FAIM-L Is an IAP-Binding Protein That Inhibits XIAP Ubiquitinylation and Protects from Fas-Induced Apoptosis

The neuronal long isoform of Fas Apoptotic Inhibitory Molecule (FAIM-L) protects from death receptor (DR)-induced apoptosis, yet its mechanism of protection remains unknown. Here, we show that FAIM-L protects rat neuronal Type II cells from Fas-induced apoptosis. XIAP has previously emerged as a molecular discriminator that is upregulated in Type II and downregulated in Type I apoptotic signaling. We demonstrate that FAIM-L requires sustained endogenous levels of XIAP to protect Type II cells as well as murine cortical neurons from Fas-induced apoptosis. FAIM-L interacts with the BIR2 domain of XIAP through an IAP-binding motif, the mutation of which impairs the antiapoptotic function of FAIM-L. Finally, we report that FAIM-L inhibits XIAP auto-ubiquitinylation and maintains its stability, thus conferring protection from apoptosis. Our results bring new understanding of the regulation of endogenous XIAP by a DR antagonist, pointing out at FAIM-L as a promising therapeutic tool for protection from apoptosis in pathological situations where XIAP levels are decreased.This work was funded by the Spanish Government Ministerio de Sanidad y Consumo (Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, CB06/05/1104 to J.X.C.), Ministerio de Economía y Competitividad (SAF2010–19953 to J.X.C.; SAF2012–31485 to V.J.Y.), Instituto de Salud Carlos III (CP11/00052 to M.F.S.), and the Generalitat de Catalunya (Suport als Grups de Recerca Consolidats 2009SGR346). F.M.-F. and L.P.-F. are supported by postgraduate fellowships from the Spanish Government Ministerio de Educación y Ciencia. J.U. is supported by a postgraduate fellowship from the Generalitat de Catalunya. R.S.M. and V.J.Y. were under the Juan de la Cierva and the Ramon y Cajal programs, respectively, from the Ministerio de Educación y Ciencia (Spain), cofinanced by the European Social Fund. M.F.S. is under the Miguel Servet program from the Instituto de Salud Carlos III and cofinanced by the European Regional Development Fund

Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program

Background Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. Methods Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. Results Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. Conclusions We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma

Key Factors Associated With Pulmonary Sequelae in the Follow-Up of Critically Ill COVID-19 Patients

Introduction: Critical COVID-19 survivors have a high risk of respiratory sequelae. Therefore, we aimed to identify key factors associated with altered lung function and CT scan abnormalities at a follow-up visit in a cohort of critical COVID-19 survivors. Methods: Multicenter ambispective observational study in 52 Spanish intensive care units. Up to 1327 PCR-confirmed critical COVID-19 patients had sociodemographic, anthropometric, comorbidity and lifestyle characteristics collected at hospital admission; clinical and biological parameters throughout hospital stay; and, lung function and CT scan at a follow-up visit. Results: The median [p25–p75] time from discharge to follow-up was 3.57 [2.77–4.92] months. Median age was 60 [53–67] years, 27.8% women. The mean (SD) percentage of predicted diffusing lung capacity for carbon monoxide (DLCO) at follow-up was 72.02 (18.33)% predicted, with 66% of patients having DLCO < 80% and 24% having DLCO < 60%. CT scan showed persistent pulmonary infiltrates, fibrotic lesions, and emphysema in 33%, 25% and 6% of patients, respectively. Key variables associated with DLCO < 60% were chronic lung disease (CLD) (OR: 1.86 (1.18–2.92)), duration of invasive mechanical ventilation (IMV) (OR: 1.56 (1.37–1.77)), age (OR [per-1-SD] (95%CI): 1.39 (1.18–1.63)), urea (OR: 1.16 (0.97–1.39)) and estimated glomerular filtration rate at ICU admission (OR: 0.88 (0.73–1.06)). Bacterial pneumonia (1.62 (1.11–2.35)) and duration of ventilation (NIMV (1.23 (1.06–1.42), IMV (1.21 (1.01–1.45)) and prone positioning (1.17 (0.98–1.39)) were associated with fibrotic lesions. Conclusion: Age and CLD, reflecting patients’ baseline vulnerability, and markers of COVID-19 severity, such as duration of IMV and renal failure, were key factors associated with impaired DLCO and CT abnormalities

Clustering COVID-19 ARDS patients through the first days of ICU admission. An analysis of the CIBERESUCICOVID Cohort

Background Acute respiratory distress syndrome (ARDS) can be classified into sub-phenotypes according to different inflammatory/clinical status. Prognostic enrichment was achieved by grouping patients into hypoinflammatory or hyperinflammatory sub-phenotypes, even though the time of analysis may change the classification according to treatment response or disease evolution. We aimed to evaluate when patients can be clustered in more than 1 group, and how they may change the clustering of patients using data of baseline or day 3, and the prognosis of patients according to their evolution by changing or not the cluster.Methods Multicenter, observational prospective, and retrospective study of patients admitted due to ARDS related to COVID-19 infection in Spain. Patients were grouped according to a clustering mixed-type data algorithm (k-prototypes) using continuous and categorical readily available variables at baseline and day 3.Results Of 6205 patients, 3743 (60%) were included in the study. According to silhouette analysis, patients were grouped in two clusters. At baseline, 1402 (37%) patients were included in cluster 1 and 2341(63%) in cluster 2. On day 3, 1557(42%) patients were included in cluster 1 and 2086 (57%) in cluster 2. The patients included in cluster 2 were older and more frequently hypertensive and had a higher prevalence of shock, organ dysfunction, inflammatory biomarkers, and worst respiratory indexes at both time points. The 90-day mortality was higher in cluster 2 at both clustering processes (43.8% [n = 1025] versus 27.3% [n = 383] at baseline, and 49% [n = 1023] versus 20.6% [n = 321] on day 3). Four hundred and fifty-eight (33%) patients clustered in the first group were clustered in the second group on day 3. In contrast, 638 (27%) patients clustered in the second group were clustered in the first group on day 3.Conclusions During the first days, patients can be clustered into two groups and the process of clustering patients may change as they continue to evolve. This means that despite a vast majority of patients remaining in the same cluster, a minority reaching 33% of patients analyzed may be re-categorized into different clusters based on their progress. Such changes can significantly impact their prognosis

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Estudio de FAIM y FLIP: dos moléculas antagonistas del receptor FAS, reguladas por NGF y con efectos promotores del crecimiento neurítico

El present treball es basa en l'estudi del efecte de dues proteïnes identificades com antagonistes de la mort induïda pel receptor Fas, FAIM i FLIP, en el sistema nerviós. Malgrat que totes dues molècules promouen el creixement neurític, existeixen diferències mecanístiques que es detallaran de forma separada. Inicialment, hem procedit a la caracterització de la proteïna FAIM (Fas Apoptosis Inhibitory Molecule), de la qual n'existeixen dues úniques referències, en la bibliografia. Hem demostrat que l'expressió forçada de FAIM no protegeix les neurones de la retirada de factors tròfics, però exerceix una clara acció promotora del creixement neurític en els diversos sistemes neuronals estudiats. D'una banda, l'expressió forçada de FAIM incrementa la longitud i el grau d'arborització de les neurites induïts pel factor de creixement nerviós (NGF), tant en la línia cel·lular PC12 com en cultius primaris de neurones del gangli cervical superior (SCG). Per altra banda, si es redueixen els nivells endògens de FAIM mitjançant la tècnica del ARN d'interferència, el creixement neurític induït pel NGF es veu dràsticament afectat de forma negativa en els dos models cel·lulars utilitzats. L'expressió exògena de FAIM promou l'activació de la via NF-&#954;B, mentre que el bloqueig d'aquesta via, mitjançant la transfecció d'una forma mutada d'I&#954;B&#945; no degradable o bé usant neurones corticals de ratolins nuls que manquen de la subunitat p65 de NF-&#954;B, evita el creixement neurític promogut per NGF. L'efecte estimulador del creixement neurític també pot ser bloquejat per la inhibició de la via de Ras/ERK. Finalment, hem demostrat que FAIM interacciona amb els dos receptors de la neurotrofina NGF, p75NTR i TrkA, d'una forma depenent de lligand. Aquests resultats revelen una nova funció de FAIM com promotor de creixement neurític mitjançant un mecanisme depenent de NF-&#954;B. En el cas de FLIP, s'ha descrit la seva funció com inhibidor endogen de la apoptosi induïda per Fas, però, també s'ha vist implicat en promoció de proliferació. El treball descriu per primera vegada una funció, fins ara desconeguda, de FLIP en el sistema nerviós. FLIP s'expressa en motoneurones, en neurones SCGs i cèl·lules PC12. Malgrat això, la seva expressió forçada, només pot protegir el cultiu de motoneurones de ratolí de la mort cel·lular induïda per la retirada de factors tròfics. De totes maneres, aquesta expressió augmenta de forma considerable el creixement neurític en els tres models després de l'estímul neurotròfic apropiat per a cadascun d'ells. De forma interessant, i sense excepció, la reducció dels nivells endògens de FLIP inhibeix la neuritogènesis en aquests models. Les vies intracel·lulars regulades per FLIP impliquen tant ERK com NF-&#954;B. L'expressió forçada de FLIP promou un increment en la seva activitat, mentre que la reducció de la seva expressió provoca una disminució d'aquesta, després de l'estímul de NGF en cèl·lules PC12. Finalment, es demostra que FLIP interacciona amb el receptor del NGF, TrkA d'una manera depenent d'estímul. Aquests resultats revelen una nova funció neuritogènica de FLIP a través d'un mecanisme que implica l'activació de les vies MAPK/ERK i NF-&#954;B, i que no està relacionada amb la seva clàssica funció antiapoptòtica.El presente trabajo se basa en el estudio del efecto de dos proteínas identificadas como antagonistas de la muerte inducida por el receptor de muerte Fas, FAIM y FLIP, en el sistema nervioso. Para ello, y pese a que comparten el fenotipo de promoción del crecimiento neurítico, existen diferencias mecanísticas que detallaremos de forma separada. Inicialmente, caracterizamos la proteína FAIM (Fas Apoptosis Inhibitory Molecule), de la cual existen dos únicas referencias en la bibliografía. Hemos demostrado que la expresión forzada de FAIM no protege las neuronas de la retirada de suporte trófico, pero ejerce una clara acción promotora del crecimiento neurítico en los diversos sistemas neuronales estudiados. Por una parte, la expresión forzada de FAIM incrementa la longitud y el grado de arborización de las neuritas inducidos por el factor de crecimiento nervioso (NGF), tanto en línea celular PC12 como en cultivos primarios de neuronas del ganglio cervical superior (SCG). Por otra parte, si se reducen los niveles endógenos de FAIM mediante la técnica del ARN de interferencia, se disminuye el crecimiento neurítico en dichas células. La expresión forzada de FAIM promueve la activación de la vía NF-&#954;B, mientras que el bloqueo de esta vía, mediante la transfección de una forma mutada de I&#954;B&#945; no degradable o bien usando neuronas corticales de ratones nulos que carecen de la subunidad p65 de NF-&#954;B, previene el crecimiento neurítico promovido por NGF. El efecto estimulador del crecimiento neurítico también puede ser bloqueado por la inhibición de la vía de Ras/ERK. Finalmente, demostramos que FAIM interacciona con los dos receptores de la neurotrofina NGF, p75NTR y TrkA, de una forma dependiente de ligando. Estos resultados revelan una nueva función de FAIM como promotor de crecimiento neurítico mediante un mecanismo dependiente de NF-&#954;B. En el caso de FLIP, se ha descrito su función como inhibidor endógeno de la apoptosis mediada por Fas, pero, también se ha visto implicado en promoción de proliferación. El trabajo describe por primera vez una función, hasta ahora desconocida, de FLIP en el sistema nervioso. FLIP se expresa en motoneuronas, en neuronas SCGs y células PC12, aunque su expresión forzada, únicamente protege de la muerte celular inducida por la retirada de factores tróficos en motoneuronas. De todos modos, dicha expresión aumenta de forma considerable el crecimiento neurítico en los tres modelos, después del estímulo neurotrófico apropiado. De forma interesante, y sin excepción, la reducción de los niveles endógenos de FLIP inhibe la neuritogénesis en estos modelos. Las vías intracelulares reguladas por FLIP implican tanto ERK como NF-&#954;B. La expresión forzada de FLIP promueve un incremento en su actividad, mientras que la reducción de su expresión provoca una disminución de ésta, después de un estímulo de NGF en células PC12. Finalmente, demostramos que FLIP interacciona con el receptor del NGF, TrkA de una manera dependiente de estímulo. Estos resultados revelan una nueva función para FLIP, la neuritogénesis, a través de un mecanismo que implica la activación de las vías Ras/ERK y NF-&#954;B, y que no está relacionada con su clásica función antiapoptótica.The present work studies the effect of two proteins identified as Fas-induced cell death antagonists, FAIM and FLIP, in the nervous system. Although they share the neuritogenesis promoting effect, there are mechanistical differences that will be detailed separately. Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. We show here that FAIM over expression fails to rescue neurons from trophic factor deprivation, but exerts a marked neurite growth -promoting action in different neuronal systems. Whereas FAIM over expression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells. FAIM over expression promoted NF-&#954;B activation, and blocking this activation by using a super repressor I&#954;B&#945; or by carrying out experiments using cortical neurons from mice that lack the p65 NF-&#954;B subunit prevented FAIM-induced neurite outgrowth. The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras-ERK pathway. Finally, we show that FAIM interacts with both TrkA and p75NTR NGF receptors in a ligand-dependent manner. These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras-ERK pathway and NF-&#954;B. Cellular FLIP (c-FLIP) is an endogenous inhibitor of the signaling pathway triggered by Fas activation implicated in apoptosis as well as in proliferation processes. Here, we demonstrate for the first time an unexpected role of FLIP in the nervous system. FLIP is expressed in motoneurons, SCGs and PC12 cells. However, its over expression is only able to protect mouse isolated motoneurons from growth factor deprivation-induced cell death. Whereas FLIP over expression greatly enhanced neurite outgrowth in the three neuronal models after appropriate neurotrophin stimuli, reduction of endogenous FLIP levels by RNAi decreased neuritogenesis in these cells. The intracellular signals regulated by FLIP implicate both ERK and NF-&#954;B pathways. Over expression of FLIP promotes an increased activity, whereas its downregulation provokes a reduction after NGF stimulus in PC12 cells. Finally, we demonstrate that FLIP interacts with TrkA receptor in a NGF dependent manner. These results reveal a new function of FLIP in neuritogenesis by a mechanism involving activation of Ras/ERK pathway and NF-&#954;B that can be separated from its classical antiapoptotic function

The prevention of the staurosporine-induced apoptosis by Bcl-XL, but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins

Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program

Altres ajuts: Asociación Española Contra el Cáncer (LABAE18009SEGU to MFS, LABAE19004LLOB to DL-N, PROYE18010POSA to FP); Generalitat de Catalunya (2017FI_B_00095 to CJ, 2017SGR799 to FP and EdN; institutional funding through CERCA Programme); State Research Agency (institutional funding through Unidad de Excelencia María de Maeztu, CEX2018-000792-M)Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma