Browsing by Author "Cueto Ureña, Cristina"

Background: Multiple myeloma (MM) is the second most common hematologic neoplasm which is characterized by proliferation and infiltration of plasmatic cells in the bone marrow. Currently, MM is considered incurable due to resistance to treatment. The CRISPR/Cas9 system has emerged as a powerful tool for understanding the role of different genetic alterations in the pathogenesis of hematologic malignancies in both cell lines and mouse models. Despite current advances of gene editing tools, the use of CRISPR/Cas9 technology for gene editing of MM have not so far been extended. In this work, we want to repress Rnd3 expression, an atypical Rho GTPase involved in several cellular processes, in MM cell lines using a CRISPR interference strategy. Results: We have designed different guide RNAs and cloning them into a lentiviral plasmid, which contains all the machinery necessary for developing the CRISPR interference strategy. We co-transfected the HEK 293T cells with this lentiviral plasmid and 3rd generation lentiviral envelope and packaging plasmids to produce lentiviral particles. The lentiviral particles were used to transduce two different multiple myeloma cell lines, RPMI 8226 and JJN3, and downregulate Rnd3 expression. Additionally, the impact of Rnd3 expression absence was analyzed by a transcriptomic analysis consisting of 3' UTR RNA sequencing. The Rnd3 knock-down cells showed a different transcriptomic profile in comparison to control cells. Conclusions: We have developed a CRISPR interference strategy to generate stable Rnd3 knockdown MM cell lines by lentiviral transduction. We have evaluated this strategy in two MM cell lines, and we have demonstrated that Rnd3 silencing works both at transcriptional and protein level. Therefore, we propose CRISPR interference strategy as an alternative tool to silence gene expression in MM cell lines. Furthermore, Rnd3 silencing produces changes in the cellular transcriptomic profile.

Autofagia es el proceso por el cual se media el direccionamiento de material citoplasmático (proteínas y orgánulos) hacia los lisosomas para su posterior degradación. Su correcto funcionamiento es clave para mantener la homeostasis celular y controlar los niveles y la calidad de los constituyentes intracelulares, contribuyendo a la obtención de energía y permitiendo la adaptación de la célula a condiciones de estrés. Se ha descrito que un mal funcionamiento en el proceso de autofagia está relacionado con numerosas enfermedades neurodegenerativas (Parkinson, Alzhéimer o esclerosis lateral amiotrófica), siendo una característica común la presencia de agregados proteicos u orgánulos disfuncionales, que, en parte, acaban explicando la aparición de los distintos síntomas clínicos. Las proteínas Rho, implicadas en la regulación de la dinámica del citoesqueleto de actina, juegan un papel clave en el desarrollo y función del sistema nervioso central. Rnd3/RhoE es un miembro de la subfamilia Rnd y se caracteriza por estar constitutivamente activada. Su función en migración, proliferación y supervivencia celular ha sido ampliamente estudiada. Rnd3 es necesario para el correcto desarrollo y funcionamiento del SNC. En nuestro grupo, la utilización de un modelo de ratón genéticamente modificado (Rnd3gt/gt) ha demostrado que la ausencia de esta proteína produce graves alteraciones motoras y de desarrollo neural, destacando la presencia de agregados proteicos y mitocondrias morfológicamente aberrantes en neuronas, un fenotipo compatible con problemas en el proceso de autofagia, tal y como se ha descrito en algunas patologías neurodegenerativas. En este estudio nos planteamos analizar el posible papel de Rnd3 en autofagia, con el objetivo de poder entender alguno de los fenotipos que aparecen en el modelo murino deficiente en esta proteína. Nuestros resultados demuestran que la ausencia de Rnd3 aumenta la tasa de degradación proteica y provoca un aumento en autofagia en condiciones basales, sugiriendo que Rnd3 es un regulador negativo de este proceso, actuando a nivel de la maduración del autofagosoma. Además, hemos demostrado un nuevo papel de Rnd3 en la homeostasis mitocondrial. La presencia de mitocondrias no funcionales en células deficientes para Rnd3 provoca una alteración del metabolismo oxidativo y una activación de la glucólisis como mecanismo alternativo para la obtención de energía. Todos estos resultados muestran un nuevo papel de Rnd3 en la regulación de autofagia, así como en la homeostasis mitocondrial, lo que podría explicar algunos de los fenotipos observados en el modelo de ratón deficiente para Rnd3. / Autophagy is the cellular process by which lysosomes contribute to the degradation of intracellular components, including proteins and organelles. The correct function of this catabolic process is necessary for cell homeostasis and controls the levels and quality of intracellular constituents, not only contributing to obtaining energy but also allowing cells to adapt to stress conditions. It has been described that an alteration in the autophagy process is related to numerous neurodegenerative diseases (Parkinson's, Alzheimer's or amyotrophic lateral sclerosis). Moreover, autophagy malfunction leads to protein aggregates and dysfunctional organelles accumulation that can explain some of the clinical symptoms. Rho proteins are involved in the regulation of the actin cytoskeleton dynamics and also have been postulated to play a key role in the development and function of the Central Nervous System. Rnd3/RhoE is a member of the Rnd subfamily, being constitutively activated. Its role in cell migration, proliferation and survival has been extensively studied. Rnd3 is necessary for the correct development and function of the CNS. In our group, the use of a genetically modified mouse model (Rnd3gt/gt) revealed that the lack of this protein produces serious motor and neural development alterations. Moreover, Rnd3 deficient neurons show protein aggregates and aberrant mitochondria, a phenotype compatible with problems in autophagy, as also described in some neurodegenerative pathologies. In this study we sought to analyze the possible role of Rnd3 in autophagy, in order to understand some of the phenotypes that appear in the Rnd3 deficient murine model. Our results demonstrate that the absence of Rnd3 promotes higher protein degradation rates and increased levels of autophagy even in basal conditions, suggesting that Rnd3 negatively regulates this process, at the level of the autophagosome maturation. In addition, we have demonstrated a new role of Rnd3 in mitochondrial homeostasis. The presence of nonfunctional mitochondria in Rnd3-deficient cells causes an alteration in the oxidative metabolism and an activation of glycolysis as an alternative mechanism to obtain energy. Altogether, these results highlight a new role of Rnd3 in the regulation of autophagy and mitochondrial homeostasis, which could explain some of the phenotypes observed in the Rnd3gt/gt mouse model.

Autophagy is a highly conserved process that mediates the targeting and degradation of intracellular components to lysosomes, contributing to the maintenance of cellular homeostasis and to obtaining energy, which ensures viability under stress conditions. Therefore, autophagy defects are common to different neurodegenerative disorders. Rnd3 belongs to the family of Rho GTPases, involved in the regulation of actin cytoskeleton dynamics and important in the modulation of cellular processes such as migration and proliferation. Murine models have shown that Rnd3 is relevant for the correct development and function of the Central Nervous System and lack of its expression produces several motor alterations and neural development impairment. However, little is known about the molecular events through which Rnd3 produces these phenotypes. Interestingly we have observed that Rnd3 deficiency correlates with the appearance of autophagy impairment profiles and irregular mitochondria. In this work, we have explored the impact of Rnd3 loss of expression in mitochondrial function and autophagy, using a Rnd3 KO CRISPR cell model. Rnd3 deficient cells show no alterations in autophagy and mitochondria turnover is not impaired. However, Rnd3 KO cells have an altered mitochondria oxidative metabolism, resembling the effect caused by oxidative stress. In fact, lack of Rnd3 expression makes these cells strictly dependent on glycolysis to obtain energy. Altogether, our results demonstrate that Rnd3 is relevant to maintain mitochondria function, suggesting a possible relationship with neurodegenerative diseases.