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Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. Abbreviations: BAF: bafilomycin A1; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; siCtrl: small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a.
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Parkinson's disease (PD) is the second most common late-onset neurodegenerative disease and the predominant cause of movement problems. PD is characterized by motor control impairment by extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). This selective dopaminergic neuronal loss is in part triggered by intracellular protein inclusions called Lewy bodies, which are composed mainly of misfolded alpha-synuclein (α-syn) protein. We previously reported insulin-like growth factor 2 (IGF2) as a key protein downregulated in PD patients. Here we demonstrated that IGF2 treatment or IGF2 overexpression reduced the α-syn aggregates and their toxicity by IGF2 receptor (IGF2R) activation in cellular PD models. Also, we observed IGF2 and its interaction with IGF2R enhance the α-syn secretion. To determine the possible IGF2 neuroprotective effect in vivo we used a gene therapy approach in an idiopathic PD model based on α-syn preformed fibrils intracerebral injection. IGF2 gene therapy revealed a significantly preventing of motor impairment in idiopathic PD model. Moreover, IGF2 expression prevents dopaminergic neuronal loss in the SN together with a decrease in α-syn accumulation (phospho-α-syn levels) in the striatum and SN brain region. Furthermore, the IGF2 neuroprotective effect was associated with the prevention of synaptic spines loss in dopaminergic neurons in vivo. The possible mechanism of IGF2 in cell survival effect could be associated with the decrease of the intracellular accumulation of α-syn and the improvement of dopaminergic synaptic function. Our results identify to IGF2 as a relevant factor for the prevention of α-syn toxicity in both in vitro and preclinical PD models.
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Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by protein accumulation in the brain as a main neuropathological hallmark. Among them, Aß42 peptides tend to aggregate and create oligomers and plaques. Macroautophagy, a form of autophagy characterized by a double-membrane vesicle, plays a crucial role in maintaining neuronal homeostasis by degrading protein aggregates and dysfunctional organelles as a quality control process. Recently, DEF8, a relatively uncharacterized protein, has been proposed as a participant in vesicular traffic and autophagy pathways. We have reported increased DEF8 levels in lymphocytes from mild cognitive impairment (MCI) and early-stage AD patients and a neuronal profile in a murine transgenic AD model. Here, we analyzed DEF8 localization and levels in the postmortem frontal cortex of AD patients, finding increased levels compared to healthy controls. To evaluate the potential function of DEF8 in the nervous system, we performed an in silico assessment of its expression and network profiles, followed by an in vivo evaluation of a neuronal Def8 deficient model using a Drosophila melanogaster model of AD based on Aß42 expression. Our findings show that DEF8 is an essential protein for maintaining cellular homeostasis in the nervous system, and it is upregulated under stress conditions generated by Aß42 aggregation. This study suggests DEF8 as a novel actor in the physiopathology of AD, and its exploration may lead to new treatment avenues.
Assuntos
Doença de Alzheimer , Animais , Humanos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Autofagia/genética , Encéfalo/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fragmentos de Peptídeos/metabolismoRESUMO
Alzheimer's disease (AD) is the most prevalent age-associated neurodegenerative disease. A decrease in autophagy during aging contributes to brain disorders by accumulating potentially toxic substrates in neurons. Rubicon is a well-established inhibitor of autophagy in all cells. However, Rubicon participates in different pathways depending on cell type, and little information is currently available on neuronal Rubicon's role in the AD context. Here, we investigated the cell-specific expression of Rubicon in postmortem brain samples from AD patients and 5xFAD mice and its impact on amyloid ß burden in vivo and neuroblastoma cells. Further, we assessed Rubicon levels in human-induced pluripotent stem cells (hiPSCs), derived from early-to-moderate AD and in postmortem samples from severe AD patients. We found increased Rubicon levels in AD-hiPSCs and postmortem samples and a notable Rubicon localization in neurons. In AD transgenic mice lacking Rubicon, we observed intensified amyloid ß burden in the hippocampus and decreased Pacer and p62 levels. In APP-expressing neuroblastoma cells, increased APP/amyloid ß secretion in the medium was found when Rubicon was absent, which was not observed in cells depleted of Atg5, essential for autophagy, or Rab27a, required for exosome secretion. Our results propose an uncharacterized role of Rubicon on APP/amyloid ß homeostasis, in which neuronal Rubicon is a repressor of APP/amyloid ß secretion, defining a new way to target AD and other similar diseases therapeutically.
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Doença de Alzheimer , Proteínas Relacionadas à Autofagia , Neuroblastoma , Doenças Neurodegenerativas , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Neuroblastoma/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismoRESUMO
Tumor necrosis factor alpha (TNF) triggers regulated necrosis of mycobacterium-infected macrophages through of mitochondrial reactive oxygen species (mitoROS) production in a RIPK1/3-dependent manner. To explain that, Roca and colleagues describe an inter-orgallenar circuit which involves the lysosomal ceramide production, mitoROS, BAX activation and RyR Ca2+ efflux from the endoplasmic reticulum into the mitochondrion.
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Cálcio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Sinalização do Cálcio , Morte Celular , Humanos , Modelos Biológicos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Endoplasmic reticulum (ER) stress is a common feature of several physiological and pathological conditions affecting the function of the secretory pathway. To restore ER homeostasis, an orchestrated signaling pathway is engaged that is known as the unfolded protein response (UPR). The UPR has a primary function in stress adaptation and cell survival; however, under irreversible ER stress a switch to pro-apoptotic signaling events induces apoptosis of damaged cells. The mechanisms that initiate ER stress-dependent apoptosis are not fully understood. Several pathways have been described where we highlight the participation of the BCL-2 family of proteins and ER calcium release. In addition, recent findings also suggest that microRNAs and oxidative stress are relevant players on the transition from adaptive to cell death programs. Here we provide a global and integrated overview of the signaling networks that may determine the elimination of a cell under chronic ER stress. This article is part of a Special Section entitled: Cell Death Pathways.
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Estresse do Retículo Endoplasmático , Animais , Morte Celular , Humanos , Modelos Biológicos , Resposta a Proteínas não DobradasRESUMO
Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.
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Apoptose/fisiologia , Ciclofilinas/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Proteína X Associada a bcl-2/fisiologia , Animais , Sangue , Caspase 9/metabolismo , Peptidil-Prolil Isomerase F , Citocromos c/metabolismo , Retículo Endoplasmático/metabolismo , Camundongos , Resposta a Proteínas não DobradasRESUMO
Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Endorribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Resposta a Proteínas não Dobradas , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Técnicas de Inativação de Genes , Imunoprecipitação , Proteínas de Membrana/genética , Camundongos , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteoma/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.
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Autofagia/genética , Endorribonucleases/metabolismo , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/genética , Ácidos/metabolismo , Animais , Sobrevivência Celular/genética , Células Cultivadas , Drosophila/genética , Endorribonucleases/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Organismos Geneticamente Modificados , Fagossomos/genética , Fagossomos/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Inanição/metabolismo , Vesículas Transportadoras/metabolismo , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
The assembling of distinct signaling protein complexes at the endoplasmic reticulum (ER) membrane controls several stress responses related to calcium homeostasis, autophagy, ER morphogenesis and protein folding. Diverse pathological conditions interfere with the function of the ER altering protein folding, a condition known as "ER stress". Adaptation to ER stress depends on the activation of the unfolded protein response (UPR) and protein degradation pathways such as autophagy. Under chronic or irreversible ER stress, cells undergo apoptosis, where the BCL-2 protein family plays a crucial role at the mitochondria to trigger cytochrome c release and apoptosome assembly. Several BCL2 family members also regulate physiological processes at the ER through dynamic interactomes. Here we provide a comprehensive view of the roles of the BCL-2 family of proteins in mediating the molecular crosstalk between the ER and mitochondria to initiate apoptosis, in addition to their emerging functions in adaptation to stress, including autophagy, UPR, calcium homeostasis and organelle morphogenesis. We envision a model where BCL-2-containing complexes may operate as stress rheostats that, beyond their known apoptosis functions at the mitochondria, determine the amplitude and kinetics of adaptive responses against ER-related injuries. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.
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Apoptose , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estresse Fisiológico , Animais , HumanosRESUMO
Apoptosis is essential for maintenance of tissue homeostasis and its deregulation results in a variety of disease conditions. The BCL-2 family of proteins is a group of evolutionarily conserved regulators of cell death that comprises both anti- and pro-apoptotic members, that operate at the mitochondrial membrane to control caspase activation. Different BCL-2-related proteins are also located in the endoplasmic reticulum (ER), where important roles in organelle physiology are proposed. Adaptation to ER stress is mediated by the activation of a complex signal transduction pathway known as the unfolded protein response (UPR). Recent reports indicate that the ER stress sensor IRE1alpha, signals through the formation of a protein complex platform at the ER membrane, here termed the "UPRosome". Alternatively, BCL-2 family members are contained in other multiprotein complexes at the ER that are involved in the control of diverse cellular processes including calcium homeostasis, autophagy and ER morphogenesis. Here we describe the emerging concept that BCL-2 family members are important regulators of essential cellular processes beyond apoptosis.
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Retículo Endoplasmático/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/ultraestrutura , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genética , Estresse Fisiológico , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
We investigated the role of Ca(2+) in generating reactive oxygen species (ROS) induced by hyposmotic stress (Hypo) and its relationship to regulatory volume decrease (RVD) in cardiomyocytes. Hypo-induced increases in cytoplasmic and mitochondrial Ca(2+). Nifedipine (Nife) inhibited both Hypo-induced Ca(2+) and ROS increases. Overexpression of catalase (CAT) induced RVD and a decrease in Hypo-induced blebs. Nife prevented CAT-dependent RVD activation. These results show a dual role of Hypo-induced Ca(2+) influx in the control of cardiomyocyte viability. Hypo-induced an intracellular Ca(2+) increase which activated RVD and inhibited necrotic blebbing thus favoring cell survival, while simultaneously increasing ROS generation, which in turn inhibited RVD and induced necrosis.
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Cálcio/metabolismo , Tamanho Celular , Miócitos Cardíacos/citologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Transporte Biológico , Cálcio/farmacologia , Catalase/metabolismo , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Regulação Enzimológica da Expressão Gênica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Necrose/metabolismo , Pressão Osmótica/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Adaptation to endoplasmic reticulum (ER) stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). Bax inhibitor-1 (BI-1) is an evolutionarily conserved ER-resident protein that suppresses cell death. Here we have investigated the role of BI-1 in the UPR. BI-1 expression suppressed IRE1alpha activity in fly and mouse models of ER stress. BI-1-deficient cells displayed hyperactivation of the ER stress sensor IRE1alpha, leading to increased levels of its downstream target X-box-binding protein-1 (XBP-1) and upregulation of UPR target genes. This phenotype was associated with the formation of a stable protein complex between BI-1 and IRE1alpha, decreasing its ribonuclease activity. Finally, BI-1 deficiency increased the secretory activity of primary B cells, a phenomenon regulated by XBP-1. Our results suggest a role for BI-1 in early adaptive responses against ER stress that contrasts with its known downstream function in apoptosis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Linfócitos B/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endorribonucleases/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imunoglobulina M/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA , Fatores de Transcrição de Fator Regulador X , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
Cells have developed compensatory mechanisms to restore cell volume, and the ability to resist osmotic swelling or shrinkage parallels their resistance to necrosis or apoptosis. There are several mechanisms by which cells adapt to hyposmotic stress including that of regulatory volume decrease. In ischemia and reperfusion, cardiomyocytes are exposed to hyposmotic stress, but little is known as to how their volume is controlled. Exposure of cultured neonatal rat cardiomyocytes to hyposmotic media induced a rapid swelling without any compensatory regulatory volume decrease. The hyposmotic stress increased the production of reactive oxygen species, mainly through NADPH oxidase. Adenoviral overexpression of catalase inhibited the hyposmosis-dependent OH(*) production, induced the regulatory volume decrease mechanism, and prevented cell death. These results suggest that hyposmotic stress of cardiomyocytes stimulates production of reactive oxygen species which are closely linked to volume regulation and cell death.