Publicação

Dissecting neuronal miRNA-124 modulation: effects on secretome-mediated microglia deregulation in mSOD1-associated amyotrophic lateral sclerosis

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Resumo:	Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons (MN) with currently limited targets and biomarkers. Neuroinflammation is a key player for disease onset/progression and the relationship between MN dysfunction and microglial activation represents a major hallmark. Our recent data demonstrated overactivation of the N9-microglia exposed to exosomes from NSC-34-MNs carrying the SOD1G93A mutation (SOD1G93A-MNs). These mutated MNs and their exosomes were enriched in microRNA(miR)-124. After internalizing these exosomes, N9-microglia exhibited time-dependent polarization and upregulated miR-124. Likewise, soluble factors released by SOD1G93A-MNs produced harmful effects on N9-microglia (upregulation of pro-inflammatory mediators/alarmins and reduced phagocytosis). Considering the paracrine effects that upregulated neuronal miR-124 may have on microglia functional properties through the influence of MN-derived secretome, we anticipate that miR-124 can be an active player for microglia immune-deregulation. We aimed to investigate whether the immune balance/function of spinal microglia is modified by wild-type (WT) or SOD1G93A-MN secretome and if it relates with MN miR- 124 upregulation. As miRNAs have been progressively associated to deregulated pathways in ALS, we explored how the modulation of miR-124 in WT/diseased MNs modifies the secretome signature of inflammatory-associated miRNA content. Next, we aimed to clarify differences in microglia feedback to the secretome from WT/mutated MNs to better understand their immunoregulatory action. Since our preliminary data showed the anti-inflammatory benefits of miR-124 modulation in SOD1G93A-MNs in the murine N9-microglia cell line, our ultimate goal was to validate such data in the mice spinal cord (SC) microglia cultures, which better resemble in vivo conditions. We used mixed glial cultures isolated from the SC of 8-day-old WT and SOD1G93A mice, that were maintained for 21 days in vitro (DIV). Microglia were then isolated and used after 2 DIV. Additionally, WT and SOD1G93A-MNs were plated and differentiated for 1 day, when SOD1G93A-MNs were transfected with anti-miR-124 for 12 h and maintained for additional 48 h. Secretomes from WT/SOD1G93A/SOD1G93A anti- miR-124 MNs were collected at 4 DIV, incubated in microglia for 4/24 h and inflammatory/functional mediators were evaluated. We demonstrated that SOD1G93A-MNs have a specific miRNA profile (increased miR-124/miR-125b, decreased miR-146a/miR-21), which was fully recapitulated in mutated MNs secretome, except for miR-125b, probably due to its intracellular retention. These effects were reversed in SOD1G93A anti-miR-124-MNs and reflected in their secretome, validating miR-124 as a driver of such deregulation. Relatively to the influence exerted by the neuronal secretome, both spinal microglia responded similarly to WT MNs secretome, displaying decreased inflammatory-associated markers upon 24 h incubation, suggesting the immunosuppressive action of this secretome. Moreover, we proved that SOD1G93A-MNs secretome has an acute and immunostimulant impact on WT microglia by increasing pro-inflammatory-associated genes and also reducing the expression of markers involved in phagocytosis and intercellular communication. In contrast, these inflammatory-associated markers were decreased in SOD1G93A microglia exposed to SOD1G93A-MN secretome, indicating their inability to mount a reparative response. As we anticipated that miR-124 elevation in SOD1G93A-MNs is partially associated with a secretome exerting microglial phenotypic aberrancies, our next results were directed to assess the microglia regenerative effects after incubation with SOD1G93A-anti-miR-124 MN secretome. Such modulation was able to prevent the upregulation of iNOS/IL-1β/IL-18/HMGB1, observed in WT microglia incubated with mutated MNs-secretome. Overall, this work highlights the impact that MNs and their secretome have in ALS microglia immune balance and dysfunction. Our findings also point out, for the first time, the benefits of downregulating miR-124 in SOD1G93A-MNs as a strategy to prevent paracrine microglia activation and ALS-associated neuroinflammation, using the SOD1G93A mice. Future studies will be performed to validate these data in MNs and microglia generated from ALS patients with familiar and sporadic forms of the disease.
Autores principais:	Colaço, Ana Rita Ambrósio
Assunto:	miR-124 modulation Amyotrophic lateral sclerosis Motor-neuron secretome mSOD1 mice and cellular models Spinal cord microglia Neurociências
Ano:	2020
País:	Portugal
Tipo de documento:	dissertação de mestrado
Tipo de acesso:	acesso aberto
Instituição associada:	Universidade de Lisboa
Idioma:	inglês
Origem:	Repositório da Universidade de Lisboa