Role of neural exosomes on microglia activation/dysfunction using AD cell line models and AD iPSCs-derived neurons and astrocytes in 2D/3D culture systems

Financiador

Organização

Publicações

Potential roles of inflammatory miRNAs and exosomes in Alzheimer’s disease pathogenesis, diagnostics and therapeutics using advanced Human models

Publication . Garcia, Gonçalo; Brites, Dora Maria Tuna de Oliveira; Malm, Tarja Maarit

Alzheimer’s disease (AD) is a complex brain disorder with a heavy socio-economic burden, affecting over 50 million people worldwide. Although aging, genetics and environmental factors constitute major risk factors, alterations in neuron-glia communication and dysregulation of microRNAs (miRNAs) dysregulation were shown to be key players in AD pathogenesis. The neural-specific miRNA(miR)-124 is a prominent player in neuronal function. We identified its consistent upregulation in SH-SY5Y-APP695 Swedish neuroblastoma cells (SH-SWE) and PSEN1 mutant iPSC-derived neurons (iNEU-PSEN), as well as in their respective exosomes. Considering the controversy around miR-124 in AD, we investigated the outcomes of modulating its levels in both neuronal cells, using specific miR-124 inhibitor/mimic. Results demonstrated that the mimic reduced APP gene expression in both models of AD. Moreover, it stimulated neurite outgrowth, mitochondria membrane potential and reduced small Aβ oligomers in SH-SWE neurons, while it inhibited tau phosphorylation in iNEU-PSEN cells. Besides, miR-124 mimic predominantly repressed the inflammatory-associated miR-125b/miR-21/miR-146a/miR-155. Oppositely, miR-124 inhibitor prompted miR-146a in the two neuronal models, while it reduced neurite outgrowth and affected mitochondrial dynamics in SH-SWE cells, and decreased dendritic spine density in iNEU-PSEN. Thereafter, we evaluated the paracrine influence of the neuronal miR-124 modulation on microglial polarization status, under an inflammatory milieu. For that, we co-cultured miR-124 modulated SWE cells with the human CHME3 microglia stimulated with interferon gamma (IFNγ-MG). Results evidenced that besides the benefits for SWE cells, miR-124 mimic reduced microglial activation (downregulated TNF-α/iNOS), inhibited extracellular MMP-2/MMP-9 activity, and led to proteomic downregulation of 72 proteins (e.g., MAP2K6) and upregulation of 21 (e.g., PAWR). In contrast, miR-124 inhibition favored microglial inflammatory signature (upregulated RAGE/HMGB1/iNOS/IL-1β; downregulated IL-10/ARG-1) and led to proteomic changes favoring the IFNγ-induced signature (e.g., TGFB1 downregulation). By blocking miRNA canonical biogenesis through Dicer1-silencing, we confirmed miR-124 uptake by microglia upon coculture with SWE cells, and upon incubation with SWE-derived exosomes. Finally, we analyzed the miRNA expression profile in cerebrospinal fluid (CSF) samples from a small cohort of Mild Cognitive Impairment (MCI) patients and specifically detected miR-21 elevation in samples from patients that progressed to AD, comparatively to those that didn’t. Such miR-21 elevation was confirmed and cross-validated in AD patient iPSC-derived microglia, neurons, and astrocytes. We further developed a new organotypic hippocampal slice model transplanted with SH or SWE cells. Remarkably, transplantation of SWE cells into hippocampal slices boosted the miR-21 expression and induced both microglia activation and astrocyte reactivity, validating the active role of miR-21 in AD. Overall, our studies support that miR-124 is a promising target with pleiotropic effects, from APP processing and maintenance of dendritic homeostasis to the reshaping of microglial plasticity in AD-associated context and should be regulated whenever necessary. Furthermore, we elucidated the potential of miR-21 as a promising disease-modifying miRNA to be explored in AD, considering its involvement in neuronal dysfunction, microglia activation, and astrocyte reactivity. Together, the novel insights provided in this Thesis may pave the way for the development of future personalized neuropharmacological strategies with miRNAs, either in the secretome or as cargo of exosomes.

2022-05Tese de doutoramento

Acesso aberto

Ver mais

Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

OE

Número da atribuição

COVID/BD/151849/2021