Papel del componente 3 del complemento (C3) como mediador de C/EBPβ en los procesos inflamatorios y excitotóxicos del sistema nervioso central

Tesis Doctoral presentada por Mª Elena Hernández Encinas, licenciado en Biología, para optar al grado de Doctor por la Universidad Autónoma de Madrid, y realizada en el Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-IIBM).-- Esta obra está bajo una licencia Creative Commons Reconocimiento-No Comercial-Sin Obra Derivada 4.0 Internacional.[ES]: CCAAT/Enhancer-binding protein (C/EBP ) es un factor de transcripción que inicialmente fue identificado como regulador de procesos de diferenciación e inflamación principalmente en tejido adiposo e hígado. Sin embargo, su función en el cerebro ha sido descrita más recientemente. Estudios previos de nuestro laboratorio revelaron que C/EBPβ estaba implicada en procesos de inflamación y daño cerebral, y que los ratones carentes de este gen eran menos susceptibles al daño excitotóxico inducido por ácido kaínico. Con el fin de analizar los mecanismos a través de los cuales C/EBPβ regula estos procesos, lo primero que hicimos fue un análisis de microarrays con RNA aislado de hipocampo de ratones C/EBP +/+ y C/EBP -/- . Algunos de los genes expresados diferencialmente, eran genes implicados en inflamación y daño cerebral. Uno de esos genes es el del componente 3 del complemento (C3), cuya expresión aumenta en los ratones C/EBPβ+/+ y que está implicado en diversos desórdenes cerebrales. En este trabajo nos hemos centrado en el posible papel de C3 como mediador de C/EBP tras un daño excitotóxico e inflamatorio. Estudios de transfección con el promotor de C3 y estudios in vitro de daño neural, mostraron que C/EBPβ regula directamente la expresión de C3 tras un daño excitotóxico e inflamatorio. En este trabajo mostramos también que la reducción de C3 mediante RNA de interferencia, reduce la producción de agentes proinflamatorios como IL-1β y COX-2, así como la de nitritos en cultivos primarios de astrocitos tratados con lipopolisacárido. Usando un modelo in vivo de excitotoxicidad mostramos que ambas proteínas aumentan principalmente en la región CA3 del hipocampo. Por último, demostramos que ratones carentes de C/EBPβ presentan una importante reducción de la expresión de C3 tras la inyección de ácido kaínico. Estos resultados sugieren que C/EBPβ podría estar implicada en enfermedades neurológicas vinculadas a procesos inflamatorios y excitotóxicos, al menos en parte a través de la regulación directa de C3.[EN]: The CCAAT/Enhancer-binding protein (C/EBP ) is a transcription factor, which was first identified as a regulator of differentiation and inflammatory processes mainly in adipose tissue and liver; however its function in the brain was largely unknown for many years. Previous studies from our laboratory indicated that C/EBP is implicated in inflammatory process and brain injury, since mice lacking this gene were less susceptible to kainic acid-induced injury. In order to further analyze the mechanisms by which C/EBP was involved in these processes, we first performed cDNA microarrays analysis using hippocampal RNA isolated from C/EBP +/+ and C/EBP -/- mice. Among the genes displaying significant changes in expression, some of them were involved in inflammatory processes and brain injury. One of those genes which expression was increased in C/EBPβ +/+ animals was complement component 3 (C3), a gene implicated in different brain disorders. This work has been focused in the possible involvement of C3 in the effects of C/EBPβ in excitotoxic injury. Transfection studies using C3 promoter and in vitro studies of neural damage, demonstrated that C/EBPβ directly regulates the expression of C3 following an excitotoxic and inflammatory injuries. In addition, we show that downregulation of C3 by siRNA reduces proinflammatory molecules such us IL-1β and COX-2 and leads to a decrease of nitrite levels in an astrocyte culture after lipopolysaccharide treatment. By using an in vivo model of excitotocicity we demonstrate an increase of C/EBP and C3 in the CA3 subfield of the hippocampus. Finally, we show that mice lacking C/EBP exhibit a marked reduction in C3 expression after kainic acid injection. These results suggest that C/EBPβ could regulate brain disorders, in which excitotocic and inflammatory processes are involved, at least in part through the direct regulation of C3.Gracias al Consejo Superior de Investigaciones Científicas por haberme concedido la beca predoctoral I3P.Peer Reviewe

CCAAT/enhancer binding protein β directly regulates the expression of the complement component 3 gene in neural cells: implications for the pro-inflammatory effects of this transcription factor

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.[Background]: The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor, which was first identified as a regulator of differentiation and inflammatory processes mainly in adipose tissue and liver; however, its function in the brain was largely unknown for many years. Previous studies from our laboratory indicated that C/EBPβ is implicated in inflammatory process and brain injury, since mice lacking this gene were less susceptible to kainic acid-induced injury. [Methods]: We first performed cDNA microarrays analysis using hippocampal RNA isolated from C/EBPβ+/+ and C/EBPβ−/− mice. Immunocytochemical and immunohistochemical studies were done to evaluate C/EBPβ and C3 levels. Transient transfection experiments were made to analyze transcriptional regulation of C3 by C/EBPβ. To knockdown C/EBPβ and C3 expression, mouse astrocytes were infected with lentiviral particles expressing an shRNA specific for C/EBPβ or an siRNA specific for C3. [Results]: Among the genes displaying significant changes in expression was complement component 3 (C3), which showed a dramatic decrease in mRNA content in the hippocampus of C/EBPβ−/− mice. C3 is the central component of the complement and is implicated in different brain disorders. In this work we have found that C/EBPβ regulates C3 levels in rodents glial in vitro and in the rat Substantia nigra pars compacta (SNpc) in vivo following an inflammatory insult. Analysis of the mouse C3 promoter showed that it is directly regulated by C/EBPβ through a C/EBPβ consensus site located at position −616/-599 of the gene. In addition, we show that depletion of C/EBPβ by a specific shRNA results in a significant decrease in the levels of C3 together with a reduction in the increased levels of pro-inflammatory agents elicited by lipopolysaccharide treatment. [Conclusions]: Altogether, these results indicate that C3 is a downstream target of C/EBPβ, and it could be a mediator of the pro-inflammatory effects of this transcription factor in neural cells.This work was supported by the Ministry of Science and Innovation Grant SAF2010-16365. JAMG was supported by CIBERNED.We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

The spectrum of intermediate SCN8A-related epilepsy

Objective: Pathogenic variants in SCN8A have been associated with a wide spectrum of epilepsy phenotypes, ranging from benign familial infantile seizures (BFIS) to epileptic encephalopathies with variable severity. Furthermore, a few patients with intellectual disability (ID) or movement disorders without epilepsy have been reported. The vast majority of the published SCN8A patients suffer from severe developmental and epileptic encephalopathy (DEE). In this study, we aimed to provide further insight on the spectrum of milder SCN8A-related epilepsies. Methods: A cohort of 1095 patients were screened using a next generation sequencing panel. Further patients were ascertained from a network of epilepsy genetics clinics. Patients with severe DEE and BFIS were excluded from the study. Results: We found 36 probands who presented with an SCN8A-related epilepsy and normal intellect (33%) or mild (61%) to moderate ID (6%). All patients presented with epilepsy between age 1.5 months and 7 years (mean = 13.6 months), and 58% of these became seizure-free, two-thirds on monotherapy. Neurological disturbances included ataxia (28%) and hypotonia (19%) as the most prominent features. Interictal electroencephalogram was normal in 41%. Several recurrent variants were observed, including Ile763Val, Val891Met, Gly1475Arg, Gly1483Lys, Phe1588Leu, Arg1617Gln, Ala1650Val/Thr, Arg1872Gln, and Asn1877Ser. Significance: With this study, we explore the electroclinical features of an intermediate SCN8A-related epilepsy with mild cognitive impairment, which is for the majority a treatable epilepsy.Peer reviewe

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Silencing phosphodiesterase 7B gene by lentiviral-shRNA interference attenuates neurodegeneration and motor deficits in hemiparkinsonian mice

42 p.-10 fig.Different studies have suggested that the nucleotide cyclic adenosine 3’, 5’- monophosphate (cAMP) can actively play an important role as neuroprotective and antiinflammatory agent after a brain injury. The phosphodiesterase 7 (PDE7) enzyme is one of the enzymes responsible for controlling specifically the intracellular levels of cAMP in the immune and central nervous systems. Therefore this enzyme could play an important role in brain inflammation and neurodegeneration. In this regard, using different chemical inhibitors of PDE7 we have demonstrated their neuroprotective and anti-inflammatory activity in different models of neurodegenerative disorders, including Parkinson disease (PD). In the present study, we have used the toxin 6- hydroxydopamine (6-OHDA) and lipopolysaccharide (LPS) to model PD and explore the protective effects of PDE7B deficiency in dopaminergic neurons cell death. Lentivirus-mediated PDE7B deprivation conferred marked in vitro and in vivo neuroprotection against 6-OHDA and LPS toxicity in dopaminergic neurons, and preserved motor function involving the dopamine system in mouse. Our results substantiate previous data and provide a validation of PDE7B enzyme as a valuable new target for therapeutic development in the treatment of PD.The authors gratefully acknowledge the financial support of MICINN (Grant SAF2010-16365, to A.P-C and GrantSAF2012-33600, to C.G.). CIBERNED is funded by the Instituto de Salud Carlos III.Peer reviewe

The mTOR pathway is necessary for survival of mice with short telomeres

Telomerase deficiency leads to age-related diseases and shorter lifespans. Inhibition of the mechanistic target of rapamycin (mTOR) delays aging and age-related pathologies. Here, we show that telomerase deficient mice with short telomeres (G2-Terc-/-) have an hyper-activated mTOR pathway with increased levels of phosphorylated ribosomal S6 protein in liver, skeletal muscle and heart, a target of mTORC1. Transcriptional profiling confirms mTOR activation in G2-Terc-/- livers. Treatment of G2-Terc-/- mice with rapamycin, an inhibitor of mTORC1, decreases survival, in contrast to lifespan extension in wild-type controls. Deletion of mTORC1 downstream S6 kinase 1 in G3-Terc-/- mice also decreases longevity, in contrast to lifespan extension in single S6K1-/- female mice. These findings demonstrate that mTOR is important for survival in the context of short telomeres, and that its inhibition is deleterious in this setting. These results are of clinical interest in the case of human syndromes characterized by critically short telomeres.We thank Sara Kozma for kindly sharing the S6K1-/- mouse. We thank A. Efeyan and A. Ortega for helpful discussions. We are indebted to D. Megias for confocal microscopy analysis. Research in the Blasco Lab is funded by the Spanish Ministry of Economy and Competitiveness Projects (SAF2013-45111-R and SAF2015-72455-EXP), the Comunidad de Madrid Project (S2017/BMD-3770), the World Cancer Research (WCR) Project (16-1177), and the Fundacion Botin (Spain).S

Additional file 1: of Complement component 3 (C3) expression in the hippocampus after excitotoxic injury: role of C/EBPβ

Double-immunofluorescence analysis showing C3 expression near microglia and blood vessels 72 h after KA injection in adult rats. All images represent the maximum intensity projection, and orthogonal views are also shown, generated by projecting z-series in the x- and y-planes. Scale bar, 5 μm. (TIF 2327 kb

CCAAT/Enhancer binding protein β silencing mitigates glial activation and neurodegeneration in a rat model of Parkinson’s disease

The CCAAT/Enhancer binding protein β (C/EBPβ) is a transcription factor involved in numerous physiological as well as pathological conditions in the brain. However, little is known regarding its possible role in neurodegenerative disorders. We have previously shown that C/EBPβ regulates the expression of genes involved in inflammatory processes and brain injury. Here, we have analyzed the effects of C/EBPβ interference in dopaminergic cell death and glial activation in the 6-hydroxydopamine model of Parkinson’s disease. Our results showed that lentivirus-mediated C/EBPβ deprivation conferred marked in vitro and in vivo neuroprotection of dopaminergic cells concomitant with a significant attenuation of the level of the inflammatory response and glial activation. Additionally, C/EBPβ interference diminished the induction of α-synuclein in the substantia nigra pars compacta of animals injected with 6-hydroxydopamine. Taking together, these results reveal an essential function for C/EBPβ in the pathways leading to inflammatory-mediated brain damage and suggest novel roles for C/EBPβ in neurodegenerative diseases, specifically in Parkinson’s disease, opening the door for new therapeutic interventions.This work was supported by MINECO (Grants SAF2010-16365 and SAF2014-52940-R to A.P-C) and MINECO (SAF-2012-34556 to J.G-C), and partially financed with FEDER funds. CIBERNED is funded by the Instituto de Salud Carlos III. J.A.M-G. is a post-doctoral fellow from CIBERNED.Peer reviewe