Implicación de la Caspasa 8 en el desarrollo embrionario

Caspase8 “knock out” - Embryo development- Dual role La apoptosis o muerte celular inducida es un proceso de muerte fisiológica implicado en numerosos eventos relacionados con el desarrollo, la homeostasis de tejidos, la educación de células, e incluso el envejecimiento. Juega un papel tan esencial en el organismo que un desequilibrio positivo podría desencadenar cáncer o manifestarse como una enfermedad autoinmune, mientras que un desequilibrio negativo podría suponer inmunodeficiencia y degeneración. Por ello, es de especial interés ahondar en el análisis de las moléculas implicadas en tan importante proceso, en nuestro caso estudiaremos la caspasa 8, una proteína de la familia de las cisteín-aspartato-proteasas encargada de iniciar la cascada inducida por receptores de muerte que desencadena la apoptosis. A pesar de que primordialmente se ha asociado a la caspasa 8 con procesos pro-apoptóticos, recientes estudios de experimentación in vivo con ratones knock-out han hallado que la supresión de esta proteasa supone la inhibición de la apoptosis y la activación de la necroptosis, un proceso de muerte celular patológica relacionada con una extensa reacción inmunogénica y consecuentemente inflamatoria. Todo ello no hace más que defender la existencia de una dualidad de funciones de la caspasa 8, la ya conocida pro-apoptótica y la pro-supervivencia. Es por ello, que, aunque primordialmente se ha asociado con la muerte, ahora queremos demostrar que desempeña un importante papel en el desarrollo embrionario, caracterizado por un fuerte proceso de formación y destrucción celular. En concreto, estudiaremos la implicación de dicha proteasa en el desarrollo, mediante el empleo de embriones deficientes para dicha caspasa y se compararán con controles con el fin de estudiar el impacto que tiene la deleción de dicha proteasa.Universidad de Sevilla. Grado en Farmaci

Brainiac Caspases: Beyond the Wall of Apoptosis

For the last two decades, caspases, a family of cysteine-aspartic proteases, have evolved from being considered solely as regulators of apoptosis or inflammation to having a wider range of functions. In this mini review, we focus on the most recent "non-apoptotic" roles of caspases in the CNS, particularly in neurons, astrocytes and oligodendrocytes. Non-apoptotic caspase functions in microglia have already been reviewed extensively elsewhere. Here we discuss the involvement of caspases in the activation of the inflammasome, autophagy, and non-apoptotic forms of cell death such as necroptosis and pyroptosis. Also, we review the involvement of caspases in synapses and the processing of aggregates key to neurodegenerative diseases such as Parkinson's, Alzheimer's and Huntington's diseases. Likewise, we mention the recently described involvement of caspases in mitochondrial biogenesis, which is a function independent of the enzymatic activity. We conclude discussing the relevance that "new" functions of caspases have in the CNS and the future of this field of researchEspaña Ministerio de Economía y Competitividad SAF2015-64171- R (MINECO/FEDER, EU)España Ministerio de Economía y Competitividad (Programa Ramón y Cajal: RYC-2017-21804

The Ubiquitin Proteasome System in Neuromuscular Disorders: Moving Beyond Movement

Neuromuscular disorders (NMDs) affect 1 in 3000 people worldwide. There are more than 150 different types of NMDs, where the common feature is the loss of muscle strength. These disorders are classified according to their neuroanatomical location, as motor neuron diseases, peripheral nerve diseases, neuromuscular junction diseases, and muscle diseases. Over the years, numerous studies have pointed to protein homeostasis as a crucial factor in the development of these fatal diseases. The ubiquitin-proteasome system (UPS) plays a fundamental role in maintaining protein homeostasis, being involved in protein degradation, among other cellular functions. Through a cascade of enzymatic reactions, proteins are ubiquitinated, tagged, and translocated to the proteasome to be degraded. Within the ubiquitin system, we can find three main groups of enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin-protein ligases). Only the ubiquitinated proteins with specific chain linkages (such as K48) will be degraded by the UPS. In this review, we describe the relevance of this system in NMDs, summarizing the UPS proteins that have been involved in pathological conditions and neuromuscular disorders, such as Spinal Muscular Atrophy (SMA), Charcot-Marie-Tooth disease (CMT), or Duchenne Muscular Dystrophy (DMD), among others. A better knowledge of the processes involved in the maintenance of proteostasis may pave the way for future progress in neuromuscular disorder studies and treatments.Ministerio de Economía y Competitividad RTI2018-098645-B-10

Reformulating Pro-Oxidant Microglia in Neurodegeneration

In neurodegenerative diseases, microglia-mediated neuroinflammation and oxidative stress are central events. Recent genome-wide transcriptomic analyses of microglial cells under different disease conditions have uncovered a new subpopulation named disease-associated microglia (DAM). These studies have challenged the classical view of the microglia polarization state's proinflammatory M1 (classical activation) and immunosuppressive M2 (alternative activation). Molecular signatures of DAM and proinflammatory microglia (highly pro-oxidant) have shown clear differences, yet a partial overlapping gene profile is evident between both phenotypes. The switch activation of homeostatic microglia into reactive microglia relies on the selective activation of key surface receptors involved in the maintenance of brain homeostasis (a.k.a. pattern recognition receptors, PRRs). Two relevant PRRs are toll-like receptors (TLRs) and triggering receptors expressed on myeloid cells-2 (TREM2), whose selective activation is believed to generate either a proinflammatory or a DAM phenotype, respectively. However, the recent identification of endogenous disease-related ligands, which bind to and activate both TLRs and TREM2, anticipates the existence of rather complex microglia responses. Examples of potential endogenous dual ligands include amyloid β, galectin-3, and apolipoprotein E. These pleiotropic ligands induce a microglia polarization that is more complicated than initially expected, suggesting the possibility that different microglia subtypes may coexist. This review highlights the main microglia polarization states under disease conditions and their leading role orchestrating oxidative stress

The Absence of Caspase-8 in the Dopaminergic System Leads to Mild Autism-like Behavior

In the last decade, new non-apoptotic roles have been ascribed to apoptotic caspases. This family of proteins plays an important role in the sculpting of the brain in the early stages of development by eliminating excessive and nonfunctional synapses and extra cells. Consequently, impairments in this process can underlie many neurological and mental illnesses. This view is particularly relevant to dopamine because it plays a pleiotropic role in motor control, motivation, and reward processing. In this study, we analyze the effects of the elimination of caspase-8 (CASP8) on the development of catecholaminergic neurons using neurochemical, ultrastructural, and behavioral tests. To do this, we selectively delete the CASP8 gene in cells that express tyrosine hydroxylase with the help of recombination through the Cre-loxP system. Our results show that the number of dopaminergic neurons increases in the substantia nigra. In the striatum, the basal extracellular level of dopamine and potassium-evoked dopamine release decreased significantly in mice lacking CASP8, clearly showing the low dopamine functioning in tissues innervated by this neurotransmitter. This view is supported by electron microscopy analysis of striatal synapses. Interestingly, behavioral analysis demonstrates that mice lacking CASP8 show changes reminiscent of autism spectrum disorders (ASD). Our research reactivates the possible role of dopamine transmission in the pathogenesis of ASD and provides a mild model of autism.Ministerio de Economía y Competitividad RTI2018-098645-B-I00, PID2019-109569GB-I00, RTI2018-099778-B-I00Junta de Andalucía P18-RT-1372, US-1264806, PI-0080-2017, PI-0009-2017, PI-0134-2018, PEMP-0008-2020, P20_00958, CTS-510Instituto de Salud Carlos III PI18/01691Instituto de Investigación e Innovación en Ciencias Biomédicas de Cádiz-INiBICA LI19/06IN-CO22, IN-C09European Union 95568

Galectin-3 Deletion Reduces LPS and Acute Colitis-Induced Pro-Inflammatory Microglial Activation in the Ventral Mesencephalon

Parkinson’s disease is a highly prevalent neurological disorder for which there is currently no cure. Therefore, the knowledge of risk factors as well as the development of new putative molecular targets is mandatory. In this sense, peripheral inflammation, especially the originated in the colon, is emerging as a predisposing factor for suffering this disease. We have largely studied the pleiotropic roles of galectin-3 in driving microglia-associated immune responses. However, studies aimed at elucidating the role of galectin-3 in peripheral inflammation in terms of microglia polarization are lacking. To achieve this, we have evaluated the effect of galectin-3 deletion in two different models of acute peripheral inflammation: intraperitoneal injection of lipopolysaccharide or gut inflammation induced by oral administration of dextran sodium sulfate. We found that under peripheral inflammation the number of microglial cells and the expression levels of pro-inflammatory mediators take place specifically in the dopaminergic system, thus supporting causative links between Parkinson’s disease and peripheral inflammation. Absence of galectin-3 highly reduced neuroinflammation in both models, suggesting an important central regulatory role of galectin-3 in driving microglial activation provoked by the peripheral inflammation. Thus, modulation of galectin-3 function emerges as a promising strategy to minimize undesired microglia polarization states.This work was supported by grants from the Spanish Ministerio de Ciencia, Innovación y Universidades (RTI 2018-098830-B-I00), from the Consejería de Economía y Conocimiento of Junta de Andalucía (P18-RT-1372 and US-1264806). MJP, MDVC and PGM were supported by a grant from the Junta de Andalucía (CTS 5884) and AEC by an associated post-doctoral grant

Rechallenge in idiosyncratic drug-induced liver injury: An analysis of cases in two large prospective registries according to existing definitions

Los datos sobre la reexposición positiva en la lesión hepática inducida por fármacos (DILI) idiosincrásica son escasos. Apuntamos a analizar la presentación clínica, el resultado y los fármacos asociados con la reexposición positiva en dos registros DILI. Se incluyeron casos de los registros DILI español y latinoamericano. De 1418 pacientes con DILI idiosincrásico, 58 casos tuvieron una nueva exposición positiva (4,1%). Pacientes con positivo la nueva exposición tuvo una duración más corta de la terapia (p=0,001) y latencia (p=0,003). En pacientes con reexposición, los niveles de aspartato transaminasa aumentaron (p=0,026) y mostraron un tiempo prolongado de recuperación (p=0,020), aunque no se observaron diferencias en términos de resultados fatales. El principal fármaco implicado en la reexposición fue amoxicilina-clavulanato (17%). La mayoría de los eventos de reexposición fueron involuntarios (71%). Usando ambos existentes definiciones de reexposición positiva, hubo cuatro casos que cumplieron exclusivamente con los criterios actuales y cinco que sólo cumplen con la definición histórica. Todos los casos de nueva impugnación positiva, independientemente del patrón de daño, cumplió con los criterios de alanina transaminasa (ALT) ≥3 veces el límite superior de lo normal (LSN) y/o fosfatasa alcalina (ALP) ≥2 veces el LSN.Funding for open access charge: Universidad de Málaga / CBU

Prior drug allergies are associated with worse outcome in patients with idiosyncratic drug-induced liver injury: A machine learning approach for risk stratification

The impact of prior drug allergies (PDA) on the clinical features and outcomes of patients who develop idiosyncratic drug-induced liver injury (DILI) is largely unknown. We aimed to assess the clinical presentation and outcomes of DILI patients based on the presence or absence of PDA and explore the association between culprit drugs responsible for DILI and allergy. We analysed a well-vetted cohort of DILI cases enrolled from the Spanish DILI Registry. Bootstrap-enhanced least absolute shrinkage operator procedure was used in variable selection, and a multivariable logistic model was fitted to predict poor outcomes in DILI. Of 912 cases with a first episode of DILI, 61 (6.7%) had documented PDA. Patients with PDA were older (p = 0.009), had higher aspartate aminotransferase (AST) levels (p = 0.047), lower platelet count (p = 0.011) and higher liver-related mortality than those without a history of drug allergies (11% vs. 1.6%, p < 0.001). Penicillin was the most common drug associated with PDA in DILI patients (32%). A model including PDA, nR-based type of liver injury, female sex, AST, total bilirubin, and platelet count showed an excellent performance in predicting poor outcome in patients from the Spanish DILI Registry (area under the ROC curve [AUC] 0.887; 95% confidence interval [CI] 0.794 – 0.981) and the LATINDILI Network (AUC 0.932; 95% CI 0.884 – 0.981). Patients with suspected DILI should be screened for PDA as they would require a close monitoring for early detection of worsening clinical course.Funding for open access charge: Universidad de Málaga/CBUA. This work was supported by grants of Instituto de Salud Carlos III (ISCIII), cofounded by Fondo Europeo de Desarrollo Regional – FEDER, cofounded by European Union (grant number PI21/01248), and by the Agencia Española de Medicamentos y Productos Sanitarios. CIBERehd is funded by ISCIII. HN holds a postdoctoral research contract funded by Junta de Andalucía (POSTDOC_21_00780). JSC holds a Juan Rodés contract (JR21/00066), JMP-B holds a Rio Hortega contract (CM21/00074), and IAA holds a Sara Borrell contract (CD20/00083), funded by ISCIII. This project has received funding from the European Horizon´s research and innovation program HORIZON-HLTH-2022-STAYHLTH-02 under agreement No 101095679. Open Access funding provided thanks to the CRUE-CSIC agreement with Elsevier

Evaluation of diagnostic and prognostic candidate biomarkers in drug-induced liver injury vs. other forms of acute liver damage

Aims Detection and characterization of idiosyncratic drug-induced liver injury (DILI) currently rely on standard liver tests, which are suboptimal in terms of specificity, sensitivity and prognosis. Therefore, DILI diagnosis can be delayed, with important consequences for the patient. In this study, we aimed to evaluate the potential of osteopontin, cytokeratin-18 (caspase-cleaved: ccK18 and total: K18), α-glutathione-S-transferase and microRNA-122 as new DILI biomarkers. Methods Serial blood samples were collected from 32 DILI and 34 non-DILI acute liver injury (ALI) cases and a single sample from 43 population controls without liver injury (HLC) and analysed using enzyme-linked immunosorbent assay (ELISA) or single-molecule arrays. Results All biomarkers differentiated DILI and ALI from HLC with an area under receiver operator characteristic curve (AUC) value of >0.75 but were less efficient in distinguishing DILI from ALI, with ccK18 (0.79) and K18 (0.76) demonstrating highest potential. However, the AUC improved considerably (0.98) for ccK18 when comparing DILI and a subgroup of autoimmune hepatitis cases. Cytokeratin-18, microRNA-122 and α-glutathione-S-transferase correlated well with traditional transaminases, while osteopontin correlated most strongly with the international normalized ratio (INR). Conclusions ccK18 appears promising in distinguishing DILI from autoimmune hepatitis but less so from other forms of acute liver injury. Osteopontin demonstrates prognostic potential with higher levels detected in more severe cases regardless of aetiology.Consejería de Salud y Familia de la Junta de Andalucía, Grant/Award Numbers: PI 0274-2016, P18-RT-3364; Instituto de Salud Carlos III (ISCIII) cofounded by Fondo Europeo de Desarrollo Regional - FEDER, Grant/Award Numbers: PI19/00883, PI18/00901, UMA18-FEDERJA-193; Universidad de Málaga/CBUA for open access charge: Universidad de Málaga / CBUA. Funding for open access charge: Universidad de Málaga / CBU

Incidence and prevalence of acute hepatitis E virus infection in patients with suspected Drug-Induced Liver Injury in the Spanish DILI Registry

Background and Aims: Drug-induced liver injury (DILI) presents with a wide phenotypic spectrum requiring an extensive differential diagnosis. Hepatitis E virus (HEV) is not systematically ruled out during acute hepatitis assessment in Spain. The aims of this study were to establish the role of HEV infection and its phenotypic presentation in patients initially suspected of DILI and to determine the anti-HEV seroprevalence rate. Methods: An analysis of 265 patients with suspected DILI and considered for enrolment in the Spanish DILI Registry and 108 controls with normal liver profiles was undertaken. Anti-HEV Immunoglobulin (Ig) G antibodies were analyzed in serum from all subjects. In those with serum samples extracted within 6 months from liver damage onset (n=144), HEV antigen (Ag) and anti- HEV IgM antibodies were tested in duplicate by ELISA. In addition, RT-PCR was performed externally in 8 patients. Results: Out of 144 patients, 12 (8%) were positive for anti-HEV IgM, mean age 61 years. Underlying hepatic diseases (OR=23.4, p20 folds upper limit of normal (OR=10.9, p=0.002) were associated with the diagnosis of acute hepatitis E. The overall anti-HEV IgG seroprevalence rate was 35%, evenly distributed between patients with suspected DILI (34%), and controls (39%). Conclusions: HEV seroprevalence and acute hepatitis E rates are relatively high in Spain. A search for active HEV infection is therefore advised in patients assessed for suspicion of DILI, particularly in patients with underlying liver diseases and high transaminase levels.The present study has been supported by grants of the Instituto de Salud Carlos III cofounded by Fondo Europeo de Desarrollo Regional FEDER (contract numbers: FIS PI0274-2016, PI-0285- 2016, PI 18-01804, PI 18-00901, PT17/0017/0020, CM17/00243, JR16/00015, B-0002-2019, UMA-18-FEDERJA-193 and by the Agencia Española del Medicamento. SCReN and CIBERehd are funded by Instituto de Salud Carlos III. European Cooperation in Science & Technology (COST) ACTION CA17112 Prospective European Drug-Induced Liver Injury Network, IMI2-Translational Safety Biomarker Pipeline (TransBioLine). The funding sources had no involvement in the study design, in the collection, analysis, and interpretation of data, in the writing of the report or in the decision to submit the manuscript for publicatio