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Role of glial cells as contributors to the onset and propagation of als disease

Publication . Cunha, Carolina; Brites, Dora, 1951-; Botelho, Ana Rita Mendonça Vaz

Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease comprehending critical neuroinflammatory pathways, where microglia and astrocytes play a crucial role. ALS onset events are largely unknown and identification of disease steps during progression and dissemination, including the possible role of exosomes, are not clarified. Several models were used to improve data validity and deepen knowledge in ALS. We identified innovative targets to regulate microglia M1 polarization, including NLRP3-inflammasome, HMGB1 alarmin and MFG-E8/lactadherin, and demonstrated the sorting of microglial microRNA(miR) 155/miR-146a into exosomes. We showed that ALS NSC-34 MNs and their exosomes are enriched in miR-124, which are captured and drive early N9-microglia M1 polarization, with later development of M1/M2 subpopulations containing increased miR-124/miR-146a/miR-155. Moving from in vitro models to the spinal cord of the SOD1G93A ALS mouse model, we observed that depressed intercellular communication and increased miR-155 were early disease events preceding the inflammatory status of the symptomatic stage. Upregulated CX3CL1-CX3CR1, connexin-43/pannexin-1 and miR-124/miR-125b/miR-146a/miR-21 emerged as candidate targets for pathological neuroinflammation. Reduced MN number, together with aberrant/reactive astrocytes showing deficient glutamate transporters and GFAP, additionally characterized such state. Differently deregulated profiles of microglia isolated from the spinal cord of 7-day old SOD1G93A mice, after short- and long-term cultures, highlighted that cells present transient phenotypes accordingly to ALS environmental progression-stimuli and ultimately acquire a less responsive phenotype to stimulation. Astrocytes isolated from these mice promoted diverse inflammatory polarized subtypes in wild-type and ALS microglia, thus accounting to microglia heterogeneous populations, while strengthened deregulated microglia-astrocyte cross-talk as part of ALS neurodegenerative mechanisms. Our studies in ALS models reveal early promising biomarkers and novel targets to control excessive neuroinflammation and spread, including exosomal microRNAs. Due to multiple microglia phenotypes induced by MNs and their exosomes, and by reactive astrocytes, in the ALS disease, differentiated and combined therapeutic approaches may be recommended.

2017Doctoral thesis

Open access

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