2. Universidad Cardenal Herrera-CEU

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.

Macrophage activation is a complex process with multiple control elements that ensures an adequate response to the aggressor pathogens and, on the other hand, avoids an excess of inflammatory activity that could cause tissue damage. In this study, we have identified RND3, a small GTP-binding protein, as a new element in the complex signaling process that leads to macrophage activation. We show that RND3 expression is transiently induced in macrophages activated through Toll receptors and potentiated by IFN-γ. We also demonstrate that RND3 increases NOTCH signaling in macrophages by favoring NOTCH1 expression and its nuclear activity; however, Rnd3 expression seems to be inhibited by NOTCH signaling, setting up a negative regulatory feedback loop. Moreover, increased RND3 protein levels seem to potentiate NFκB and STAT1 transcriptional activity resulting in increased expression of proinflammatory genes, such as Tnf-α, Irf-1, or Cxcl-10. Altogether, our results indicate that RND3 seems to be a new regulatory element which could control the activation of macrophages, able to fine tune the inflammatory response through NOTCH.

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.

Autophagy is a fine-tuned proteolytic pathway that moves dysfunctional/aged cellular components into the lysosomal compartment for degradation. Over the last 3 decades, global research has provided evidence for the protective role of autophagy in different brain cell components. Autophagic capacities decline with age, which contributes to the accumulation of obsolete/damaged organelles and proteins and, ultimately, leads to cellular aging in brain tissues. It is thus well-accepted that autophagy plays an essential role in brain homeostasis, and malfunction of this catabolic system is associated with major neurodegenerative disorders. Autophagy function can be modulated by different types of stress, including glycative stress. Glycative stress is defined as a cellular status with abnormal and accelerated accumulation of advanced glycation end products (AGEs). It occurs in hyperglycemic states, both through the consumption of high-sugar diets or under metabolic conditions such as diabetes. In recent years, glycative stress has gained attention for its adverse impact on brain pathology. This is because glycative stress stimulates insoluble, proteinaceous aggregation that is linked to the malfunction of different neuropathological proteins. Despite the emergence of new literature suggesting that autophagy plays a major role in fighting glycation-derived damage by removing cytosolic AGEs, excessive glycative stress might also negatively impact autophagic function. In this mini-review, we provide insight on the status of present knowledge regarding the role of autophagy in brain physiology and pathophysiology, with an emphasis on the cytoprotective role of autophagic function to ameliorate the adverse effects of glycation-derived damage in neurons, glia, and neuron-glia interactions.