MS-based proteomics and network analysis of Lipotoxicity caused by palmitic acid in normal human astrocytes and the response of tibolone treatment

La exposición crónica a grandes cantidades de ácido palmítico (pal) causa daño celular y muerte, un fenómeno que se conoce como lipotoxicidad. La acumulación de pal en el cerebro induce las características distintivas de algunas enfermedades neurodegenerativas (NDs), y causa daño en los diferentes tipos de células cerebrales; particularmente los astrocitos reaccionan volviéndose reactivos e induciendo un bloqueo inicial de la autofagia que, acompañado de otras alteraciones como la inflamación o el estrés de retículo endoplásmico, disminuye la viabilidad de los astrocitos. Al tener diversas funciones en el sistema nervioso como captación de glutamato, soporte metabólico y estructural para el cerebro, liberación de factores neurotróficos y participación en la respuesta inmune en el cerebro, entre otras, es importante la búsqueda de estrategias que permitan proteger a los astrocitos del efecto lipotóxico de pal. Diferentes compuestos esteroidales han sido estudiados como agentes efectivos en el control de enfermedades inflamatorias, sin embargo, su prescripción es limitada debido a efectos adversos como el aumento de la incidencia de cáncer de útero y mama. Algunos esteroides sintetizados en el sistema nervioso llamados “neuroesteroides”, tienen propiedades neuroprotectoras, que son de particular interés en el tratamiento de enfermedades como demencia, isquemia, trauma cráneo encefálico, epilepsia, enfermedad de Alzheimer (EA) y Parkinson (EP). Debido a estos efectos protectores a nivel cerebral, la investigación se ha centrado en fármacos que generen esos efectos protectores de los estrógenos, pero sin aumentar la incidencia de cáncer de útero y mama, entre estos fármacos se han destacado los (SERMs) por su sigla en inglés Selective Estrogen Receptor Modulators y los (STEARs) por su sigla en inglés Specific Tissue Estrogenic Activity Regulators. Entre estos últimos se resalta la tibolona, un fármaco que cuenta con amplios estudios de sus efectos en la salud y siendo promisorio como agente neuroprotector. A pesar de que existen amplias investigaciones en neurodegeneración y al tener en cuenta que son enfermedades que pueden activar diferentes vías de señalización haciendo necesarios estudios menos reduccionistas para entender estas enfermedades. Hasta la fecha no se tienen suficientes aproximaciones que evalúen desde una perspectiva holística los cambios a nivel de productos funcionales de proteoma que tienen lugar durante el daño lipotóxico por pal, que nos permitan identificar biomarcadores y proteínas o rutas metabólicas como posibles blancos de terapias, o que 11 analicen la respuesta a compuestos que potencialmente tienen actividad neuroprotectora con el objetivo de identificar proteínas y sus rutas metabólicas asociadas, relevantes en el proceso de neuroprotección. Asimismo, en la actualidad son escasos los medicamentos o tratamientos efectivos para los procesos neurodegenerativos típicos de estas lesiones, y más allá, es limitado el entendimiento del daño lipotóxico y la respuesta protectora de la tibolona en los diferentes tipos de células del cerebro (neuronas, oligodendrocitos, microglía y astrocitos), haciendo relevante su estudio como posible agente neuroprotector ante un daño lipotóxico inducido por pal. En general, existe la necesidad de mejorar la calidad de vida de dichos pacientes, aliviar los costos asociados tanto a los diferentes proveedores de salud y las familias que asumen gran parte de los cuidados y del soporte económico. Considerando este panorama que impone un claro problema de investigación, en este estudio se realizó un análisis proteómico exhaustivo del daño lipotóxico desencadeno por pal y la evaluación del efecto neuroprotector del esteroide sintético tibolona, en una línea celular de astrocitos humanos. Los resultados indican la expresión diferencial de proteínas no consideradas anteriormente como las principales causas de daño por pal en los astrocitos humanos. También, se identificaron 27 proteínas expresadas diferencialmente con alto grado de conectividad. A partir del desarrollo del presente estudio, se encontró que pal alteró la expresión de proteínas vinculadas a la síntesis y transporte de proteínas, respuesta viral, y la tibolona moduló la expresión de algunas de esas proteínas a niveles similares a los del control, evito disrupciones de la autofagia y redujo la expresión de proteínas vinculadas a señales proapoptóticas, demostrando así que la tibolona podría revertir algunas de las alteraciones a nivel de proteínas generada por pal en los astrocitos humanos.ColcienciasPontificia Universidad JaverianaChronic exposure to large amounts of palmitic acid (pal) causes cell damage and death, a phenomenon known as lipotoxicity. The accumulation of pal in the brain induces the distinctive characteristics of some neurodegenerative diseases (NDs) and causes damage in the different types of brain cells; particularly astrocytes that become reactive and inducing an initial block of autophagy that, accompanied by other alterations such as inflammation or endoplasmic reticulum stress, decrease the viability of astrocytes. As it has various functions in the nervous system such 12 as glutamate uptake, metabolic and structural support for the brain, the release of neurotrophic factors, and participation in the immune response in the brain, among others, it is important to search for strategies to protect astrocytes of the lipotoxic effect of pal. Different steroidal compounds have been studied as effective agents in the control of inflammatory diseases, however, their prescription is limited due to adverse effects such as the increase in the incidence of uterine and breast cancer. Some steroids synthesized in the nervous system called "neurosteroids" have neuroprotective properties, which are of particular interest in the treatment of diseases such as dementia, ischemia, head trauma, epilepsy, Alzheimer's disease (AD), and Parkinson's disease (PD). Due to these protective effects at the brain level, research has focused on drugs that generate these protective effects of estrogens, but without increasing the incidence of uterine and breast cancer, among these drugs the Selective Estrogen Receptor Modulators (SERMs) and the Specific Tissue Estrogenic Activity Regulators (STEARs) have been highlighted. Among the latter, tibolone stands out, a drug that has extensive studies of its effects on health and is promising as a neuroprotective agent. Even though there is extensive research in neurodegeneration and considering that they are diseases that can activate different signaling pathways, making less reductionist studies necessary to understand these diseases. To date, there are not enough approaches that evaluate from a holistic perspective the changes at the level of functional proteome products that take place during lipotoxic damage by pal, which allow us to identify biomarkers and proteins or metabolic pathways as possible targets for therapies, or that analyze the response to compounds that potentially have a neuroprotective activity to identify proteins and their associated metabolic pathways, relevant in the neuroprotection process. Likewise, effective drugs or treatments for the neurodegenerative processes typical of these lesions are currently scarce, and the understanding of lipotoxic damage and the protective response of tibolone in different types of brain cells (neurons, oligodendrocytes, microglia, and astrocytes), making its study relevant as a possible neuroprotective agent against lipotoxic damage induced by pal. In general, there is a need to improve the quality of life of these patients, alleviate the costs associated with both the different health providers and the families that assume a large part of the 13 care and financial support. Considering this panorama that imposes a clear research problem, in this study an exhaustive proteomic analysis of the lipotoxic damage triggered by pal and the evaluation of the neuroprotective effect of the synthetic steroid tibolone, in a human astrocyte cell line, was carried out. The results indicate the differential expression of proteins not previously considered as the main causes of pal damage in human astrocytes. Also, 27 differentially expressed proteins with a high degree of connectivity were identified. From the development of the present study, it was found that pal altered the expression of proteins linked to protein synthesis and transport, viral response, and tibolone modulated the expression of some of these proteins at levels similar to those of the control, avoiding disruptions of autophagy and reduced the expression of proteins linked to pro-apoptotic signals, thus demonstrating that tibolone could reverse some of the protein-level alterations generated by pal in human astrocytes.Magíster en Ciencias BiológicasMaestríahttps://orcid.org/0000-0003-1531-3862https://scholar.google.com/citations?user=cRxUANEAAAAJ&hl=eshttps://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=000165624

Neuroprotective role of hypothermia in hypoxic-ischemic brain injury: Combined therapies using estrogen

Hypoxic-ischemic brain injury is a complex network of factors, which is mainly characterized by a decrease in levels of oxygen concentration and blood flow, which lead to an inefficient supply of nutrients to the brain. Hypoxic-ischemic brain injury can be found in perinatal asphyxia and ischemic-stroke, which represent one of the main causes of mortality and morbidity in children and adults worldwide. Therefore, knowledge of underlying mechanisms triggering these insults may help establish neuroprotective treatments. Selective Estrogen Receptor Modulators and Selective Tissue Estrogenic Activity Regulators exert several neuroprotective effects, including a decrease of reactive oxygen species, maintenance of cell viability, mitochondrial survival, among others. However, these strategies represent a traditional approach of targeting a single factor of pathology without satisfactory results. Hence, combined therapies, such as the administration of therapeutic hypothermia with a complementary neuroprotective agent, constitute a promising alternative. In this sense, the present review summarizes the underlying mechanisms of hypoxic-ischemic brain injury and compiles several neuroprotective strategies, including Selective Estrogen Receptor Modulators and Selective Tissue Estrogenic Activity Regulators, which represent putative agents for combined therapies with therapeutic hypothermia.Fil: Toro Urrego, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Vesga Jiménez, Diego Julián. Pontificia Universidad Javeriana; ColombiaFil: Herrera, María Inés. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Psicología y Psicopedagogía. Centro de Investigaciones en Psicología y Psicopedagogía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Luaces, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Capani, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina. Universidad Argentina "John F. Kennedy"; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Psicología y Psicopedagogía; Argentina. Universidad Autónoma de Chile; Chil

Tibolone pre-treatment ameliorates the dysregulation of protein translation and transport generated by palmitic acid-induced lipotoxicity in human astrocytes: A label-free MS-based proteomics and network analysis

Excessive accumulation and release of fatty acids (FAs) in adipose and non-adipose tissue are characteristic of obesity and are associated with the leading causes of death worldwide. Chronic exposure to high concentrations of FAs such as palmitic acid (pal) is a risk factor for developing different neurodegenerative diseases (NDs) through several mechanisms. In the brain, astrocytic dysregulation plays an essential role in detrimental processes like metabolic inflammatory state, oxidative stress, endoplasmic reticulum stress, and autophagy impairment. Evidence shows that tibolone, a synthetic steroid, induces neuroprotective effects, but its molecular mechanisms upon exposure to pal remain largely unknown. Due to the capacity of identifying changes in the whole data-set of proteins and their interaction allowing a deeper understanding, we used a proteomic approach on normal human astrocytes under supraphysiological levels of pal as a model to induce cytotoxicity, finding changes of expression in proteins related to translation, transport, autophagy, and apoptosis. Additionally, tibolone pre-treatment showed protective effects by restoring those same pal-altered processes and increasing the expression of proteins from cell survival processes. Interestingly, ARF3 and IPO7 were identified as relevant proteins, presenting a high weight in the protein-protein interaction network and significant differences in expression levels. These proteins are related to transport and translation processes, and their expression was restored by tibolone. This work suggests that the damage caused by pal in astrocytes simultaneously involves different mechanisms that the tibolone can partially revert, making tibolone interesting for further research to understand how to modulate these damages. </p