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Age-dependent microglia inflammatory profile in the spinal cord of the ALS mSOD1G93A mice
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Abstract(s)
A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa fatal e heterogénea, caracterizada pela perda progressiva de neurónios motores e neuroinflamação. A microglia-associada-a-doença (MAD) desempenha um papel crucial, adotando fenótipos neurotóxicos ao longo da progressão de patologias. Além disso, existe evidência de que a disfunção microglial está relacionada com variações, dependentes da idade, em marcadores inflamatórios. Contudo, essa variação e os seus mecanismos moleculares subjacentes permanecem pouco compreendidos na patogénese da ELA.
Neste contexto, é essencial investigar os microRNAs (miRNAs) associados à inflamação, uma vez que são reguladores importantes da inflamação durante a progressão da doença e têm potencial terapêutico como alvos de modulação. O objetivo deste estudo é investigar o perfil inflamatório de miRNAs e os estádios reativos, dependentes da idade, na microglia espinhal de murganhos com mutação G93A no gene da superóxido dismutase-1, modelo mSOD1G93A de ELA. Assim, foram utilizadas culturas primárias de microglia espinhal de murganhos recém-nascidos mSOD1G93A (TG) e selvagens (WT) e analisados miRNAs inflamatórios (miR-21, miR-124, miR-125b, miR-146a e miR-155) e marcadores inflamatórios/fagocíticos (TNF-α, ARG1, iNOS, TREM2 e MFG-E8) aos 2, 10 e 16 dias in vitro (DIV), simulando as fases jovem/reativa, madura e envelhecida/senescentes, respetivamente. Os resultados obtidos revelam alterações dinâmicas na expressão de miRNAs com a idade, que se correlacionam com mudanças funcionais da microglia, de um estado reativo para um estado pouco responsivo. A microglia mSOD1 aos 2 DIV apresentou sobre-expressão do miR-124 e um fenótipo pró-inflamatório (sobre-expressão de iNOS e MFG-E8 e sub-expressão de ARG1). Aos 10 DIV, a microglia mSOD1 apresentou sub-expressão de miR-125b e miR-155a, associada à expressão aumentada de TNF-α e TREM2, possivelmente contribuindo para um perfil neurotóxico e inflamação crónica. Aos 16 DIV, encontrou-se um padrão misto na microglia mSOD1, caracterizado pela diminuição de miR-146a e sub-expressão de marcadores pró- e anti-inflamatórios, respetivamente TNF-α e ARG1.
Em suma, estes resultados demonstram um perfil complexo de miRNAs, dependente da idade, na microglia mSOD1, que transita de uma reatividade pró-inflamatória para um fenótipo mais disfuncional e de inflamação mista. A modulação específica destes miRNAs, regulando essas transições e restabelecendo a neuroprotecção, pode fornecer terapêuticas promissoras para a ELA.
Amyotrophic lateral sclerosis (ALS) is a fatal and heterogeneous neurodegenerative disorder characterized by progressive motor neuron loss and related neuroinflammation. Disease-associated microglia (DAM) play an important role in the ALS pathogenesis, adopting neurotoxic phenotypes throughout disease progression, with microglial dysfunction linked to age-related shifts in inflammatory signaling; however, their underlying molecular mechanism is not-yet well-understood. In this context, inflammatory-associated-microRNAs (miRNAs) are key parameters to identify during disease progression since they are important regulators of inflammation and hold therapeutic potential as targets for modulation. This study aims to investigate the age-dependent inflammatory miRNA signature and microglial reactivity stages in spinal microglia from the ALS mice (G93A mutation in the superoxide dismutase-1 gene, mSOD1G93A model). For that we used spinal microglial primary cultures from mSOD1G93A (TG) and aged matched wild-type (WT) mice pups and analyzed inflammatory miRNAs (miR-21, miR-124, miR-125b, miR-146a, and miR-155) and inflammatory/phagocytic markers [Tumor Necrosis Factor-alpha (TNF-α), Arginase 1 (ARG1), Inducible Nitric Oxide Synthase (iNOS), Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), and Milk Fat Globule-Epidermal Growth Factor 8 (MFG-E8)] at 2, 10, and 16 days in vitro (DIV) microglia, to mimic young/reactive, mature and aged/senescent stage, respectively. Our findings reveal dynamic changes in miRNA expression with age that correlate with microglial functional shifts from a reactive state to an unresponsive state. mSOD1 microglia at 2 DIV were characterized by overexpression of miR-124 and a pro-inflammatory phenotype, as indicated by the upregulation of iNOS and MFG-E8 and downregulation of ARG1. At 10 DIV, mSOD1 microglia exhibited downregulation of both miR-125b and miR-155, coinciding with sustained expression of TNF-α and TREM2, likely contributing to chronic inflammation that may lead to a more neurotoxic profile. At 16 DIV, mixed microglial phenotypes seemed to emerge, characterized by the decline of miR-146a and an upregulation of both pro- and anti-inflammatory markers, respectively TNF-α and ARG1. Overall, our results demonstrate a unique, age-dependent complex miRNA signature in mSOD1 microglia, shifting from pro-inflammatory reactivity to a more dysfunctional and mixed phenotypes. Targeting specific miRNAs to modulate these transitions could provide promising therapeutic avenues for re-establishing neuroprotection in ALS.
Amyotrophic lateral sclerosis (ALS) is a fatal and heterogeneous neurodegenerative disorder characterized by progressive motor neuron loss and related neuroinflammation. Disease-associated microglia (DAM) play an important role in the ALS pathogenesis, adopting neurotoxic phenotypes throughout disease progression, with microglial dysfunction linked to age-related shifts in inflammatory signaling; however, their underlying molecular mechanism is not-yet well-understood. In this context, inflammatory-associated-microRNAs (miRNAs) are key parameters to identify during disease progression since they are important regulators of inflammation and hold therapeutic potential as targets for modulation. This study aims to investigate the age-dependent inflammatory miRNA signature and microglial reactivity stages in spinal microglia from the ALS mice (G93A mutation in the superoxide dismutase-1 gene, mSOD1G93A model). For that we used spinal microglial primary cultures from mSOD1G93A (TG) and aged matched wild-type (WT) mice pups and analyzed inflammatory miRNAs (miR-21, miR-124, miR-125b, miR-146a, and miR-155) and inflammatory/phagocytic markers [Tumor Necrosis Factor-alpha (TNF-α), Arginase 1 (ARG1), Inducible Nitric Oxide Synthase (iNOS), Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), and Milk Fat Globule-Epidermal Growth Factor 8 (MFG-E8)] at 2, 10, and 16 days in vitro (DIV) microglia, to mimic young/reactive, mature and aged/senescent stage, respectively. Our findings reveal dynamic changes in miRNA expression with age that correlate with microglial functional shifts from a reactive state to an unresponsive state. mSOD1 microglia at 2 DIV were characterized by overexpression of miR-124 and a pro-inflammatory phenotype, as indicated by the upregulation of iNOS and MFG-E8 and downregulation of ARG1. At 10 DIV, mSOD1 microglia exhibited downregulation of both miR-125b and miR-155, coinciding with sustained expression of TNF-α and TREM2, likely contributing to chronic inflammation that may lead to a more neurotoxic profile. At 16 DIV, mixed microglial phenotypes seemed to emerge, characterized by the decline of miR-146a and an upregulation of both pro- and anti-inflammatory markers, respectively TNF-α and ARG1. Overall, our results demonstrate a unique, age-dependent complex miRNA signature in mSOD1 microglia, shifting from pro-inflammatory reactivity to a more dysfunctional and mixed phenotypes. Targeting specific miRNAs to modulate these transitions could provide promising therapeutic avenues for re-establishing neuroprotection in ALS.
Description
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2024, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Amyotrophic lateral sclerosis Inflammatory-microRNAs Neuroinflammation Spinal microglia SOD1G93A mouse model Mestrado Integrado - 2024