Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder affecting motor neurons (MNs). Evidences indicate that ALS is a non-cell autonomous disease in which glial cells participate in both disease onset and progression. Exosomal transfer of mutant copper-zinc superoxide dismutase (mSOD1) from cell-to-cell was suggested to contribute to disease dissemination. Data from our group and others showed that exosomes from activated cells contain inflammatory-related microRNAs (inflamma-miRNAs) that recapitulate the donor cell. While glia-derived exosomes and their effects in neurons have been addressed by several studies, only a few investigated the influence of motor neuron (MN)-derived exosomes in other cell function, the aim of the present study. We assessed a set of inflamma-miRs in NSC-34 MN-like cells transfected with mutant SOD1(G93A) and extended the study into their derived exosomes (mSOD1 exosomes). Then, the effects produced by mSOD1 exosomes in the activation and polarization of the recipient N9 microglial cells were investigated. Exosomes in coculture with N9 microglia and NSC-34 cells (either transfected or not) showed to be transferred into N9 cells. Increased miR-124 expression was found in mSOD1 NSC-34 cells and in their derived exosomes. Incubation of mSOD1 exosomes with N9 cells determined a sustained 50% reduction in the cell phagocytic ability. It also caused a persistent NF-KappaB activation and an acute generation of NO, MMP-2 and MMP-9 activation, as well as upregulation of IL-1β, TNF-α, MHC-II and iNOS gene expression, suggestive of induced M1 polarization. Marked elevation of IL-10, Arginase 1, TREM2, RAGE and TLR4 mRNA levels, together with increased miR-124, miR-146a and miR-155, at 24 h incubation, suggest the switch to mixed M1 and M2 subpopulations in the exosome-treated N9 microglial cells. Exosomes from mSOD1 NSC-34 MNs also enhanced the number of senescent-like positive N9 cells. Data suggest that miR-124 is translocated from the mSOD1 MNs to exosomes, which determine early and late phenotypic alterations in the recipient N9-microglial cells. In conclusion, modulation of the inflammatory-associated miR-124, in mSOD1 NSC-34 MNs, with potential benefits in the cargo of their exosomes may reveal a promising therapeutic strategy in halting microglia activation and associated effects in MN degeneration.

MiRNA(miR)-124 is an important regulator of neurogenesis, but its upregulation in SOD1G93A motor neurons (mSOD1 MNs) was shown to associate with neurodegeneration and microglia activation. We used pre-miR-124 in wild-type (WT) MNs and anti-miR-124 in mSOD1 MNs to characterize the miR-124 pathological role. miR-124 overexpression in WT MNs produced a miRNA profile like that of mSOD1 MNs (high miR-125b; low miR-146a and miR-21), and similarly led to early apoptosis. Alterations in mSOD1 MNs were abrogated with anti-miR-124 and changes in their miRNAs mostly recapitulated by their secretome. Normalization of miR-124 levels in mSOD1 MNs prevented the dysregulation of neurite network, mitochondria dynamics, axonal transport, and synaptic signaling. Same alterations were observed in WT MNs after pre-miR-124 transfection. Secretome from mSOD1 MNs triggered spinal microglia activation, which was unno-ticed with that from anti-miR-124-modulated cells. Secretome from such modulated MNs, when added to SC organotypic cultures from mSOD1 mice in the early symptomatic stage, also coun-teracted the pathology associated to GFAP decrease, PSD-95 and CX3CL1-CX3CR1 signaling im-pairment, neuro-immune homeostatic imbalance, and enhanced miR-124 expression levels. Data suggest that miR-124 is implicated in MN degeneration and paracrine-mediated pathogenicity. We propose miR-124 as a new therapeutic target and a promising ALS biomarker in patient sub-populations.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with short life expectancy and no effective therapy. We previously identified upregulated miR-124 in NSC-34-motor neurons (MNs) expressing human SOD1-G93A (mSOD1) and established its implication in mSOD1 MN degeneration and glial cell activation. When anti-miR-124-treated mSOD1 MN (preconditioned) secretome was incubated in spinal cord organotypic cultures from symptomatic mSOD1 mice, the dysregulated homeostatic balance was circumvented. To decipher the therapeutic potential of such preconditioned secretome, we intrathecally injected it in mSOD1 mice at the early stage of the disease (12-week-old). Preconditioned secretome prevented motor impairment and was effective in counteracting muscle atrophy, glial reactivity/dysfunction, and the neurodegeneration of the symptomatic mSOD1 mice. Deficits in corticospinal function and gait abnormalities were precluded, and the loss of gastrocnemius muscle fiber area was avoided. At the molecular level, the preconditioned secretome enhanced NeuN mRNA/protein expression levels and the PSD-95/TREM2/IL-10/arginase 1/MBP/PLP genes, thus avoiding the neuronal/glial cell dysregulation that characterizes ALS mice. It also prevented upregulated GFAP/Cx43/S100B/vimentin and inflammatory-associated miRNAs, specifically miR-146a/miR-155/miR-21, which are displayed by symptomatic animals. Collectively, our study highlights the intrathecal administration of the secretome from anti-miR-124-treated mSOD1 MNs as a therapeutic strategy for halting/delaying disease progression in an ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neurons (MNs) loss and glial reactivity in brain and spinal cord (SC). A dysregulated expression of microRNAs (miRNAs) have been implicated in neurodegeneration and neuroinflammation in ALS. MiRNA(miR)-124 is elevated in the cerebrospinal fluid of ALS patients and in the SC of the ALS mice with the G93A mutation in human superoxide dismutase-1 (mSOD1). We demonstrated that this miRNA is upregulated in MN-NSC-34 overexpressing mSOD1 and in small extracellular vesicles isolated from cell secretome [1]. We also observed miR-155 upregulation in the SC of pe-symptomatic mSOD1 mice [2], often associated with mSOD1 microglia [3], but with unknown effect when expressed by astrocytes. In contrast, miR-146a was downregulated either in the cortical brain (CB) of symptomatic mSOD1 mice and in isolated astrocytes [4], suggesting distinct miRNA deregulation depending on the CNS region. Here, we evaluated the effects of miR-155/146a/124 modulation in astrocytic and MN function. For that, we used primary astrocytes from mSOD1 mice pups (CB or SC) and mSOD1 MN-NSC-34. Cells were transfected with anti-miR-155 (SC astrocytes), premiR-146a (CB astrocytes) or pre/anti-miR-124 (MN). We also explored whether changes in MN-miR-124 expression impact on secretome-mediated astrocytic function. Downregulation of miR-155 in mSOD1 SC astrocytes counteracted the inflammatory milieu, by decreasing TNF-α/IL-1β/HMGB1/miR-124, and increasing IL-6/IL-10/SOCS1/miR-146a expression, without changing the astrocytic aberrant phenotype (GFAPlow/Cx43high/S100Bhigh). However, miR-146a upregulation in mSOD1 CB astrocytes neutralized both the phenotypic aberrancy and the inflammatory profile. The upregulation of miR-124 in WT-MNs mimicked the pathological features of mSOD1-MNs, namely inflammatory-associated-miRNAs (increased miR-125b/decreased miR-146a/-21), synaptic dysfunction (increased synaptophysin/decreased PDS-95), neurite loss, reduced mitochondria viability and axonal transport deficits (increased dynein/decreased kinesin), while miR-124 downregulation was able to prevent those dysfunctionalities in mSOD1-MNs. The astrocytic aberrant phenotype of mSOD1 SC astrocytes was observed in WT cells upon incubation with secretome from mSOD1-MNs or pre-miR-124-WT-MN, but prevented in the presence of the secretome of anti-miR-124-mSOD1-MN. More importantly, secretome derived from anti-miR-124-mSOD1-MN rescued the aberrant phenotype of mSOD1 SC astrocytes by increasing GFAP/GLT-1 expression. Overall, our results highlight that combined modulation of specific miRNAs in different target cells may represent a promising therapeutic potential in ALS disease.