Abstract
Accumulation of mutated superoxide dismutase 1 (mSOD1) in amyotrophic lateral sclerosis (ALS) involves injury to motor neurons (MNs), activation of glial cells and immune unbalance. However, neuroinflammation, besides its detrimental effects, also plays beneficial roles in ALS pathophysiology. Therefore, the targeting of microglia to modulate the release of inflammatory neurotoxic mediators and their exosomal dissemination, while strengthening cell neuroprotective properties, has gained growing interest. We used the N9 microglia cell line to identify phenotype diversity upon the overexpression of wild-type (WT; hSOD1WT) and mutated G93A (hSOD1G93A) protein. To investigate how each transduced cell respond to an inflammatory stimulus, N9 microglia were treated with lipopolysaccharide (LPS). Glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS), known to exert neuroprotective properties, were tested for their immunoregulatory properties. Reduced Fizz1, IL-10 and TLR4 mRNAs were observed in both transduced cells. However, in contrast with hSOD1WT-induced decreased of inflammatory markers, microglia transduced with hSOD1G93A showed upregulation of pro-inflammatory (TNF-α/IL-1β/HMGB1/S100B/iNOS) and membrane receptors (MFG-E8/RAGE). Importantly, their derived exosomes were enriched in HMGB1 and SOD1. When inflammatory-associated miRNAs were evaluated, increased miR-146a in cells with overexpressed hSOD1WT was not recapitulated in their exosomes, whereas hSOD1G93A triggered elevated exosomal miR-155/miR-146a, but no changes in cells. LPS stimulus increased M1/M2 associated markers in the naïve microglia, including MFG-E8, miR-155 and miR-146a, whose expression was decreased in both hSOD1WT and hSOD1G93A cells treated with LPS. Treatment with GUDCA or VS led to a decrease of TNF-α, IL-1β, HMGB1, S100B and miR-155 in hSOD1G93A microglia. Only GUDCA was able to increase cellular IL-10, RAGE and TLR4, together with miR-21, while decreased exosomal miR-155 cargo. Conversely, VS reduced MMP-2/MMP-9 activation, as well as upregulated MFG-E8 and miR-146a, while producing miR-21 shuttling into exosomes. The current study supports the powerful role of overexpressed hSOD1WT in attenuating M1/M2 activation, and that of hSOD1G93A in switching microglia from the steady state into a reactive phenotype with low responsiveness to stimuli. This work further reveals GUDCA and VS as promising modulators of microglia immune response by eliciting common and compound-specific molecular mechanisms that may promote neuroregeneration.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron (MN) disease that affects both upper MNs, in the motor cortex, and lower MNs, in the brainstem and spinal cord
To achieve superoxide dismutase 1 (SOD1) overexpression in microglia, N9 naïve cells were transduced with hSOD1WT and hSOD1G93A coupled with a green fluorescent protein (GFP) tail, as detailed in the methods section, which allowed us to monitor protein expression over time
In N9 hSOD1WT and hSOD1G93A microglia, hSOD1 expression was verified at ∼48 kDa, consistent with the weight of hSOD1 coupled with the GFP tail (Figure 2A)
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron (MN) disease that affects both upper MNs, in the motor cortex, and lower MNs, in the brainstem and spinal cord. Microglia activation was described as having both beneficial and injurious effects in ALS, depending on the relative prevalence of harmful and protective genes, on the ALS disease model and on the state of disease progression (Liao et al, 2012; Brites and Vaz, 2014; Gravel et al, 2016) In this sense, while the reduction of microgliosis was shown to slow ALS progression in the mutated SOD1 mice (Martínez-Muriana et al, 2016), reactive microglia was protective to MN degeneration in a mouse model of TDP-43 proteinopathy (Spiller et al, 2018), reinforcing the relevance of microglia reactivity and function in the ALS context
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