Recovery of Depleted miR-146a in ALS Cortical Astrocytes Reverts Cell Aberrancies and Prevents Paracrine Pathogenicity on Microglia and Motor Neurons

Abstract

Reactive astrocytes in Amyotrophic Lateral Sclerosis (ALS) change their molecular expression pattern and release toxic factors that contribute to neurodegeneration and microglial activation. We and others identified a dysregulated inflammatory miRNA profile in ALS patients and in mice models suggesting that they represent potential targets for therapeutic intervention. Such cellular miRNAs are known to be released into the secretome and to be carried by small extracellular vesicles (sEVs), which may be harmful to recipient cells. Thus, ALS astrocyte secretome may disrupt cell homeostasis and impact on ALS pathogenesis. Previously, we identified a specific aberrant signature in the cortical brain of symptomatic SOD1-G93A (mSOD1) mice, as well as in astrocytes isolated from the same region of 7-day-old mSOD1 mice, with upregulated S100B/HMGB1/Cx43/vimentin and downregulated GFAP. The presence of downregulated miR-146a on both cases suggests that it can be a promising target for modulation in ALS. Here, we upregulated miR-146a with pre-miR-146a, and tested glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS) for their immunoregulatory properties. VS was more effective in restoring astrocytic miR-146a, GFAP, S100B, HMGB1, Cx43, and vimentin levels than GUDCA, which only recovered Cx43 and vimentin mRNA. The miR-146a inhibitor generated typical ALS aberrancies in wild type astrocytes that were abolished by VS. Similarly, pre-miR-146a transfection into the mSOD1 astrocytes abrogated aberrant markers and intracellular Ca2+ overload. Such treatment counteracted miR-146a depletion in sEVs and led to secretome-mediated miR-146a enhancement in NSC-34-motor neurons (MNs) and N9-microglia. Secretome from mSOD1 astrocytes increased early/late apoptosis and FGFR3 mRNA in MNs and microglia, but not when derived from pre-miR-146a or VS-treated cells. These last strategies prevented the impairment of axonal transport and synaptic dynamics by the pathological secretome, while also averted microglia activation through either secretome, or their isolated sEVs. Proteomic analysis of the target cells indicated that pre-miR-146a regulates mitochondria and inflammation via paracrine signaling. We demonstrate that replenishment of miR-146a in mSOD1 cortical astrocytes with pre-miR-146a or by VS abrogates their phenotypic aberrancies and paracrine deleterious consequences to MNs and microglia. These results propose miR-146a as a new causal and emerging therapeutic target for astrocyte pathogenic processes in ALS.

Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

“…In the past years, it was demonstrated that miRNAs are not only present intracellularly, but are also detectable in substantial amounts extracellularly, including in biological fluids, being present either in exosomes or as soluble components in the vesicle-free secretome [83]. This horizontal transfer of miRNAs is part of autocrine and paracrine signaling mechanisms involved in cell-to-cell communication and in the propagation of inflammatory mediators, including miRNAs [11,84,85]. In this study, we have shown that SH-WT and SH-SWE cells overexpressing miR-124 are responsible for its enhanced expression in donor cells, when using a non-contact co-culture system, thus attesting the cell-to-cell transfer of this miRNA [64].…”

Astrocytes in Amyotrophic Lateral Sclerosis

“…mechanisms (15,17), modulation of such mechanisms with specific miRNAbased strategies may prevent cell-to-cell paracrine dysregulation and MN degeneration (81,86).…”

“…In contrast, a decreased cargo in miR-494-3p was found in C9ORF72 astrocyte-derived exosomes with harmful consequences in neurite network in ALS (89). Depleted levels of miR-146a were also recognized in exosomes from the cortical astrocytes of mSOD1 mice (15), and its cellular replenishment abrogated the astrocyte aberrant phenotype, characterized by increased S100B and Cx43 levels, together with decreased GFAP, while leading to a secretome with paracrine neuroprotective properties (81). Exosomes from both cortical and spinal mSOD1 astrocytes were deficient in miR-155, miR-21 and miR-146a (12).…”

“…Several studies have shown that TGF-β signaling is involved in ALS and that TGF-β1 release from astrocytes accelerates disease progression in ALS mice (23). Extracellular vesicles with ~40-160 nm, denominated exosomes, are released from mSOD1 astrocytes and were shown to contain mutant SOD1 and dysregulated cargo in miRNAs, accounting for MN pathology and homeostatic imbalance (80,81). miRNAs are small non-coding RNAs that control posttranscriptional expression of target genes (82).…”

Astrocyte-Derived Extracellular Vesicles (ADEVs): Deciphering their Influences in Aging

“…In mouse models of ALS, SOD1 mutant astrocytes release EVs that increase microglial expression of inducible nitric oxide synthase (iNOS) and TNF-alpha. These changes are associated with microglial activation, but the ADEVs were also found to induce apoptosis of microglia [ 73 ]. It is well recognized that microglia are important activators of astrocyte responses in a range of human neurological diseases, these ADEV focused studies also indicate a reciprocal communication from astrocyte to microglia, which may have similar influence and impact on neuropathology and function.…”