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ALS pathogenesis: role of motor neuron-derived exosomes in microglia activation and dysfunction

Publication . Pinto, Sara Filipa Castro da Costa; Brites, Dora; Botelho, Ana Rita Mendonça Vaz

Exosomes are nanosized (30-100 nm) extracellular vesicles that are formed by nearly all types of cells and derive from the endocytic pathway and intracellular multivesicular bodies. They are released when the multivesicular bodies fuse with the plasma membrane. Exosomes mediate intercellular communication and have an important role in the spreading of neurodegenerative diseases, probably also of Amyotrophic Lateral Sclerosis (ALS). Recently, release of exosomes derived from motor neuron (MN)-like NSC-34 cells overexpressing human superoxide dismutase 1 mutated in G93A (mSOD1), was suggested to be implicated in cell-to-cell transfer of mSOD1 toxicity. However, how the uptake of such exosomes by receptor cells, such as microglia, contributes to their activation or loss of function was never investigated. Here we evaluated a selected set of promising markers and mediators of inflammatory response to establish: (i) the pro- and anti-inflammatory microRNA profile in NSC-34 MN-like cell line and in their derived exosomes; (ii) alterations in microglia function and generated polarized microglia subtypes triggered by the mSOD1 MN-derived exosomes; and (iii) the cellular distribution of labelled exosomes in a MN-microglia co-culture system. For that, we used mouse NSC-34 cells expressing either wild-type SOD1 (wt) or the G93A mutation (mSOD1) and the mouse N9 microglial cell line. Exosomes were isolated from the cell culture medium by differential ultracentrifugation and incubated with microglia for 2, 4 and 24 hours, or with the MN-microglia co-cultures for 24 hours. We assessed microglia phagocytic ability and senescence, nitric oxide (NO) production, matrix metalloproteinase (MMP)-2 and MMP-9 activity in the extracellular media, nuclear factor-kappa B (NF-κB) activation, and gene and microRNA expression by quantitative Real-Time PCR. We observed that the overexpression of microRNA (miR)-124 in mSOD1 MNs was reproduced in their derived exosomes. Such exosomes led to a loss of microglia phagocytic ability, acute release of NO, MMP-2 and MMP-9, and interleukin (IL)-1β and tumor necrosis factor (TNF)-α expression, together with lasting NF-κB activation and delayed increase of senescent-like cells. Interestingly, the early decrease in miR-124 and miR-146a expression induced by both types of exosomes was followed by their increase after 24 hours of incubation with the mSOD1 MN-derived exosomes, where enhanced miR-155 expression was similarly observed. Finally, we observed that the distribution of exosomes was preferentially towards microglia than to MNs, in the co-culture system. Preliminary data also suggest that mSOD1-associated exosomes increase the microglial expression of IL-1β and TNF-α, together with that of alarmin HMGB1. However, further studies are needed to confirm and assess the relevance of these pilot results. Overall, data highlight exosomes from mSOD1 MNs as inducers of microglia activation and dysfunction, different microglia subsets and inflammatory mediators’ production.

2016-11-23Master thesis

Open access

Role of microRNA in microglial phenotype during the progression of Alzheimer’s disease

Publication . Monteiro, Mafalda Aurélio; Fernandes, Adelaide Maria Afonso; Brites, Dora

Alzheimer’s disease (AD) is the most prevalent form of dementia and its impact in society has been aggravating throughout years. Due to its progressive nature and lack of marked treatment benefits, many efforts have been done to unveil AD pathogenesis seeking for novel therapeutic targets or biomarkers. Current view on AD pathogenesis attributes significant importance to neuroinflammation, where microglia play a pivotal role. Under normal conditions, microglia exhibit a quiescent/vigilant state and perform the brain surveillance. After an injury, microglia initiate the immune defense of the brain and acquire a pro-inflammatory or anti-inflammatory phenotype depending on stimuli. After the inflammation resolution, the brain homeostasis is restored. Various conditions such as the presence of amyloid β-peptide (Aβ) and aging can deregulate microglial response, though it remains unclear how microglial deregulation affect the course of AD. Furthermore, it was established that some microRNAs (miRNAs or miRs) that are known to promote microglial quiescence (miR-124) or regulate microglial activation states (miR-155 and miR-146a) are deregulated in AD. However, it has not been established whether the deregulation of these miRNAs can influence microglial phenotype and response in AD, particularly concerning human microglia. With this work, we proposed to analyze the temporal response of human CHME3 microglia when co-cultured with two Aβ-expressing human neuroblastoma cells, SH-SY5Y APP695 or SH-SY5Y APP695 Swe cells. We assessed microglia for miRNAs (miR-124, miR-155 and miR-146a) and their targets, as well as for pro-inflammatory (IL-1β, IL-6 and TNF-α), anti-inflammatory (TGFβ, IL-10 and Arginase 1) and immune (iNOS and MHC class II) markers, and additionally for phagocytic capacity and senescence. We found that when CHME3 microglia are co-cultured with SH-SY5Y APP695 Swe cells they exhibit a more pronounced response than when co-cultured with other neuroblastoma cells. Indeed, in the presence of SH-SY5Y APP695 Swe cells CHME3 microglia initially exhibit a miR-124low/miR-155high/miR-146ahigh profile like activated cells but gradually switch to a miR-124high/miR-155low/miR-146alow profile typical of a gradual shift towards an alternative activated/deactivated phenotype that ultimately give rise to quiescent cells. The pro-inflammatory markers are robustly expressed in microglia during the whole time, but the expression of the anti-inflammatory markers is gradually enhanced suggesting an immunoregulatory response. With regards to immunity, microglia rapidly express the innate immune marker iNOS followed by a later induction of the adaptive immune marker MHC class II. Altogether, we demonstrated that the CHME3 / SH-SY5Y APP695 Swe co-culture is the most adequate in vitro AD model to study human microglial response and possibly to assay new microglia-targeted therapeutic strategies.

2016-05-25Master thesis

Open access

Astrocyte-specific Transcriptomic Response to PGC-1a Isoforms

Publication . Ribeiro, Mariana Messias de Jesus Rufino; Rodrigues, Elsa; Ruas, Jorge Lira

Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) transcriptional coactivators are key regulators of energy metabolism-related genes and are expressed in energy-demanding tissues. There are several PGC-1α variants that exert differential and specific biological functions. In the brain, PGC-1α1 has been implicated in reactive oxygen species detoxification but our understanding of the role of the other isoforms is far from complete. Previous results have shown that PGC-1α isoform expression levels can be modulated in the brain by stress conditions, suggesting a functional role for these proteins. Moreover, we have observed that increased expression of PGC-1α3 in astrocytes could be deleterious to neurons. In the adult brain, astrocyte-neuron crosstalk is crucial for neurite outgrowth, dendritic spine maintenance and synaptic function. The main objective of this study was to characterize the molecular pathways controlled by PGC-1α3 in in vitro neuronal and astrocytic experimental models. In parallel, we analysed the transcriptome of astrocytes transduced with a viral vector encoding PGC-1α3 by massively parallel sequencing (RNA-seq) in order to identify its downstream targets. The most striking hits, as well as the expression of other astrocyte-specific genes that could be involved in the neuronal response to PGC-1α3-overexpressing astrocytes, were determined by qPCR. We also used the Ingenuity Pathway Analysis suite to determine which pathways were being modulated as a result of differential expression of the identified hits. Our results show that PGC-1α3 can trigger Gq-coupled protein-mediate calcium dysregulation within astrocytes, inducing secretory pathways and neuroinflammation. In addition, our observations suggest a severe impairment of cell migration. Since proteins secreted by astrocytes play an important role in different neuronal physiological functions, we analyzed our RNA-seq data in order to predict the astrocytic secretome profile. Using a suite of bioinformatic tools we were able to identify candidate proteins putatively secreted through both canonical and non-canonical pathways. Combining these results with the analysis of the differentially expressed genes, led to the identification of Serpini1, Angptl4, galectin 9 and TGFβ as putative PGC-1α3 target genes. Interestingly, an extensive number of transcripts regulated by PGC-1α3 map to pseudogenes, which could potentially include miR sequences. Our results identify for the first-time an astrocyte-specific transcriptomic response to PGC-1α3 expression and highlight PGC-1α isoforms as novel therapeutic targets for the treatment of neurodegenerative diseases.

2018-11-15Master thesis

Open access

Deciphering the involvement of S100B-rage axis in imflammation-associated myelin damage

Publication . Santos, Gisela Filipa Assunção, 1988-; Borralho, Adelaide Maria Afonso Fernandes; Brites, Dora, 1951-

Oligodendrocytes (OL) are responsible for the production of the myelin sheath involving the neuronal axons, providing nutritional support and fastening the transmission of electrical signals. Oligodendrogenesis impairment or myelin damage, either during neurodevelopment or later in life, may result in impaired brain functions leading to cognitive and/or motor deficits. Increased levels of S100B have been described in several conditions associated with both OL and myelin damage, being associated with a poorer disease prognosis. Moreover, S100B toxic effects have been mainly correlated with engagement of its receptor for advanced glycation end-products (RAGE), which is also highly expressed under these conditions. Thus, the major goal of this thesis was to decipher the involvement of the S100B-RAGE axis in the inflammatory-associated myelin damage, both during neurodevelopmental white matter damage, and upon demyelinating disorders. First, we showed that excessive S100B levels impaired oligodendrogenesis, either in primary cultures of OL, or in maturating organotypic cultures of cerebellar slices. Indeed, high S100B levels impaired OL differentiation and promoted morphological maturation arrest in pure cultures that was corroborated by deficient myelination in cerebellar slices. Additionally, S100B also compromised neuronal and synaptic integrity, while inducing astrogliosis, nuclear factor (NF)-kB activation and inflammation. Interestingly, the use of the RAGE antagonist FPS-ZM1 prevented S100B effects, indicating that RAGE engagement is necessary for this S100B detrimental role during neurodevelopment. We next observed that demyelination of mature organotypic cultures of cerebellar slices with lysophosphatidylcholine (LPC) markedly enhanced the expression of S100B and RAGE. In parallel, there was an elevation of markers of immature oligodendrocytes, compromised neuronal networks, and a marked gliosis with augmented inflammatory response also via NF-kB induction. Once again the use of the RAGE antagonist FPS-ZM1 prevented LPC effects, suggesting that S100B-RAGE axis have as well an injurious action during demyelination. Finally, we observed that the S100B-RAGE axis was highly expressed in the in vivo animal model of multiple sclerosis, the experimental autoimmune encephalomyelitis. These animals, besides showing a chronic elevated clinical score also displayed a great loss of myelin, downregulation of OL and synaptic specific markers, in addition to a potentiated expression of proinflammatory cytokines and inhibition of anti-inflammatory response. Interestingly, when the animals where treated with dimethyl fumarate (DMF), a drug used in clinical practice for multiple sclerosis treatment, although no changes were observed in their clinical score, there was a mild improvement in their cognitive performance. In addition, we observed a marked improvement in myelination, a restoration of oligodendrocyte and synaptic markers, as well as a reduction of inflammation, including a decreased expression of S100B and RAGE. Our studies imply a toxic role of S100B-RAGE axis in oligodendrogenesis, either during myelin formation, or following demyelination, along with impaired neuronal and synaptic integrity, glia activation and establishment of an inflammatory milieu. Most importantly, this work supports that S100B-RAGE interaction may constitute a new and more specific target for therapeutic intervention strategies to reduce brain injury associated with inflammation-associated myelin diseases.

2018Doctoral thesis

Open access

Exploring mek5-erk5 signaling in colorectal cancer : A therapeutic target for chemosensitization

Publication . Pereira, Diane M.; Rodrigues, Cecília M. P., 1968-; Borralho, Pedro Miguel Martinho, 1979-

Colorectal cancer remains one of the leading causes of cancer morbidity and mortality in the world. Despite significant advances in early detection and multimodal care, drug resistance, recurrence, and metastasis persist as major challenges in the management of the disease, calling for the identification of alternative targets for therapeutic intervention. Interestingly, in human colorectal adenomas and adenocarcinomas, there is ample evidence that aberrant MEK5-ERK5 expression and/or activation correlates with tumor-node-metastasis stage. As such, the studies presented in this thesis were conducted with the purpose of defining the contribution of ERK5-mediated signaling to the regulation of hallmark traits associated with disease progression, namely tumor cell resistance to 5-fluorouracil (5-FU), the cornerstone of systemic colorectal cancer treatment; and malignant stem cell-like pools, major players in therapy-refractory disease. Using colorectal cancer cell lines as model, we showed that exposure to 5-FU impairs endogenous MEK5-ERK5 signaling, whereas ectopic expression of a constitutively active MEK5 increases resistance to this drug. Conversely, blocking ERK5 in combination with 5-FU results in impaired tumor cell survival and growth in vitro and in subcutaneous xenografts. Further studies established that ERK5 inhibition promotes 5-FU–induced apoptosis in a TP53-wild-type but not a TP53-null background, implying a p53-dependent axis mediating 5-FU sensitization. In parallel, we demonstrated that MEK5-ERK5 phosphorylation levels are increased in three-dimensional sphere cultures enriched for neoplastic stem-like cells. Further, targeting ERK5 suppresses the rates of tumorsphere formation and the expression/activity of representative markers of immature cancer cell fractions, while sensitizing to 5-FU–based chemotherapy. Moreover, downregulation of NF-κB–mediated IL-8 expression might be a crucial event for the impact of ERK5 inhibition on malignant stem-like phenotypes. Finally, analysis of publicly available databases revealed that increased ERK5 expression correlates with shorter overall survival in colorectal cancer patients, reinforcing the clinical relevance of the MEK5-ERK5 axis. Overall, our findings indicate that upregulated MEK5-ERK5 signaling in colorectal carcinoma cells contributes to a shift to an undifferentiated state, whilst providing a route for cancer (stem) cells to escape cytotoxic insults inflicted by classical chemotherapy, therefore encouraging future investigations on the translational potential of ERK5-targeted agents for antineoplastic treatment and chemosensitization.

2019Doctoral thesis

Restricted access