RESUMEN
Parkinson's disease (PD) is a multifactorial, chronic, and progressive neurodegenerative disorder inducing movement alterations as a result of the loss of dopaminergic (DAergic) neurons of the pars compacta in the substantia nigra and protein aggregates of alpha synuclein (α-Syn). Although its etiopathology agent has not yet been clearly established, environmental and genetic factors have been suggested as the major contributors to the disease. Mutations in the glucosidase beta acid 1 (GBA1) gene, which encodes the lysosomal glucosylceramidase (GCase) enzyme, are one of the major genetic risks for PD. We found that the GBA1 K198E fibroblasts but not WT fibroblasts showed reduced catalytic activity of heterozygous mutant GCase by -70% but its expression levels increased by 3.68-fold; increased the acidification of autophagy vacuoles (e.g., autophagosomes, lysosomes, and autolysosomes) by +1600%; augmented the expression of autophagosome protein Beclin-1 (+133%) and LC3-II (+750%), and lysosomal-autophagosome fusion protein LAMP-2 (+107%); increased the accumulation of lysosomes (+400%); decreased the mitochondrial membrane potential (∆Ψm) by -19% but the expression of Parkin protein remained unperturbed; increased the oxidized DJ-1Cys106-SOH by +900%, as evidence of oxidative stress; increased phosphorylated LRRK2 at Ser935 (+1050%) along with phosphorylated α-synuclein (α-Syn) at pathological residue Ser129 (+1200%); increased the executer apoptotic protein caspase 3 (cleaved caspase 3) by +733%. Although exposure of WT fibroblasts to environmental neutoxin rotenone (ROT, 1 µM) exacerbated the autophagy-lysosomal system, oxidative stress, and apoptosis markers, ROT moderately increased those markers in GBA1 K198E fibroblasts. We concluded that the K198E mutation endogenously primes skin fibroblasts toward autophagy dysfunction, OS, and apoptosis. Our findings suggest that the GBA1 K198E fibroblasts are biochemically and molecularly equivalent to the response of WT GBA1 fibroblasts exposed to ROT.
Asunto(s)
Apoptosis , Autofagia , Fibroblastos , Glucosilceramidasa , Mitocondrias , Estrés Oxidativo , Glucosilceramidasa/metabolismo , Glucosilceramidasa/genética , Humanos , Fibroblastos/metabolismo , Autofagia/genética , Mitocondrias/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Piel/metabolismo , Piel/patología , Lisosomas/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , MutaciónRESUMEN
Lysosomes are involved in a myriad of cellular functions, such as degradation of macromolecules, endocytosis and exocytosis, modulation of several signaling pathways, and regulation of cell metabolism. To fulfill these diverse functions, lysosomes can undergo several dynamic changes in their content, size, pH, and location within cells. Here, we studied some of these parameters during embryonic chick skeletal muscle cells. We used an anti-lysosome-associated membrane protein 2 (LAMP2) antibody to specifically determine the intracellular localization of lysosomes in these cells. Our data shows that lysosomes are highly enriched in the perinuclear region of chick embryonic muscle cells. We also showed that the wingless signaling pathway (Wnt)/ß-catenin signaling pathway can modulate the location of LAMP2 in chick myogenic cells. Our results highlight the role of lysosomes during muscle differentiation and particularly the presence of a subcellular population of lysosomes that are concentrated in the perinuclear region of muscle cells.
Asunto(s)
Lisosomas , Desarrollo de Músculos , Animales , Lisosomas/metabolismo , Desarrollo de Músculos/fisiología , Embrión de Pollo , Diferenciación Celular/fisiología , Vía de Señalización Wnt/fisiología , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Núcleo Celular/metabolismo , Pollos , beta Catenina/metabolismo , Células Musculares/metabolismo , Células Musculares/citología , Células CultivadasRESUMEN
Safety and effectiveness are the cornerstone objectives of nanomedicine in developing nanotherapies. It is crucial to understand the biological interactions between nanoparticles and immune cells. This study focuses on the manufacture by the microfluidic technique of N-trimethyl chitosan/protein nanocarriers and their interaction with J774 cells to elucidate the cellular processes involved in absorption and their impact on the immune system, mainly through endocytosis, activation of lysosomes and intracellular degradation. TEM of the manufactured nanoparticles showed spherical morphology with an average diameter ranging from 36 ± 16 nm to 179 ± 92 nm, depending on the concentration of the cargo protein (0, 12, 55 µg/mL). FTIR showed the crosslinking between N-trimethyl chitosan and the sodium tripolyphosphate and the α-helix binding loss of BSA. TGA revealed an increase in the thermal stability of N-trimethyl chitosan/protein nanoparticles compared with the powder. The encapsulation of the cargo protein used was demonstrated using XPS. Their potential to improve cell permeability and use as nanocarriers in future vaccine formulations was demonstrated. The toxicity of the nanoparticles in HaCaT and J774 cells was studied, as well as the importance of evaluating the differentiation status of J774 cells. Thus, possible endocytosis pathways and their impact on the immune response were discussed. This allowed us to conclude that N-trimethyl chitosan nanoparticles show potential as carriers for the immune system. Still, more studies are required to understand their effectiveness and possible use in therapies.
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Quitosano , Endocitosis , Lisosomas , Nanopartículas , Quitosano/química , Lisosomas/metabolismo , Endocitosis/efectos de los fármacos , Nanopartículas/química , Animales , Ratones , Línea Celular , Humanos , Portadores de Fármacos/química , Tamaño de la Partícula , Albúmina Sérica Bovina/química , Supervivencia Celular/efectos de los fármacosRESUMEN
Cholesterol is crucial for the proper functioning of eukaryotic cells, especially neurons, which rely on cholesterol to maintain their complex structure and facilitate synaptic transmission. However, brain cells are isolated from peripheral cholesterol by the blood-brain barrier and mature neurons primarily uptake the cholesterol synthesized by astrocytes for proper function. This study aimed to investigate the effect of aging on cholesterol trafficking in astrocytes and its delivery to neurons. We found that aged astrocytes accumulated high levels of cholesterol in the lysosomal compartment, and this cholesterol buildup can be attributed to the simultaneous occurrence of two events: decreased levels of the ABCA1 transporter, which impairs ApoE-cholesterol export from astrocytes, and reduced expression of NPC1, which hinders cholesterol release from lysosomes. We show that these two events are accompanied by increased microR-33 in aged astrocytes, which targets ABCA1 and NPC1. In addition, we demonstrate that the microR-33 increase is triggered by oxidative stress, one of the hallmarks of aging. By coculture experiments, we show that cholesterol accumulation in astrocytes impairs the cholesterol delivery from astrocytes to neurons. Remarkably, we found that this altered transport of cholesterol could be alleviated through treatment with endocannabinoids as well as cannabidiol or CBD. Finally, according to data demonstrating that aged astrocytes develop an A1 phenotype, we found that cholesterol buildup is also observed in reactive C3+ astrocytes. Given that reduced neuronal cholesterol affects synaptic plasticity, the ability of cannabinoids to restore cholesterol transport from aged astrocytes to neurons holds significant implications in aging and inflammation.
Asunto(s)
Transportador 1 de Casete de Unión a ATP , Astrocitos , Cannabinoides , Colesterol , Lisosomas , Neuronas , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Animales , Colesterol/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Transportador 1 de Casete de Unión a ATP/metabolismo , Cannabinoides/farmacología , Cannabinoides/metabolismo , Células Cultivadas , Proteína Niemann-Pick C1 , Ratones , Envejecimiento/metabolismo , Técnicas de Cocultivo , Ratones Endogámicos C57BLRESUMEN
The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail. The transport activity of organelles is usually coupled to the transport of charged species; therefore, it is not only associated with the metabolic landscape but also entangled with membrane potentials. In this context, the targeted expression of fluorescent probes for interrogating organellar membrane potential (Ψorg) emerges as a powerful approach, offering less-invasive conditions and technical simplicity to interrogate cellular signalling and metabolism. Different research groups have made remarkable progress in adapting a variety of optical methods for measuring and monitoring Ψorg. These approaches include using potentiometric dyes, genetically encoded voltage indicators, hybrid fluorescence resonance energy transfer sensors and photoinduced electron transfer systems. These studies have provided consistent values for the resting potential of single-membrane organelles, such as lysosomes, the Golgi and the endoplasmic reticulum. We can foresee the use of dynamic measurements of Ψorg to study fundamental problems in organellar physiology that are linked to serious cellular disorders. Here, we present an overview of the available techniques, a survey of the resting membrane potential of internal membranes and, finally, an open-source mathematical model useful to interpret and interrogate membrane-bound structures of small volume by using the lysosome as an example.
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Lisosomas , Orgánulos , Potenciales de la Membrana , Orgánulos/metabolismo , Lisosomas/metabolismo , Retículo Endoplásmico/metabolismo , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismoRESUMEN
Endolysosomes perform a wide range of cellular functions, including nutrient sensing, macromolecule digestion and recycling, as well as plasma membrane repair. Because of their high activity in cancerous cells, endolysosomes are attractive targets for the development of novel cancer treatments. Light-activated compounds termed photosensitizers (PS) can catalyze the oxidation of specific biomolecules and intracellular organelles. To selectively damage endosomes and lysosomes, HT-29 colorectal cancer cells were incubated with nanomolar concentrations of meso-tetraphenylporphine disulfonate (TPPS2a), an amphiphilic PS taken up via endocytosis and activated by green light (522 nm, 2.1 J.cm-1). Several cellular responses were characterized by a combination of immunofluorescence and immunoblotting assays. We showed that TPPS2a photosensitization blocked autophagic flux without extensive endolysosomal membrane rupture. Nevertheless, there was a severe functional failure of endolysosomes due to a decrease in CTSD (cathepsin D, 55%) and CTSB (cathepsin B, 52%) maturation. PSAP (prosaposin) processing (into saposins) was also considerably impaired, a fact that could be detrimental to glycosphingolipid homeostasis. Therefore, photosensitization of HT-29 cells previously incubated with a low concentration of TPPS2a promotes endolysosomal dysfunction, an effect that can be used to improve cancer therapies.
Asunto(s)
Autofagia , Lisosomas , Fármacos Fotosensibilizantes , Humanos , Células HT29 , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Autofagia/efectos de los fármacos , Autofagia/efectos de la radiación , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/química , Endosomas/metabolismo , Endosomas/efectos de los fármacos , Catepsinas/metabolismo , Catepsinas/antagonistas & inhibidores , Luz , Porfirinas/farmacología , Porfirinas/química , Catepsina D/metabolismo , Catepsina B/metabolismoRESUMEN
Parkinson's disease is a neurodegenerative disorder primarily known for typical motor features that arise due to the loss of dopaminergic neurons in the substantia nigra. However, the precise molecular aetiology of the disease is still unclear. Several cellular pathways have been linked to Parkinson's disease, including the autophagy-lysosome pathway, α-synuclein aggregation and mitochondrial function. Interestingly, the mechanistic link between GBA1, the gene that encodes for lysosomal ß-glucocerebrosidase (GCase), and Parkinson's disease lies in the interplay between GCase functions in the lysosome and mitochondria. GCase mutations alter mitochondria-lysosome contact sites. In the lysosome, reduced GCase activity leads to glycosphingolipid build-up, disrupting lysosomal function and autophagy, thereby triggering α-synuclein accumulation. Additionally, α-synuclein aggregates reduce GCase activity, creating a self-perpetuating cycle of lysosomal dysfunction and α-synuclein accumulation. GCase can also be imported into the mitochondria, where it promotes the integrity and function of mitochondrial complex I. Thus, GCase mutations that impair its normal function increase oxidative stress in mitochondria, the compartment where dopamine is oxidized. In turn, the accumulation of oxidized dopamine adducts further impairs GCase activity, creating a second cycle of GCase dysfunction. The oxidative state triggered by GCase dysfunction can also induce mitochondrial DNA damage which, in turn, can cause dopaminergic cell death. In this review, we highlight the pivotal role of GCase in Parkinson's disease pathogenesis and discuss promising examples of GCase-based therapeutics, such as gene and enzyme replacement therapies, small molecule chaperones and substrate reduction therapies, among others, as potential therapeutic interventions.
Asunto(s)
Glucosilceramidasa , Lisosomas , Mitocondrias , Enfermedad de Parkinson , Humanos , Glucosilceramidasa/metabolismo , Glucosilceramidasa/genética , Lisosomas/metabolismo , Mitocondrias/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Animales , alfa-Sinucleína/metabolismo , Autofagia/fisiología , MutaciónRESUMEN
HIV-associated neurocognitive disorders (HAND) affect 15-55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND.
Asunto(s)
Cannabinoides , Infecciones por VIH , VIH-1 , Humanos , Femenino , FN-kappa B/metabolismo , Proteómica , Espectrometría de Masas en Tándem , Macrófagos/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Estrés Oxidativo , Exocitosis , Lisosomas/metabolismoRESUMEN
SIDT2 is a lysosomal protein involved in the degradation of nucleic acids and the transport of cholesterol between membranes. Previous studies identified two "cholesterol recognition/interaction amino acid consensus" (CRAC) motifs in SIDT1 and SIDT2 members. We have previously shown that the first CRAC motif (CRAC-1) is essential for protein translocation to the PM upon cholesterol depletion in the cell. In the present study, we show that SIDT2 and the apolipoprotein A1 (ApoA1) form a complex which requires the second CRAC-2 motif in SIDT2 to be established. The overexpression of SIDT2 and ApoA1 results in enhanced ApoA1 secretion by HepG2 cells. This is not observed when overexpressing the SIDT2 with the CRAC-2 domain mutated to render it unfunctional. All these results provide evidence of a novel role for SIDT2 as a protein forming a complex with ApoA1 and enhancing its secretion to the extracellular space.
Asunto(s)
Apolipoproteína A-I , Hepatocitos , Transporte de Proteínas , Hepatocitos/metabolismo , Colesterol/metabolismo , Lisosomas/metabolismoRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra and the intraneuronal presence of Lewy bodies (LBs), composed of aggregates of phosphorylated alpha-synuclein at residue Ser129 (p-Ser129α-Syn). Unfortunately, no curative treatment is available yet. To aggravate matters further, the etiopathogenesis of the disorder is still unresolved. However, the neurotoxin rotenone (ROT) has been implicated in PD. Therefore, it has been widely used to understand the molecular mechanism of neuronal cell death. In the present investigation, we show that ROT induces two convergent pathways in HEK-293 cells. First, ROT generates H2O2, which, in turn, either oxidizes the stress sensor protein DJ-Cys106-SH into DJ-1Cys106SO3 or induces the phosphorylation of the protein LRRK2 kinase at residue Ser395 (p-Ser395 LRRK2). Once active, the kinase phosphorylates α-Syn (at Ser129), induces the loss of mitochondrial membrane potential (ΔΨm), and triggers the production of cleaved caspase 3 (CC3), resulting in signs of apoptotic cell death. ROT also reduces glucocerebrosidase (GCase) activity concomitant with the accumulation of lysosomes and autophagolysosomes reflected by the increase in LC3-II (microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate II) markers in HEK-293 cells. Second, the exposure of HEK-293 LRRK2 knockout (KO) cells to ROT displays an almost-normal phenotype. Indeed, KO cells showed neither H2O2, DJ-1Cys106SO3, p-Ser395 LRRK2, p-Ser129α-Syn, nor CC3 but displayed high ΔΨm, reduced GCase activity, and the accumulation of lysosomes and autophagolysosomes. Similar observations are obtained when HEK-293 LRRK2 wild-type (WT) cells are exposed to the inhibitor GCase conduritol-ß-epoxide (CBE). Taken together, these observations imply that the combined development of LRRK2 inhibitors and compounds for recovering GCase activity might be promising therapeutic agents for PD.
Asunto(s)
Glucosilceramidasa , Enfermedad de Parkinson , Humanos , Glucosilceramidasa/genética , Rotenona/farmacología , Rotenona/metabolismo , Células HEK293 , Peróxido de Hidrógeno/metabolismo , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/metabolismo , Lisosomas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismoRESUMEN
Calcium is a crucial messenger of intracellular and extracellular signals, regulating a great variety of cellular processes such as cell death, proliferation, and metabolism. Inside the cell, calcium signaling is one of the main interorganelle communication mediators, with central functional roles at the endoplasmic reticulum (ER), mitochondria, Golgi complex, and lysosomes. Lysosomal function is highly dependent on lumenal calcium and most of the lysosomal membrane-localised ion channels regulate several lysosomal functions and properties such as lumenal pH. One of these functions configures a specific type of cell death involving lysosomes, named lysosome-dependent cell death (LDCD), which contributes to maintenance of tissue homeostasis, development and pathology when deregulated. Here, we cover the fundamental aspects of LDCD with a special focus on recent advances in calcium signaling in LDCD.
Asunto(s)
Señalización del Calcio , Calcio , Calcio/metabolismo , Muerte Celular , Lisosomas/metabolismo , Membranas Intracelulares/metabolismoRESUMEN
The mechanisms of autophagy have been related to Alzheimer's disease (AD) pathogenesis by the endosomal-lysosomal system, having a critical function in forming amyloid-ß (Aß) plaques. Nevertheless, the exact mechanisms mediating disease pathogenesis remain unclear. The transcription factor EB (TFEB), a primary transcriptional autophagy regulator, improves gene expression, mediating lysosome function, autophagic flux, and autophagosome biogenesis. In this review, we present for the first time the hypothesis of how TFEB, autophagy, and mitochondrial function are interconnected in AD, providing a logical foundation for unraveling the critical role of chronic physical exercise in this process. Aerobic exercise training promotes Adiponectin Receptor 1 (AdipoR1)/AMP-activated protein kinase (AMPK)/TFEB axis activation in the brain of the AD animal model, which contributes to alleviated Aß deposition and neuronal apoptosis while improving cognitive function. Moreover, TFEB upregulates Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear factor erythroid 2-related factor 2 (NRF-2), improving mitochondrial biogenesis and redox status. In addition, tissue contraction activates calcineurin in skeletal muscle, which induces TFEB nuclear translocation, raising the hypothesis that the same would occur in the brain. Thus, a deep and comprehensive exploration of the TFEB could provide new directions and strategies for preventing AD. We conclude that chronic exercise can be an effective TFEB activator, inducing autophagy and mitochondrial biogenesis, representing a potential nonpharmacological strategy contributing to brain health.
Asunto(s)
Enfermedad de Alzheimer , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Animales , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Ejercicio Físico , Lisosomas/metabolismo , Músculo Esquelético/metabolismoRESUMEN
Identification of genetic modulators of lysosomal enzyme activities and glycosphingolipids (GSLs) may facilitate the development of therapeutics for diseases in which they participate, including Lysosomal Storage Disorders (LSDs). To this end, we used a systems genetics approach: we measured 11 hepatic lysosomal enzymes and many of their natural substrates (GSLs), followed by modifier gene mapping by GWAS and transcriptomics associations in a panel of inbred strains. Unexpectedly, most GSLs showed no association between their levels and the enzyme activity that catabolizes them. Genomic mapping identified 30 shared predicted modifier genes between the enzymes and GSLs, which are clustered in three pathways and are associated with other diseases. Surprisingly, they are regulated by ten common transcription factors, and their majority by miRNA-340p. In conclusion, we have identified novel regulators of GSL metabolism, which may serve as therapeutic targets for LSDs and may suggest the involvement of GSL metabolism in other pathologies.
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Glicoesfingolípidos , Enfermedades por Almacenamiento Lisosomal , Animales , Ratones , Glicoesfingolípidos/metabolismo , Enfermedades por Almacenamiento Lisosomal/metabolismo , Hidrolasas/metabolismo , Lisosomas/metabolismoRESUMEN
The coronavirus disease pandemic, which profoundly reshaped the world in 2019 (COVID-19), and is currently ongoing, has affected over 200 countries, caused over 500 million cumulative cases, and claimed the lives of over 6.4 million people worldwide as of August 2022. The causative agent is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Depicting this virus' life cycle and pathogenic mechanisms, as well as the cellular host factors and pathways involved during infection, has great relevance for the development of therapeutic strategies. Autophagy is a catabolic process that sequesters damaged cell organelles, proteins, and external invading microbes, and delivers them to the lysosomes for degradation. Autophagy would be involved in the entry, endo, and release, as well as the transcription and translation, of the viral particles in the host cell. Secretory autophagy would also be involved in developing the thrombotic immune-inflammatory syndrome seen in a significant number of COVID-19 patients that can lead to severe illness and even death. This review aims to review the main aspects that characterize the complex and not yet fully elucidated relationship between SARS-CoV-2 infection and autophagy. It briefly describes the key concepts regarding autophagy and mentions its pro- and antiviral roles, while also noting the reciprocal effect of viral infection in autophagic pathways and their clinical aspects.
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COVID-19 , SARS-CoV-2 , Humanos , Autofagia , Antivirales/farmacología , Lisosomas/metabolismoRESUMEN
Huntington's disease (HD) is a progressive neurodegenerative disease characterized by mutations in the huntingtin gene (mHtt), causing an unstable repeat of the CAG trinucleotide, leading to abnormal long repeats of polyglutamine (poly-Q) in the N-terminal region of the huntingtin, which form abnormal conformations and aggregates. Alterations in Ca2+ signaling are involved in HD models and the accumulation of mutated huntingtin interferes with Ca2+ homeostasis. Lysosomes are intracellular Ca2+ storages that participate in endocytic and lysosomal degradation processes, including autophagy. Nicotinic acid adenine dinucleotide phosphate (NAADP) is an intracellular second messenger that promotes Ca2+ release from the endo-lysosomal system via Two-Pore Channels (TPCs) activation. Herein, we show the impact of lysosomal Ca2+ signals on mHtt aggregation and autophagy blockade in murine astrocytes overexpressing mHtt-Q74. We observed that mHtt-Q74 overexpression causes an increase in NAADP-evoked Ca2+ signals and mHtt aggregation, which was inhibited in the presence of Ned-19, a TPC antagonist, or BAPTA-AM, a Ca2+ chelator. Additionally, TPC2 silencing revert the mHtt aggregation. Furthermore, mHtt has been shown co-localized with TPC2 which may contribute to its effects on lysosomal homeostasis. Moreover, NAADP-mediated autophagy was also blocked since its function is dependent on lysosomal functionality. Taken together, our data show that increased levels of cytosolic Ca2+ mediated by NAADP causes mHtt aggregation. Additionally, mHtt co-localizes with the lysosomes, where it possibly affects organelle functions and impairs autophagy.
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Canales de Calcio , Enfermedades Neurodegenerativas , Ratones , Animales , Canales de Calcio/metabolismo , Astrocitos/metabolismo , Enfermedades Neurodegenerativas/metabolismo , NADP/metabolismo , Lisosomas/metabolismo , Autofagia , Calcio/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismoRESUMEN
Mitochondrial activity and quality control are essential for neuronal homeostasis as neurons rely on glucose oxidative metabolism. The ketone body, D-ß-hydroxybutyrate (D-BHB), is metabolized to acetyl-CoA in brain mitochondria and used as an energy fuel alternative to glucose. We have previously reported that D-BHB sustains ATP production and stimulates the autophagic flux under glucose deprivation in neurons; however, the effects of D-BHB on mitochondrial turnover under physiological conditions are still unknown. Sirtuins (SIRTs) are NAD+-activated protein deacetylases involved in the regulation of mitochondrial biogenesis and mitophagy through the activation of transcription factors FOXO1, FOXO3a, TFEB and PGC1α coactivator. Here, we aimed to investigate the effect of D-BHB on mitochondrial turnover in cultured neurons and the mechanisms involved. Results show that D-BHB increased mitochondrial membrane potential and regulated the NAD+/NADH ratio. D-BHB enhanced FOXO1, FOXO3a and PGC1α nuclear levels in an SIRT2-dependent manner and stimulated autophagy, mitophagy and mitochondrial biogenesis. These effects increased neuronal resistance to energy stress. D-BHB also stimulated the autophagic-lysosomal pathway through AMPK activation and TFEB-mediated lysosomal biogenesis. Upregulation of SIRT2, FOXOs, PGC1α and TFEB was confirmed in the brain of ketogenic diet (KD)-treated mice. Altogether, the results identify SIRT2, for the first time, as a target of D-BHB in neurons, which is involved in the regulation of autophagy/mitophagy and mitochondrial quality control.
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NAD , Sirtuina 2 , Animales , Ratones , Ácido 3-Hidroxibutírico/farmacología , Ácido 3-Hidroxibutírico/metabolismo , Autofagia , Glucosa/metabolismo , Cuerpos Cetónicos/metabolismo , Cuerpos Cetónicos/farmacología , Lisosomas/metabolismo , Mitocondrias/metabolismo , NAD/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Sirtuina 2/metabolismoRESUMEN
Chagas disease is caused by the parasite Trypanosoma cruzi (T. cruzi) and, among all the chronic manifestations of the disease, Chronic Chagas Cardiomyopathy (CCC) is the most severe outcome. Despite high burden and public health importance in Latin America, there is a gap in understanding the molecular mechanisms that results in CCC development. Previous studies showed that T. cruzi uses the host machinery for infection and replication, including the repurposing of the responses to intracellular infection such as mitochondrial activity, vacuolar membrane, and lysosomal activation in benefit of parasite infection and replication. One common signaling upstream to many responses to parasite infection is mTOR pathway, previous associated to several downstream cellular mechanisms including autophagy, mitophagy and lysosomal activation. Here, using human iPSC derived cardiomyocytes (hiPSCCM), we show the mTOR pathway is activated in hiPSCCM after T. cruzi infection, and the inhibition of mTOR with rapamycin reduced number of T. cruzi 48 h post infection (hpi). Rapamycin treatment also reduced lysosome migration from nuclei region to cell periphery resulting in less T. cruzi inside the parasitophorous vacuole (PV) in the first hour of infection. In addition, the number of parasites leaving the PV to the cytoplasm to replicate in later times of infection was also lower after rapamycin treatment. Altogether, our data suggest that host's mTOR activation concomitant with parasite infection modulates lysosome migration and that T. cruzi uses this mechanism to achieve infection and replication. Modulating this mechanism with rapamycin impaired the success of T. cruzi life cycle independent of mitophagy.
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Enfermedad de Chagas , Trypanosoma cruzi , Humanos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/parasitología , Enfermedad de Chagas/parasitología , Trypanosoma cruzi/fisiología , Serina-Treonina Quinasas TOR , Lisosomas/metabolismo , Lisosomas/parasitología , Sirolimus/metabolismoRESUMEN
Lysosomes are highly dynamic organelles involved in the breakdown and recycling of macromolecules, cell cycle, cell differentiation, and cell death, among many other functions in eukaryotic cells. Recently, lysosomes have been identified as cellular hubs for the modulation of intracellular signaling pathways, such as the Wnt/beta-catenin pathway. Here we analyzed morphological and functional characteristics of lysosomes in muscle and non-muscle cells during chick myogenesis, as well as their modulation by the Wnt/beta-catenin pathway. Our results show that (i) muscle and non-muscle cells show differences in lysosomal size and its distribution, (ii) lysosomes are found in spherical structures in myoblasts and fibroblasts and tubular structures in myotubes, (iii) lysosomes are found close to the plasma membrane in fibroblasts and close to the nucleus in myoblasts and myotubes, (iv) lysosomal distribution and size are dependent on the integrity of microtubules and microfilaments in myogenic cells, (v) alterations in lysosomal function, in the expression of LAMP2, and in Wnt/beta-catenin pathway affect the distribution and size of lysosomes in myogenic cells, (vi) the effects of the knockdown of LAMP2 on myogenesis can be rescued by the activation of the Wnt/beta-catenin pathway, and (vii) the chloroquine Lys05 is a potent inhibitor of both the Wnt/beta-catenin pathway and lysosomal function. Our data highlight the involvement of the Wnt/beta-catenin pathway in the regulation of the positioning, size, and function of lysosomes during chick myogenesis.
Asunto(s)
Desarrollo de Músculos , beta Catenina , beta Catenina/metabolismo , Desarrollo de Músculos/fisiología , Vía de Señalización Wnt , Fibras Musculares Esqueléticas/metabolismo , Citoesqueleto/metabolismo , Lisosomas/metabolismoRESUMEN
Transcription factor EB (TFEB) is considered the master transcriptional regulator of autophagy and lysosomal biogenesis, which regulates target gene expression through binding to CLEAR motifs. TFEB dysregulation has been linked to the development of numerous pathological conditions; however, several other lines of evidence show that TFEB might be a point of convergence of diverse signaling pathways and might therefore modulate other important biological processes such as cellular senescence, DNA repair, ER stress, carbohydrates, and lipid metabolism and WNT signaling-related processes. The regulation of TFEB occurs predominantly at the post-translational level, including phosphorylation, acetylation, SUMOylating, PARsylation, and glycosylation. It is noteworthy that TFEB activation is context-dependent; therefore, its regulation is subjected to coordinated mechanisms that respond not only to nutrient fluctuations but also to stress cell programs to ensure proper cell homeostasis and organismal health. In this review, we provide updated insights into novel post-translational modifications that regulate TFEB activity and give an overview of TFEB beyond its widely known role in autophagy and the lysosomal pathway, thus opening the possibility of considering TFEB as a potential therapeutic target.