Downregulated Glia Interplay and Increased miRNA-155 as Promising Markers to Track ALS at an Early Stage

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause. Absence of specific targets and biomarkers compromise the development of new therapeutic strategies and of innovative tools to stratify patients and assess their responses to treatment. Here, we investigate changes in neuroprotective-neuroinflammatory actions in the spinal cord of SOD1 mice, at presymptomatic and symptomatic stages to identify stage-specific biomarkers and potential targets. Results showed that in the presymptomatic stage, there are alterations in both astrocytes and microglia, which comprise decreased expression of GFAP and S100B and upregulation of GLT-1, as well as reduced expression of CD11b, M2-phenotype markers, and a set of inflammatory mediators. Reduced levels of Connexin-43, Pannexin-1, CCL21, and CX3CL1 further indicate the existence of a compromised intercellular communication. In contrast, in the symptomatic stage, increased markers of inflammation became evident, such as NF-κB/Nlrp3-inflammasome, Iba1, pro-inflammatory cytokines, and M1-polarizion markers, together with a decreased expression of M2-phenotypic markers. We also observed upregulation of the CX3CL1-CX3CR1 axis, Connexin-43, Pannexin-1, and of microRNAs (miR)-124, miR-125b, miR-146a and miR-21. Reduced motor neuron number and presence of reactive astrocytes with decreased GFAP, GLT-1, and GLAST further characterized this inflammatory stage. Interestingly, upregulation of miR-155 and downregulation of MFG-E8 appear as consistent biomarkers of both presymptomatic and symptomatic stages. We hypothesize that downregulated cellular interplay at the early stages may represent neuroprotective mechanisms against inflammation, SOD1 aggregation, and ALS onset. The present study identified a set of inflamma-miRNAs, NLRP3-inflammasome, HMGB1, CX3CL1-CX3CR1, Connexin-43, and Pannexin-1 as emerging candidates and promising pharmacological targets that may represent potential neuroprotective strategies in ALS therapy.

Cortical Neurotoxic Astrocytes with Early ALS Pathology and miR-146a Deficit Replicate Gliosis Markers of Symptomatic SOD1G93A Mouse Model

“…Among other miRNAs suspected to contribute to ALS pathology is the altered expression of miR-146a and miR-21in the SC and brainstem of ALS transgenic mice [81]. Similarly to these authors we also found reduced miR-146a levels in the cortical homogenates, a feature not observed in the SC from the same mSOD1 mice model, in either stage 1 or stage 2 [19]. Again, our results contrast with other studies showing an elevated miR-146a expression in patient SC samples [82], suggesting the existence of phenotypic changes in ALS patients and rodent models, while supporting the new label of precision medicine.…”

“…1b). WT mice only exhibited one band corresponding to the native mouse SOD1, as previously described [19]. Now considering the phosphorylated eIF2α and the transcription factor ATF4, we observed that both were reduced, but only in mSOD1 mice at stage 2 (p < 0.01, Fig.…”

“…This finding together with the signal transmission failure deriving from the impaired synaptic protein expression, will account to aggravate neurodegeneration. In what concerns miR-155, whose upregulation is associated with microglia activation in the SC, and pointed as a promising therapeutic target in ALS [22,19], we were unable to identify any alteration in the cortical tissue samples. Actually, no microglia activation based on Cd11b expression was even detected in cortical homogenates suggesting the presence of regional variants and microglia phenotypic variability in ALS, which may have implications when deciding therapeutic strategies.…”

“…Expression of miRNAs was also assessed by qRT-PCR. After RNA quantification, cDNA conversion for miRNA quantification was performed with the universal cDNA Synthesis Kit (Exiqon), as described [19], using 5 ng total RNA. qRT-PCR was performed using the above indicated 7300 Real Time PCR System and 96-well plates.…”

“…Lately, we recognized immune-enriched microRNAs (miRNA), namely miRNA(miR)-124, miR-125b, miR-146a, miR-21 and miR-155 in the SC of symptomatic mSOD1 mice, together with upregulated nuclear factor kappa B (NF-kB)/NLRP3-inflammasome, HMGB1and Cx43, and with downregulated glial fibrillary acidic protein (GFAP), glutamate aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) [19]. Differentially expressed miR-22, miR-155, miR-125b and miR-146b were found among the most highly modulated in microglia isolated from the brain cortex of SOD1G93A neonatal mice [20].…”