Ana L. Cardoso

miR‐155 modulates microglia‐mediated immune response by down‐regulating SOCS‐1 and promoting cytokine and nitric oxide production

Innate immunity constitutes the first line of defence against both external and endogenous threats in the brain, and microglia cells are considered key mediators of this process. Recent studies have shown that microRNAs (miRNAs) may play a determinant role in the regulation of gene expression during innate immune responses. The major goal of this work was to investigate the contribution of a specific miRNA – miR‐155 – to the modulation of the microglia‐mediated immune response. For this purpose, in vitro studies were performed in N9 microglia cells to evaluate changes in the levels of this miRNA following microglia activation. A strong up‐regulation of miR‐155 expression was observed following microglia exposure to lipopolysaccharide, which was consistent with a decrease in the levels of the suppressor of cytokine signalling 1 (SOCS‐1) protein, a key inhibitor of the inflammatory process and a predicted target of miR‐155. The miR‐155 knockdown by anti‐miRNA oligonucleotides up‐regulated SOCS‐1 mRNA and protein levels and significantly decreased the production of nitric oxide and the expression of inflammatory cytokines and inducible nitric oxide synthase. Finally, treatment of neuronal primary cultures with conditioned medium obtained from microglia cells, in which miR‐155 was inhibited before cell activation, decreased inflammatory‐mediated neuronal cell death. Overall, our results show that miR‐155 has a pro‐inflammatory role in microglia and is necessary for the progression of the immune response through the modulation of SOCS‐1, suggesting that, in a chronic inflammatory context, miR‐155 inhibition can have a neuroprotective effect.

siRNA delivery by a transferrin-associated lipid-based vector: a non-viral strategy to mediate gene silencing

RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation, protection, and extra‐ and intracellular delivery of these nucleic acids. Here, we evaluated the potential of transferrin (Tf)‐associated liposomes for siRNA complexation and gene silencing.

Tumor-targeted Chlorotoxin-coupled Nanoparticles for Nucleic Acid Delivery to Glioblastoma Cells: A Promising System for Glioblastoma Treatment

The present work aimed at the development and application of a lipid-based nanocarrier for targeted delivery of nucleic acids to glioblastoma (GBM). For this purpose, chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells while showing no affinity for non-neoplastic cells, was covalently coupled to liposomes encapsulating antisense oligonucleotides (asOs) or small interfering RNAs (siRNAs). The resulting targeted nanoparticles, designated CTX-coupled stable nucleic acid lipid particles (SNALPs), exhibited excellent features for in vivo application, namely small size (<180 nm) and neutral surface charge. Cellular association and internalization studies revealed that attachment of CTX onto the liposomal surface enhanced particle internalization into glioma cells, whereas no significant internalization was observed in noncancer cells. Moreover, nanoparticle-mediated miR-21 silencing in U87 human GBM and GL261 mouse glioma cells resulted in increased levels of the tumor suppressors PTEN and PDCD4, caspase 3/7 activation and decreased tumor cell proliferation. Preliminary in vivo studies revealed that CTX enhances particle internalization into established intracranial tumors. Overall, our results indicate that the developed targeted nanoparticles represent a valuable tool for targeted nucleic acid delivery to cancer cells. Combined with a drug-based therapy, nanoparticle-mediated miR-21 silencing constitutes a promising multimodal therapeutic approach towards GBM.

MiRNA-21 silencing mediated by tumor-targeted nanoparticles combined with sunitinib: A new multimodal gene therapy approach for glioblastoma.

Malignant brain tumors, including glioblastoma (GBM), are among the most lethal human cancers, due to their tremendous invasive capacity and limited therapeutic options. Despite remarkable advances in cancer theranostics, which resulted in significant improvement of clinical outcomes, GBM relapse is very frequent and patient survival remains under one year. The elucidation of the role of abnormally-expressed miRNAs in different steps of GBM pathogenesis and in tumor resistance to therapy paved the way for the development of new miRNA-based therapeutic approaches targeting this disease, aiming at increasing specific tumor cell killing and, ultimately, cancer eradication. Here, we demonstrate that intravenously-administered chlorotoxin (CTX)-coupled (targeted) stable nucleic acid lipid particle (SNALP)-formulated anti-miR-21 oligonucleotides accumulate preferentially within brain tumors and promote efficient miR-21 silencing, which results in increased mRNA and protein levels of its target RhoB, while showing no signs of systemic immunogenicity. Decreased tumor cell proliferation and tumor size, as well as enhanced apoptosis activation and, to a lesser extent, improvement of animal survival, were also observed in GBM-bearing mice upon systemic delivery of targeted nanoparticle-formulated anti-miR-21 oligonucleotides and exposure to the tyrosine kinase inhibitor sunitinib. Overall, our results provide evidence that CTX-coupled SNALPs are a reliable and efficient system for systemic delivery of anti-miRNA oligonucleotides. Moreover, although further studies are still necessary to demonstrate a therapeutic benefit in a clinical context, our findings suggest that miRNA modulation by the targeted nanoparticles combined with anti-angiogenic chemotherapy may hold promise as an attractive approach towards GBM treatment.

Tf-lipoplexes for neuronal siRNA delivery: A promising system to mediate gene silencing in the CNS

Although RNAi-based gene silencing holds a great potential for treatment of neurological disorders, its application to the CNS has been restricted by low levels of tissue distribution and cellular uptake. In this work we report that cationic lipid-based vectors can enhance siRNA delivery to neurons both in vitro and in vivo. DOTAP:Chol liposomes associated with transferrin (Tf) and complexed with siRNAs (Tf-lipoplexes) were delivered to primary cultures of luciferase-expressing cortical neurons. Confocal microscopy studies revealed efficient cellular uptake of Cy3-labelled siRNAs after Tf-lipoplex delivery, which was reduced but not completely inhibited by blocking the Tf-receptor with excess Tf. Gene silencing was also evaluated after delivery of anti-luciferase or anti-c-Jun siRNAs. Our results demonstrate that Tf-lipoplexes achieve up to 50% luciferase and c-Jun knockdown, 48 h after transfection, without significant cytotoxicity. Similar results were observed in vivo, where a 40% reduction of luciferase activity was found in the striatum of luciferase mice. In addition, fluorescence microscopy studies showed extensive local distribution and internalization of Tf-lipoplex-associated Cy3-siRNAs without tissue toxicity. Overall, our results demonstrate that Tf-lipoplexes can mediate efficient gene silencing in neuronal cells, both in vitro an in vivo, which may prove useful in therapeutic approaches to neuronal protection and repair.

Tf-lipoplex-mediated NGF gene transfer to the CNS: neuronal protection and recovery in an excitotoxic model of brain injury

The development of efficient systems for in vivo gene transfer to the central nervous system (CNS) may provide a useful therapeutic strategy for the alleviation of several neurological disorders. In this study, we evaluated the feasibility of nonviral gene therapy to the CNS mediated by cationic liposomes. We present evidence of the successful delivery and expression of both a reporter and a therapeutic gene in the rodent brain, as evaluated by immunohistochemical assays. Our results indicate that transferrin-associated cationic liposome/DNA complexes (Tf-lipoplexes) allow a significant enhancement of transfection activity as compared to plain complexes, and that 8/1 (+/−) Tf-lipoplexes constitute the best formulation to mediate in vivo gene transfer. We demonstrated that Tf-lipoplex-mediated nerve growth factor transgene expression attenuates the morphological damages of the kainic acid-induced lesion as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) vital staining. These findings suggest the usefulness of these lipid-based vectors in mediating the delivery of therapeutic genes to the CNS.

Early miR-155 upregulation contributes to neuroinflammation in Alzheimer's disease triple transgenic mouse model

MicroRNAs (miRNAs) have emerged as a class of small, endogenous, regulatory RNAs that exhibit the ability to epigenetically modulate the translation of mRNAs into proteins. This feature enables them to control cell phenotypes and, consequently, modify cell function in a disease context. The role of inflammatory miRNAs in Alzheimers disease (AD) and their ability to modulate glia responses are now beginning to be explored. In this study, we propose to disclose the functional role of miR-155, one of the most well studied immune-related miRNAs in AD-associated neuroinflammatory events, employing the 3xTg AD animal model. A strong upregulation of miR-155 levels was observed in the brain of 12-month-old 3xTg AD animals. This event occurred simultaneously with an increase of microglia and astrocyte activation, and before the appearance of extracellular Aβ aggregates, suggesting that less complex Aβ species, such as Aβ oligomers may contribute to early neuroinflammation. In addition, we investigated the contribution of miR-155 and the c-Jun transcription factor to the molecular mechanisms that underlie Aβ-mediated activation of glial cells. Our results suggest early miR-155 and c-Jun upregulation in the 3xTg AD mice, as well as in Aβ-activated microglia and astrocytes, thus contributing to the production of inflammatory mediators such as IL-6 and IFN-β. This effect is associated with a miR-155-dependent decrease of suppressor of cytokine signaling 1. Furthermore, since c-Jun silencing decreases the levels of miR-155 in Aβ-activated microglia and astrocytes, we propose that miR-155 targeting can constitute an interesting and promising approach to control neuroinflammation in AD.

Involvement of microRNA in microglia-mediated immune response.

MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that play an important role in the regulation of gene expression at the posttranscriptional level. Due to their ability to simultaneously modulate the fate of different genes, these molecules are particularly well suited to act as key regulators during immune cell differentiation and activation, and their dysfunction can contribute to pathological conditions associated with neuroinflammation. Recent studies have addressed the role of miRNAs in the differentiation of progenitor cells into microglia and in the activation process, aiming at clarifying the origin of adult microglia cells and the contribution of the central nervous system (CNS) environment to microglia phenotype, in health and disease. Altered expression of several miRNAs has been associated with Alzheimers disease, multiple sclerosis, and ischemic injury, hence strongly advocating the use of these small molecules as disease markers and new therapeutic targets. This review summarizes the recent advances in the field of miRNA-mediated regulation of microglia development and activation. We discuss the role of specific miRNAs in the maintenance and switching of microglia activation states and illustrate the potential of this class of nucleic acids both as biomarkers of inflammation and new therapeutic tools for the modulation of microglia behavior in the CNS.

MicroRNA-21 silencing enhances the cytotoxic effect of the antiangiogenic drug sunitinib in glioblastoma

Highly malignant glioblastoma (GBM) is characterized by high genetic heterogeneity and infiltrative brain invasion patterns, and aberrant miRNA expression has been associated with hallmark malignant properties of GBM. The lack of effective GBM treatment options prompted us to investigate whether miRNAs would constitute promising therapeutic targets toward the generation of a gene therapy approach with clinical significance for this disease. Here, we show that microRNA-21 (miR-21) is upregulated and microRNA-128 (miR-128) is downregulated in mouse and human GBM samples, a finding that is corroborated by analysis of a large set of human GBM data from The Cancer Genome Atlas. Moreover, we demonstrate that oligonucleotide-mediated miR-21 silencing in U87 human GBM cells resulted in increased levels of the tumor suppressors PTEN and PDCD4, caspase 3/7 activation and decreased tumor cell proliferation. Cell exposure to pifithrin, an inhibitor of p53 transcriptional activity, reduced the caspase activity associated with decreased miR-21 expression. Finally, we demonstrate for the first time that miR-21 silencing enhances the antitumoral effect of the tyrosine kinase inhibitor sunitinib, whereas no therapeutic benefit is observed when coupling miR-21 silencing with the first-line drug temozolomide. Overall, our results provide evidence that miR-21 is uniformly overexpressed in GBM and constitutes a highly promising target for multimodal therapeutic approaches toward GBM.

Tf-lipoplex-mediated c-Jun silencing improves neuronal survival following excitotoxic damage in vivo.

Excitotoxicity is one of the main features responsible for neuronal cell death after acute brain injury and in several neurodegenerative disorders, for which only few therapeutic options are currently available. In this work, RNA interference was employed to identify and validate a potential target for successful treatment of excitotoxic brain injury, the transcription factor c-Jun. The nuclear translocation of c-Jun and its upregulation are early events following glutamate-induced excitotoxic damage in primary neuronal cultures. We present evidence for the efficient knockdown of this transcription factor using a non-viral vector consisting of cationic liposomes associated to transferrin (Tf-lipoplexes). Tf-lipoplexes were able to deliver anti-c-Jun siRNAs to neuronal cells in culture, resulting in efficient silencing of c-Jun mRNA and protein and in a significant decrease of cell death following glutamate-induced damage or oxygen-glucose deprivation. This formulation also leads to a significant c-Jun knockdown in the mouse hippocampus in vivo, resulting in the attenuation of both neuronal death and inflammation following kainic acid-mediated lesion of this region. Furthermore, a strong reduction of seizure activity and cytokine production was observed in animals treated with anti-c-Jun siRNAs. These findings demonstrate the efficient delivery of therapeutic siRNAs to the brain by Tf-lipoplexes and validate c-Jun as a promising therapeutic target in neurodegenerative disorders involving excitotoxic lesions.