Exosomes from NSC-34 Cells Transfected with hSOD1-G93A Are Enriched in miR-124 and Drive Alterations in Microglia Phenotype.

Sara Pinto,Ana R Vaz,Marta Barbosa,Carolina Cunha,Dora Brites

doi:10.3389/fnins.2017.00273

Abstract

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 1 (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 with either wild-type (wt) human SOD1 or mutant SOD1(G93A)] 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-kB 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.

Highlights

Since the beginning of the last decade, exosomes, and their role in the central nervous system (CNS), namely in the pathophysiology of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), have been of increased interest in the science community
NSC-34 cells were grown in proliferation media [Dulbecco’s modified Eagle’s medium (DMEM) high glucose with glutamine, w/o pyruvate, supplemented with 10% of fetal bovine serum (FBS) and 1% of Penicillin/Streptomycin] and selection was made with Geneticin 418 sulfate (G418) at 0.5 mg/ml (Vaz et al, 2015)
It is important to refer that similar total RNA concentrations were found for exosomes isolated from wt or mutant copper-zinc superoxide dismutase 1 (mSOD1) cells (Figure 1E)

Summary

Introduction

Since the beginning of the last decade, exosomes, and their role in the central nervous system (CNS), namely in the pathophysiology of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), have been of increased interest in the science community. Among the several potential pathogenic genes in fALS and sporadic cases (sALS), the most frequent are C9orf (40% of fALS and 5–6% of sALS cases) and SOD1 (20% of fALS and 3% of sALS cases) (Kruger et al, 2016). This fatal and progressive neurodegenerative disease affects motor neurons (MNs) in the spinal cord and motor cortex. It was shown that astrocyte-derived exosomes may transfer mSOD1 to MNs contributing to neurodegeneration and disease spread (Basso et al, 2013). It was demonstrated that both mSOD1 and misfolded wild-type (wt) SOD1 from NSC-34 MN-like cells are transferred on the surface of exosomes and delivered to neighboring MN cells by macropinocytosis (Grad et al, 2014b)

Methods

Results

Conclusion