Protective Signature of IFNγ-Stimulated Microglia Relies on miR-124-3p Regulation From the Secretome Released by Mutant APP Swedish Neuronal Cells

Abstract

Microglia-associated inflammation and miRNA dysregulation are key players in Alzheimer’s disease (AD) pathophysiology. Previously, we showed miR-124 upregulation in APP Swedish SH-SY5Y (SWE) and PSEN1 iPSC-derived neurons and its propagation by the secretome (soluble and exosomal fractions). After modulation with miR-124 mimic/inhibitor, we identified common responsive mechanisms between such models. We also reported miR-124 colocalization with microglia in AD patient hippocampi. Herein, we determined how miR-124 modulation in SWE cells influences microglia polarized subtypes in the context of inflammation. We used a coculture system without cell-to-cell contact formed by miR-124 modulated SWE cells and human CHME3 microglia stimulated with interferon-gamma (IFNγ-MG), in which we assessed their adopted gene/miRNA profile and proteomic signature. The increase of miR-124 in SWE cells/secretome (soluble and exosomal) was mimicked in IFNγ-MG. Treatment of SWE cells with the miR-124 inhibitor led to RAGE overexpression and loss of neuronal viability, while the mimic caused RAGE/HMGB1 downregulation and prevented mitochondria membrane potential loss. When accessing the paracrine effects on microglia, SWE miR-124 inhibitor favored their IFNγ-induced inflammatory signature (upregulated RAGE/HMGB1/iNOS/IL-1β; downregulated IL-10/ARG-1), while the mimic reduced microglia activation (downregulated TNF-α/iNOS) and deactivated extracellular MMP-2/MMP-9 levels. Microglia proteomics identified 113 responsive proteins to SWE miR-124 levels, including a subgroup of 17 proteins involved in immune function/inflammation and/or miR-124 targets. A total of 72 proteins were downregulated (e.g., MAP2K6) and 21 upregulated (e.g., PAWR) by the mimic, while the inhibitor also upregulated 21 proteins and downregulated 17 (e.g., TGFB1, PAWR, and EFEMP1). Other targets were associated with neurodevelopmental mechanisms, synaptic function, and vesicular trafficking. To examine the source of miR-124 variations in microglia, we silenced the RNase III endonuclease Dicer1 to block miRNA canonical biogenesis. Despite this suppression, the coculture with SWE cells/exosomes still raised microglial miR-124 levels, evidencing miR-124 transfer from neurons to microglia. This study is pioneer in elucidating that neuronal miR-124 reshapes microglia plasticity and in revealing the relevance of neuronal survival in mechanisms underlying inflammation in AD-associated neurodegeneration. These novel insights pave the way for the application of miRNA-based neuropharmacological strategies in AD whenever miRNA dysregulated levels are identified during patient stratification.

Intrathecal Injection of the Secretome from ALS Motor Neurons Regulated for miR-124 Expression Prevents Disease Outcomes in SOD1-G93A Mice

“…In our prior studies, we demonstrated that miR-124 levels in the secretome recapitulate the levels observed in cells, either non-modulated or treated with its mimics and inhibitors [ 9 , 10 , 11 , 13 ]. Here, we intended to test whether the intrathecal injection of the secretome from anti-miR-124 mSOD1 MNs in pre-symptomatic mSOD1 mice was able to reduce the miR-124 expression in the lumbar SC of ALS mice when assessed at 15 weeks of age.…”

“…miR-124 is the most abundant miRNA in the adult brain and has been described as a regulator of microglia-mediated inflammation in neurological diseases [ 10 , 60 ]. It is mainly expressed in neuronal cells, showing important functions in neuronal development, differentiation, synaptic plasticity, and even in memory signalling molecules [ 61 ].…”

“…It is mainly expressed in neuronal cells, showing important functions in neuronal development, differentiation, synaptic plasticity, and even in memory signalling molecules [ 61 ]. The elevated expression of miR-124 in Alzheimer’s disease (AD) was shown to benefit the neurite outgrowth, mitochondria activation, small Aβ oligomer reduction, and beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) in experimental models and patients [ 9 , 10 , 62 ]. However, in ALS, the upregulation of miR-124 in mSOD1 MNs was shown to impair the neurite network, mitochondria dynamics, axonal transport, and synaptic signalling [ 13 ].…”

“…microRNAs (miRNAs) have been defined as important regulatory molecules for gene expression, and their dysregulation in ALS can be modulated toward the recovery of cell function and an increase in mSOD1 mouse lifespan, as reviewed in [ 7 ]. Furthermore, these molecules can be released into the secretome as free species or encapsulated in small extracellular vesicles (sEVs) [ 8 , 9 , 10 , 11 ] and internalised by neighbouring cells, also having the ability to influence distant cells and the extracellular environment [ 11 , 12 ]. Since ALS is characterized as a neuroinflammatory and neurodegenerative disease, we have focused on the study of inflammatory-associated (inflamma)-miRNAs, including miR-124, miR-155, and miR-146a, which were shown to be differentially expressed at the cellular and regional levels [ 12 , 13 ].…”

Exercise Intervention Modulates Synaptic Plasticity by Inhibiting Excessive Microglial Activation via Exosomes

“…Exosomes can regulate synaptic plasticity through microglia. Studies have shown that exosomes can regulate neuroplasticity (Xin et al, 2013 ) and neuronal development and maintain myelin and synaptic function in peripheral brain infarcts by enhancing neurite remodeling (Domingues et al, 2020 ), thus affecting the morphology and function of microglia (Vogel et al, 2018 ; Garcia et al, 2022 ) and the density of dendritic spines (Sobue et al, 2018 ). Combined with our previous studies, we speculate that exercise can regulate synaptic plasticity by inhibiting the excessive activation of microglia after exosomes enter the brain.…”