Regulation of a prion-induced immune response by miRNA-146a
Prion diseases are curious neurodegenerative diseases characterized by the conversion of a cellular protein, PrPC, into an infectious isoform, PrPSc. One of the earliest hallmarks of disease and concurrent with prion deposition, is the activation of the brain’s principal immune effector cells, microglia. In prion disease, activated microglia synthesize fairly low levels of pro-inflammatory cytokines, presumably to ameliorate the severe pathology that can arise in host tissue as a result of an acute inflammatory response. The specific stimuli and signaling pathways that lead to this modulation of function are as yet unknown. However, the involvement of miRNAs, a recently identified class of regulatory molecules, is likely. Recently, miR-146a was found to be upregulated in the brains of prion infected mice. In addition, its expression was found to be enriched in cells of microglial origin. It was hypothesized that, given the immunomodulatory role ascribed to miR-146a in macrophages, upregulation of miR-146a may function to attenuate the microglial immune response to prion infection. The first objective was to identify inflammatory related miRNAs associated with prion disease in microglia. Using Taqman Low Density Arrays, allowing for the detection of hundreds of miRNAs at once, the miRNAs of microglia treated with inflammatory agonists were profiled. The miRNA profile of activated microglia was found to be similar to that of macrophages. Furthermore, among the miRNAs profiled, miR-146a and miR-155 were the most highly induced and persistently expressed over 24 hours. The second objective was to investigate miR-146a induction. Therefore, microglia were treated with various agonists and miR-146a expression was determined using Taqman miR-146a assays. Although treatment with a PrP-mimic did not induce miR-146a expression, stimulation of TLRs 1, 2, 4, and 5, resulted in significant over-expression similar to what has been described previously. Moreover, in contrast to the rapid and transient induction of inflammatory mediators, miR-146a follows alternate kinetics functioning to prolong the dampening of the innate immune response following activation via TLR4 and TLR2. By employing a functional proteomic strategy, the third objective was to identify miR-146a regulated proteins. First, miR-146a expression was manipulated using miR-146a mimics and miR-146a inhibitors. Secondly, the functional model was validated by confirming decreased expression of IL6 by ELISA in miR-146a over-expressing microglia cells. Lastly, using Tandem Mass Tag labels to discriminate between treatment group (miR-146a mimic and TLR2 agonist) and control group (scrambled-miR and TLR2 agonist), the effect of miR-146a on the proteome was determined. In total, 172 proteins were identified as being miR-146a regulated and gene ontology assignment resulted in an over-representation of proteins involved in cellular dynamics capable of altering the activation state of microglia. After filtering for bioinformatically predicted targets and those implicated in a similar genomic study, it was decided to further investigate proteins ARF6, RhoA and NOS2 based on their role in modulating the phagocytic potential of microglia. The final objective was to validate miR-146a putative direct targets identified from the proteomics analysis. Luciferase expression of the 3’UTR of targets upon transfection with miR-146a were determined. Based on luciferase analysis, NOS2 appears to be directly targeted by miR-146a and this was also confirmed by western blot. While production of NOS2 by microglia under an acute activation state serves to support and protect CNS homeostasis, chronic expression of this factor can lead to neurotoxicity. Therefore, miR-146a appears to have an overarching role in altering microglial activation during prion disease thus protecting neurons from bystander damage. Taken together, these results suggest that miR-146a could play an important role in the prion disease process by specifically attenuating the microglial induced immune response. Therefore, manipulation of miR-146a may represent a novel therapeutic strategy. Furthermore, given that miR-146a de-regulation has been observed in other neurodegenerative diseases, these results may well extend beyond the realm of prion disease. Lastly, although practical limitations relating to the sensitivity of the comparative proteomics methodology meant that it alone were not sufficient to identify miRNA targets, an integrated approach that takes into consideration genomic and bioinformatic strategies is most promising.
Prion disease, Innate immunity, Microglia, MiRNA, Neurodegeneration