolismo , TransfecciónRESUMEN
Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show nonneural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood. We developed multiple models to investigate the impact of CNS stressors on motor function and found that Escherichia coli infections and SARS-CoV-2 protein expression caused reactive oxygen species (ROS) to accumulate in the brain. ROS induced expression of the cytokine Unpaired 3 (Upd3) in Drosophila and its ortholog, IL-6, in mice. CNS-derived Upd3/IL-6 activated the JAK-STAT pathway in skeletal muscle, which caused muscle mitochondrial dysfunction and impaired motor function. We observed similar phenotypes after expressing toxic amyloid-ß (Aß42) in the CNS. Infection and chronic disease therefore activate a systemic brain-muscle signaling axis in which CNS-derived cytokines bypass the connectome and directly regulate muscle physiology, highlighting IL-6 as a therapeutic target to treat disease-associated muscle dysfunction.
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Encéfalo , COVID-19 , Músculo Esquelético , Transducción de Señal , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Transducción de Señal/inmunología , Ratones , Músculo Esquelético/inmunología , Músculo Esquelético/metabolismo , COVID-19/inmunología , Enfermedad Crónica , Interleucina-6/metabolismo , Interleucina-6/inmunología , Infecciones por Escherichia coli/inmunología , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/inmunología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/inmunología , Proteínas de Drosophila/genética , SARS-CoV-2/inmunología , Drosophila melanogaster/inmunología , Péptidos beta-Amiloides/metabolismo , Humanos , Ratones Endogámicos C57BLRESUMEN
Alzheimer's disease (AD) is an incurable neurodegenerative disorder clinically characterized by progressive cognitive impairment. A prominent pathologic hallmark in the AD brain is the abnormal accumulation of the amyloid-ß 1-42 peptide (Aß), but the exact pathways mediating Aß neurotoxicity remain enigmatic. Endoplasmic reticulum (ER) stress is induced during AD, and has been indirectly implicated as a mediator of Aß neurotoxicity. We report here that Aß activates the ER stress response factor X-box binding protein 1 (XBP1) in transgenic flies and in mammalian cultured neurons, yielding its active form, the transcription factor XBP1s. XBP1s shows neuroprotective activity in two different AD models, flies expressing Aß and mammalian cultured neurons treated with Aß oligomers. Trying to identify the mechanisms mediating XBP1s neuroprotection, we found that in PC12 cells treated with Aß oligomers, XBP1s prevents the accumulation of free calcium (Ca(2+)) in the cytosol. This protective activity can be mediated by the downregulation of a specific isoform of the ryanodine Ca(2+) channel, RyR3. In support of this observation, a mutation in the only ryanodine receptor (RyR) in flies also suppresses Aß neurotoxicity, indicating the conserved mechanisms between the two AD models. These results underscore the functional relevance of XBP1s in Aß toxicity, and uncover the potential of XBP1 and RyR as targets for AD therapeutics.
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Péptidos beta-Amiloides/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila/genética , Retículo Endoplásmico/metabolismo , Fragmentos de Péptidos/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Animales Modificados Genéticamente/genética , Calcio/metabolismo , Ojo/patología , Femenino , Masculino , Neuronas/metabolismo , Neuronas/patología , Células PC12 , Fragmentos de Péptidos/metabolismo , Pliegue de Proteína , Empalme del ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , TransfecciónRESUMEN
Several vectors for gene expression are available in Drosophila, a hub for genetics and genomics innovation. However, the vectors for ubiquitous expression have a complex structure, including coding exons, that makes in-frame cloning of cDNAs very complicated. In this report we describe a new Drosophila expression vector (p∆TubHA4C) for ubiquitous expression of coding sequences under the control of a minimal 0.9 kb promoter of α1 tubulin (α1t). This plasmid was designed to include optimized multiple cloning sites (polylinker) to provide flexibility in cloning strategies. We also added the option of double labeling the expressed proteins with two C-terminal tags, the viral epitope hemagglutinin and a synthetic tetracysteine (4C) tag that binds small fluorescent compounds. This dual tag allows both in situ and biochemical detection of the desired protein. In particular, the new 4C tag technology combines easy fluorescent labeling with small arsenical compounds in live or fixed cells and tissues, while producing minimal alterations to the tagged protein due to its small size. To demonstrate the potent and ubiquitous expression under the control of the ∆Tub promoter, bacterial lacZ was expressed and monitored in cell culture and transgenic flies. We found that the modified 0.9 kb ΔTub promoter induced similar expression levels to the intact 2.6 kb α1t promoter, supporting the inclusion of all critical regulatory elements in the new and flexible ∆TubHA4C vector.
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Drosophila/genética , Expresión Génica/genética , Vectores Genéticos/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Cartilla de ADN/genética , Drosophila/metabolismo , Fluorescencia , Técnica del Anticuerpo Fluorescente , Galactósidos , Vectores Genéticos/metabolismo , Hemaglutininas/genética , Indoles , Datos de Secuencia Molecular , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Tubulina (Proteína)/genéticaRESUMEN
Alzheimer's disease (AD) is the most prevalent neurodegenerative dementia in older adults worldwide. Sadly, there are no disease-modifying therapies available for treatment due to the multifactorial complexity of the disease. AD is pathologically characterized by extracellular deposition of amyloid beta (Aß) and intracellular neurofibrillary tangles composed of hyperphosphorylated tau. Increasing evidence suggest that Aß also accumulates intracellularly, which may contribute to the pathological mitochondrial dysfunction observed in AD. According with the mitochondrial cascade hypothesis, mitochondrial dysfunction precedes clinical decline and thus targeting mitochondria may result in new therapeutic strategies. Unfortunately, the precise mechanisms connecting mitochondrial dysfunction with AD are largely unknown. In this review, we will discuss how the fruit fly Drosophila melanogaster is contributing to answer mechanistic questions in the field, from mitochondrial oxidative stress and calcium dysregulation to mitophagy and mitochondrial fusion and fission. In particular, we will highlight specific mitochondrial insults caused by Aß and tau in transgenic flies and will also discuss a variety of genetic tools and sensors available to study mitochondrial biology in this flexible organism. Areas of opportunity and future directions will be also considered.
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OBJECTIVE: To identify potential diagnostic and prognostic biomarkers for clinical management and clinical trials in amyotrophic lateral sclerosis. METHODS: We analysed proteomics data of ALS patient-induced pluripotent stem cell-derived motor neurons available through the AnswerALS consortium. After stratifying patients using clinical ALSFRS-R and ALS-CBS scales, we identified differentially expressed proteins indicative of ALS disease severity and progression rate as candidate ALS-related and prognostic biomarkers. Pathway analysis for identified proteins was performed using STITCH. Protein sets were correlated with the effects of drugs using the Connectivity Map tool to identify compounds likely to affect similar pathways. RNAi screening was performed in a Drosophila TDP-43 ALS model to validate pathological relevance. A statistical classification machine learning model was constructed using ridge regression that uses proteomics data to differentiate ALS patients from controls. RESULTS: We identified 76, 21, 71 and 1 candidate ALS-related biomarkers and 22, 41, 27 and 64 candidate prognostic biomarkers from patients stratified by ALSFRS-R baseline, ALSFRS-R progression slope, ALS-CBS baseline and ALS-CBS progression slope, respectively. Nineteen proteins enhanced or suppressed pathogenic eye phenotypes in the ALS fly model. Nutraceuticals, dopamine pathway modulators, statins, anti-inflammatories and antimicrobials were predicted starting points for drug repurposing using the connectivity map tool. Ten diagnostic biomarker proteins were predicted by machine learning to identify ALS patients with high accuracy and sensitivity. INTERPRETATION: This study showcases the powerful approach of iPSC-motor neuron proteomics combined with machine learning and biological confirmation in the prediction of novel mechanisms and diagnostic and predictive biomarkers in ALS.
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Esclerosis Amiotrófica Lateral , Progresión de la Enfermedad , Proteómica , Humanos , Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Biomarcadores , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Fenotipo , Aprendizaje Automático , Modelos Animales de Enfermedad , Drosophila/genética , Drosophila/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteómica/métodosRESUMEN
BACKGROUND: The accumulation of amyloid beta (Aß) peptides in fibrils is prerequisite for Alzheimer's disease (AD). Our understanding of the proteins that promote Aß fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. METHODS: To investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of Aß immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic Aß42 flies, and Aß42 seeded cultured neurons. RESULTS: Amyloid fibrils are primarily composed by Aß42 and unexpectedly harbor Aß38 but generally lack Aß40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 20 new amyloid-associated proteins. Notably, we confirmed 57 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic Aß42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Here, we could functionally validate 20 genes as modifiers of Aß42 toxicity in vivo. CONCLUSIONS: These discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.
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Enfermedad de Alzheimer , Amiloide , Humanos , Animales , Ratones , Péptidos beta-Amiloides , Proteoma , Proteómica , Proteínas Amiloidogénicas , EncéfaloRESUMEN
The prion protein (PrP) is best known for its association with prion diseases. However, a controversial new role for PrP in Alzheimer disease (AD) has recently emerged. In vitro studies and mouse models of AD suggest that PrP may be involved in AD pathogenesis through a highly specific interaction with amyloid-ß (Aß42) oligomers. Immobilized recombinant human PrP (huPrP) also exhibited high affinity and specificity for Aß42 oligomers. Here we report the novel finding that aggregated forms of huPrP and Aß42 are co-purified from AD brain extracts. Moreover, an anti-PrP antibody and an agent that specifically binds to insoluble PrP (iPrP) co-precipitate insoluble Aß from human AD brain. Finally, using peptide membrane arrays of 99 13-mer peptides that span the entire sequence of mature huPrP, two distinct types of Aß binding sites on huPrP are identified in vitro. One specifically binds to Aß42 and the other binds to both Aß42 and Aß40. Notably, Aß42-specific binding sites are localized predominantly in the octapeptide repeat region, whereas sites that bind both Aß40 and Aß42 are mainly in the extreme N-terminal or C-terminal domains of PrP. Our study suggests that iPrP is the major PrP species that interacts with insoluble Aß42 in vivo. Although this work indicated the interaction of Aß42 with huPrP in the AD brain, the pathophysiological relevance of the iPrP/Aß42 interaction remains to be established.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Priones/metabolismo , Anciano , Anciano de 80 o más Años , Sitios de Unión , Encéfalo/metabolismo , Estudios de Casos y Controles , Humanos , Persona de Mediana Edad , Unión Proteica , SolubilidadRESUMEN
Several neurodegenerative disorders are characterized by protein misfolding, a phenomenon that results in perturbation of cellular homeostasis. We recently identified the protective activity of the ER stress response factor XBP1 (X-box binding protein 1) against Amyloid-ß1-42 (Aß42) neurotoxicity in cellular and Drosophila models of Alzheimer's disease. Additionally, subtoxic concentrations of Aß42 soluble aggregates (oligomers) induced accumulation of spliced (active) XBP1 transcripts, supporting the involvement of the ER stress response in Aß42 neurotoxicity. Here, we tested the ability of three additional disease-related amyloidogenic proteins to induce ER stress by analyzing XBP1 activation at the RNA level. Treatment of human SY5Y neuroblastoma cells with homogeneous preparations of α-Synuclein (α-Syn), Prion protein (PrP106-126), and British dementia amyloid peptide (ABri1-34) confirmed the high toxicity of oligomers compared to monomers and fibers. Additionally, α-Syn oligomers, but not monomers or fibers, demonstrated potent induction of XBP1 splicing. On the other hand, PrP106-126 and ABri1-34 did not activate XBP1. These results illustrate the biological complexity of these structurally related assemblies and argue that oligomer toxicity depends on the activation of amyloid-specific cellular responses.
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Proteínas de Unión al ADN/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Fragmentos de Péptidos/farmacología , Priones/farmacología , Factores de Transcripción/metabolismo , alfa-Sinucleína/farmacología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Bases , Proteínas de Unión al ADN/efectos de los fármacos , Humanos , Multimerización de Proteína , Deficiencias en la Proteostasis/fisiopatología , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/efectos de los fármacos , Células Tumorales Cultivadas , Proteína 1 de Unión a la X-BoxRESUMEN
Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP(C)) converts into a misfolded isoform (PrP(Sc)) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP(C); in older flies, PrP misfolds, acquires biochemical and structural properties of PrP(Sc), and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP(Sc)-specific conformational epitopes. In contrast to PrP(Sc) from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP(Sc). Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP(Sc)-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP(Sc) is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.
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Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas PrPSc/química , Animales , Animales Modificados Genéticamente , Cricetinae , Drosophila/genética , Drosophila/metabolismo , Humanos , Proteínas PrPSc/metabolismo , Priones , Pliegue de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show a number of non-neural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood, so we developed three models to investigate the impact of neuroinflammation on muscle performance. We found that bacterial infection, COVID-like viral infection, and expression of a neurotoxic protein associated with Alzheimer' s disease promoted the accumulation of reactive oxygen species (ROS) in the brain. Excessive ROS induces the expression of the cytokine Unpaired 3 (Upd3) in insects, or its orthologue IL-6 in mammals, and CNS-derived Upd3/IL-6 activates the JAK/Stat pathway in skeletal muscle. In response to JAK/Stat signaling, mitochondrial function is impaired and muscle performance is reduced. Our work uncovers a brain-muscle signaling axis in which infections and chronic diseases induce cytokine-dependent changes in muscle performance, suggesting IL-6 could be a therapeutic target to treat muscle weakness caused by neuroinflammation.
RESUMEN
BACKGROUND: Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-ß1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. METHODS: We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. RESULTS: We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. CONCLUSION: Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Animales , Ratones , Transporte Activo de Núcleo Celular , Autopsia , Proteínas de Unión al ADN , Proteínas de Complejo Poro Nuclear , Humanos , DrosophilaRESUMEN
Prion diseases are neurodegenerative disorders caused by misfolding of the normal prion protein (PrP) into a pathogenic "scrapie" conformation. To better understand the cellular and molecular mechanisms that govern the conformational changes (conversion) of PrP, we compared the dynamics of PrP from mammals susceptible (hamster and mouse) and resistant (rabbit) to prion diseases in transgenic flies. We recently showed that hamster PrP induces spongiform degeneration and accumulates into highly aggregated, scrapie-like conformers in transgenic flies. We show now that rabbit PrP does not induce spongiform degeneration and does not convert into scrapie-like conformers. Surprisingly, mouse PrP induces weak neurodegeneration and accumulates small amounts of scrapie-like conformers. Thus, the expression of three highly conserved mammalian prion proteins in transgenic flies uncovered prominent differences in their conformational dynamics. How these properties are encoded in the amino acid sequence remains to be elucidated.
Asunto(s)
Drosophila melanogaster/genética , Síndromes de Neurotoxicidad/etiología , Enfermedades por Prión/patología , Priones/química , Priones/genética , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Western Blotting , Cromatografía en Gel , Secuencia Conservada , Cricetinae , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Locomoción/fisiología , Masculino , Ratones , Datos de Secuencia Molecular , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/patología , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Priones/metabolismo , Conformación Proteica , ARN Mensajero/genética , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Nuclear depletion, abnormal modification, and cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43) are linked to a group of fatal neurodegenerative diseases called TDP-43 proteinopathies, which include amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Although our understanding of the physiological function of TDP-43 is rapidly advancing, the molecular mechanisms associated with its pathogenesis remain poorly understood. Accumulating evidence suggests that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are important players in TDP-43 pathology. However, while neurons derived from autopsied ALS and FTLD patients revealed TDP-43 deposits in the ER and displayed UPR activation, data originated from in vitro and in vivo TDP-43 models produced contradictory results. In this review, we will explore the complex interplay between TDP-43 pathology, ER stress, and the UPR by breaking down the evidence available in the literature and addressing the reasons behind these discrepancies. We also highlight underexplored areas and key unanswered questions in the field. A better synchronization and integration of methodologies, models, and mechanistic pathways will be crucial to discover the true nature of the TDP-43 and ER stress relationship and, ultimately, to uncover the full therapeutic potential of the UPR.
RESUMEN
Transactive response DNA binding protein 43 (TDP-43) is a DNA/RNA binding protein involved in transcriptional regulation and RNA processing. It is linked to sporadic and familial amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 is predominantly nuclear, but it translocates to the cytoplasm under pathological conditions. Cytoplasmic accumulation, phosphorylation, ubiquitination and truncation of TDP-43 are the main hallmarks of TDP-43 proteinopathies. Among these processes, the pathways leading to TDP-43 fragmentation remain poorly understood. We review here the molecular and biochemical properties of several TDP-43 fragments, the mechanisms and factors mediating their production, and their potential role in disease progression. We also address the presence of TDP-43 C-terminal fragments in several neurological disorders, including Alzheimer's disease, and highlight their respective implications. Finally, we discuss features of animal models expressing TDP-43 fragments as well as recent therapeutic strategies to approach TDP-43 truncation.