Avances en el diagnóstico y en el conocimiento genético de la enfermedad de Niemann Pick tipo C

La enfermedad de Niemann Pick tipo C (NP-C), es una enfermedad de herencia recesiva y con una baja incidencia poblacional, por lo que se encuentra englobada entre las enfermedades denominadas raras. Se debe a la presencia de variantes asociadas a patogenicidad en los genes NPC1 o NPC2, que codifican para proteínas lisosomales, la primera transmembrana y la segunda una proteína soluble. La principal función de éstas es el transporte del colesterol no esterificado fuera del lisosoma, de modo que una disfunción, provoca su acúmulo en el interior de este orgánulo y en consecuencia la acumulación de otros sustratos y el mal funcionamiento celular. La gran heterogeneidad fenotípica que presenta esta enfermedad, así como la complejidad del estudio de los genes causantes hace muy complejo el diagnóstico de una enfermedad que si no se trata de forma temprana provoca un rápido deterioro neurológico.Esta tesis doctoral se centra en implementar nuevos sistemas para mejorar el diagnóstico de la enfermedad y en la caracterización genética de sujetos con sospecha de la enfermedad, además de buscar otros mecanismos que puedan justificar la aparición de sintomatología en sujetos portadores de una única variante patogénica en heterozigosidad.En base a esto, se ha implementado el test de resistencia osmótica eritrocitaria como un nuevo método rápido, económico y fácil para descartar rápidamente la enfermedad en sujetos con sospecha, además de valorar la capacidad combinada de tres biomarcadores para orientar el diagnóstico. A nivel molecular se han detectado variantes no asociadas a patogenicidad no descritas en población española, así como una alteración en la frecuencia de la variante patogénica más común en población europea. El estudio ha permitido clasificar seis variantes de significado incierto como benignas y describir 5 nuevas variantes asociadas a patogenicidad, pero no ha detectado otros genes asociados a la expresión fenotípica de la NP-C o alteraciones en la secuencia del miRNA que regula la expresión del gen NPC1.<br /

Circulating extracellular vesicle proteins and microRNA profiles in subcortical and cortical-subcortical ischaemic stroke

In order to investigate the role of circulating extracellular vesicles (EVs), proteins, and microRNAs as damage and repair markers in ischaemic stroke depending on its topography, subcor-tical (SC), and cortical-subcortical (CSC) involvement, we quantified the total amount of EVs using an enzyme-linked immunosorbent assay technique and analysed their global protein content using proteomics. We also employed a polymerase chain reaction to evaluate the circulating microRNA profile. The study included 81 patients with ischaemic stroke (26 SC and 55 CSC) and 22 healthy controls (HCs). No differences were found in circulating EV levels between the SC, CSC, and HC groups. We detected the specific expression of C1QA and Casp14 in the EVs of patients with CSC ischaemic stroke and the specific expression of ANXA2 in the EVs of patients with SC involvement. Patients with CSC ischaemic stroke showed a lower expression of miR-15a, miR-424, miR-100, and miR-339 compared with those with SC ischaemic stroke, and the levels of miR-339, miR-100, miR-199a, miR-369a, miR-424, and miR-15a were lower than those of the HCs. Circulating EV proteins and microRNAs from patients with CSC ischaemic stroke could be considered markers of neurite outgrowth, neurogenesis, inflammation process, and atherosclerosis. On the other hand, EV proteins and microRNAs from patients with SC ischaemic stroke might be markers of an anti-inflammatory process and blood–brain barrier disruption reduction.This work was sponsored by a grant from Miguel Servet (CP15/00069; CPII20/00002 to María Gutiérrez-Fernández), Miguel Servet (CP20/00024 to Laura Otero-Ortega), a predoctoral fellowship (FI17/00188 to Mari Carmen Gómez-de Frutos; FI18/00026 to Fernando Laso-García), a Sara Borrell postdoctoral fellowship (CD19/00033 to María Pérez-Mato), a Río Hortega (CM20/00047 to Elisa Alonso-López) and the INVICTUS PLUS network grant (RD16/0019/0005) from the Carlos III Health Institute Health Care Research Fund and was co-funded by the European Regional Development Fund (ERDF)

Similarities and differences in extracellular vesicle profiles between ischaemic stroke and myocardial infarction

Extracellular vesicles (EVs) are involved in intercellular signalling through the transfer of molecules during physiological and pathological conditions, such as ischaemic disease. EVs might therefore play a role in ischaemic stroke (IS) and myocardial infarction (MI). In the present study, we analysed the similarities and differences in the content of circulating EVs in patients with IS and MI. This prospective observational study enrolled 140 participants (81 patients with IS, 37 with MI and 22 healthy controls [HCs]). We analysed the protein and microRNA content from EVs using proteomics and reverse transcription quantitative real-time polymerase chain reaction and compared it between the groups. In the patients with IS and MI, we identified 14 common proteins. When comparing IS and MI, we found differences in the protein profiles (apolipoprotein B, alpha-2-macroglobulin, fibronectin). We also found lower levels of miR-340 and miR-424 and higher levels of miR-29b in the patients with IS and MI compared with the HCs. Lastly, we found higher miR-340 levels in IS than in MI. In conclusion, proteomic and miRNA analyses suggest a relationship between circulating EV content and the patient’s disease state. Although IS and MI affect different organs (brain and heart) with distinct histological characteristics, certain EV proteins and miRNAs appear to participate in both diseases, while others are present only in patients with ISThis work was sponsored by a grant from Miguel Servet (CP15/00069 to María Gutiérrez- Fernández), a predoctoral fellowship (FI17/00188 to Mari Carmen Gómez-de Frutos;FI18/00026 to Fernando Laso-García), a Sara Borrell postdoctoral fellowship (CD19/00033 to María Pérez-Mato), the INVICTUS PLUS network grant (RD16/0019/0005) from the Carlos III Health Institute Health Care Research Fund and was co-funded by the European Regional Development Fund (ERDF) and Juan de la Cierva postdoctoral fellowship (IJCI-2017-33505 to Laura Otero-Ortega, Spanish State Research Agency) and the Spanish Ministry of Science and Innovatio

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Ibeta pathway activation

15 p.Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs.We have demonstrated that H-rasgene deletion produces mice hypotensionviaa soluble guanylate cyclase-proteinkinase G (PKG)–dependent mechanism. In this study, we analyzed the consequences of H-rasdeletiononcardiacremodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Leftventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H-ras2/2) and control wild-type (H-ras+/+) mice, as were extracellular matrix proteinexpression. Increased cardiac PKG-Ibprotein expression in H-ras2/2mice suggests the involvement of this proteinin heart protection.Ex vivoexperiments on cardiac explants could support this mechanism, as PKG blockadeblunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H-ras2/2mice. Geneticmodulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3b-dependent activation ofthe transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iboverexpressionin H-ras2/2mouse embryonic fibroblasts. This study demonstrates that H-rasdeletion protects against AngII-induced cardiac remodeling, possiblyviaa mechanism in which PKG-Iboverexpression could play a partial role, andpoints to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.Instituto de Salud Carlos IIIUniversidad de AlcaláFundación SenefroFEDE

Brain and immune system-derived extracellular vesicles mediate regulation of complement system, extracellular matrix remodeling, brain repair and antigen tolerance in Multiple sclerosis

Background: Multiple sclerosis (MS) is an immune-mediated central nervous system disease whose course is unpredictable. Finding biomarkers that help to better comprehend the disease's pathogenesis is crucial for supporting clinical decision-making. Blood extracellular vesicles (EVs) are membrane-bound particles secreted by all cell types that contain information on the disease's pathological processes. Purpose: To identify the immune and nervous system-derived EV profile from blood that could have a specific role as biomarker in MS and assess its possible correlation with disease state. Results: Higher levels of T cell-derived EVs and smaller size of neuron-derived EVs were associated with clinical relapse. The smaller size of the oligodendrocyte-derived EVs was related with motor and cognitive impairment. The proteomic analysis identified mannose-binding lectin serine protease 1 and complement factor H from immune system cell-derived EVs as autoimmune disease-associated proteins. We observed hepatocyte growth factor-like protein in EVs from T cells and inter-alpha-trypsin inhibitor heavy chain 2 from neurons as white matter injury-related proteins. In patients with MS, a specific protein profile was found in the EVs, higher levels of alpha-1-microglobulin and fibrinogen β chain, lower levels of C1S and gelsolin in the immune system-released vesicles, and Talin-1 overexpression in oligodendrocyte EVs. These specific MS-associated proteins, as well as myelin basic protein in oligodendrocyte EVs, correlated with disease activity in the patients with MS. Conclusion: Neural-derived and immune-derived EVs found in blood appear to be good specific biomarkers in MS for reflecting the disease state.This work was sponsored by a grant from Miguel Servet (CP20/00024 to Laura Otero-Ortega), Miguel Servet (CPII20/00002 to María Gutiérrez-Fernández), a predoctoral fellowship (FI18/00026 to Fernando Laso-García), a Río-Hortega grant (CM22/00065 to Gabriel Torres Iglesias and CM20/00047 to Elisa Alonso-López) and by Research Project (PI21/00918) from the Instituto de Salud Carlos III and co-funded by the European Union and by a grant CA1/RSUE/2021-00753 to Dolores Piniella funded by Ministerio de Universidades, Plan de Recuperación, Transformación y Resiliencia y la Universidad Autónoma de Madrid.S

Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase

Soluble Transferrin Receptor as Iron Deficiency Biomarker: Impact on Exercise Capacity in Heart Failure Patients

The soluble transferrin receptor (sTfR) is a marker of tissue iron status, which could indicate an increased iron demand at the tissue level. The impact of sTfR levels on functional capacity and quality of life (QoL) in non-anemic heart failure (HF) patients with otherwise normal systemic iron status has not been evaluated. We conducted an observational, prospective, cohort study of 1236 patients with chronic HF. We selected patients with normal hemoglobin levels and normal systemic iron status. Tissue iron deficiency (ID) was defined as levels of sTfR > 75th percentile (1.63 mg per L). The primary endpoints were the distance walked in the 6 min walking test (6MWT) and the overall summary score (OSS) of the Minnesota Living with Heart Failure Questionnaire (MLHFQ). The final study cohort consisted of 215 patients. Overall QoL was significantly worse (51 +/- 27 vs. 39 +/- 20, p-value = 0.006, respectively), and the 6 MWT distance was significantly worse in patients with tissue ID when compared to patients without tissue ID (206 +/- 179 m vs. 314 +/- 155, p-value < 0.0001, respectively). Higher sTfR levels, indicating increased iron demand, were associated with a shorter distance in the 6 MWT (standardized beta = -0.249, p < 0.001) and a higher MLHFQ OSS (standardized fi = 0.183, p-value = 0.008). In this study, we show that in patients with normal systemic iron parameters, higher levels of sTfR are strongly associated with an impaired submaximal exercise capacity and with worse QoL

Protein content of blood-derived extracellular vesicles: An approach to the pathophysiology of cerebral hemorrhage

Introduction: Extracellular vesicles (EVs) participate in cell-to-cell paracrine signaling and can be biomarkers of the pathophysiological processes underlying disease. In intracerebral hemorrhage, the study of the number and molecular content of circulating EVs may help elucidate the biological mechanisms involved in damage and repair, contributing valuable information to the identification of new therapeutic targets.Methods: The objective of this study was to describe the number and protein content of blood-derived EVs following an intracerebral hemorrhage (ICH). For this purpose, an experimental ICH was induced in the striatum of Sprague-Dawley rats and EVs were isolated and characterized from blood at baseline, 24 h and 28 days. The protein content in the EVs was analyzed by mass spectrometric data-dependent acquisition; protein quantification was obtained by sequential window acquisition of all theoretical mass spectra data and compared at pre-defined time points.Results: Although no differences were found in the number of EVs, the proteomic study revealed that proteins related to the response to cellular damage such as deubiquitination, regulation of MAP kinase activity (UCHL1) and signal transduction (NDGR3), were up-expressed at 24 h compared to baseline; and that at 28 days, the protein expression profile was characterized by a higher content of the proteins involved in healing and repair processes such as cytoskeleton organization and response to growth factors (COR1B) and the regulation of autophagy (PI42B).Discussion: The protein content of circulating EVs at different time points following an ICH may reflect evolutionary changes in the pathophysiology of the disease