MicroRNA 146a (miR-146a) Is Over-Expressed during Prion Disease and Modulates the Innate Immune Response and the Microglial Activation State

Increasing evidence supports the involvement of microRNAs (miRNAs) in inflammatory and immune processes in prion neuropathogenesis. MiRNAs are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. We established miR-146a over-expression in prion-infected mouse brain tissues concurrent with the onset of prion deposition and appearance of activated microglia. Expression profiling of a variety of central nervous system derived cell-lines revealed that miR-146a is preferentially expressed in cells of microglial lineage. Prominent up-regulation of miR-146a was evident in the microglial cell lines BV-2 following TLR2 or TLR4 activation and also EOC 13.31 via TLR2 that reached a maximum 24–48 hours post-stimulation, concomitant with the return to basal levels of transcription of induced cytokines. Gain- and loss-of-function studies with miR-146a revealed a substantial deregulation of inflammatory response pathways in response to TLR2 stimulation. Significant transcriptional alterations in response to miR-146a perturbation included downstream mediators of the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-κB) and the JAK-STAT signaling pathway. Microarray analysis also predicts a role for miR-146a regulation of morphological changes in microglial activation states as well as phagocytic mediators of the oxidative burst such as CYBA and NOS3. Based on our results, we propose a role for miR-146a as a potent modulator of microglial function by regulating the activation state during prion induced neurodegeneration

Listening to a personal music player is associated with fewer but more serious injuries among snowboarders in a terrain park : a case-control study

Background Some snowboarders listen to music on a personal music player and the objective was to determine if listening to music was associated with injury in a terrain park. Methods A case–control study was conducted at a terrain park in Alberta, Canada during the 2008–2009 and 2009–2010 winter seasons. Cases were snowboarders who were injured in the terrain park and presented to either the ski patrol and/or a nearby emergency department (ED). Demographic, environmental and injury characteristics were collected from standardised ski patrol Accident Report Forms, ED medical records and telephone interviews. Controls were uninjured snowboarders using the same terrain park and were interviewed as they approached the lift-line on randomly selected days and times. Multivariable logistic regression determined if listening to music was associated with the odds of snowboard injury. Results Overall, 333 injured cases and 1261 non-injured controls were enrolled; 69 (21%) cases and 425 (34%) controls were listening to music. Snowboarders listening to music had significantly lower odds of injury compared with those not listening to music (adjusted odds ratio (OR) 0.68; 95% CI 0.48 to 0.98). Snowboarders listening to music had significantly higher odds of presenting to the ED versus ski patrol only compared with those not listening to music (adjusted OR 2.09; 95% CI 1.07 to 4.05). Conclusions While listening to music decreased the odds of any injury in the terrain park, it increased the odds of an injury resulting in ED presentation

EOC 13.31 cells were stimulated with 100 ng/ml semi-pure LPS and temporal changes in cytokine gene expression relative to mock-treated control were measured by microarray analysis.

<p>MiR-146a expression relative to mock-treated control was measured by TaqMan® qRT-PCR and expression is shown as grey bars.</p

Analysis of genes dysregulated by over-expression, or knock-down, in resting EOC 13.31 cells using either miR-146a mimics or anti-miRs.

<p>Genes listed in bold are those that are bioinformatically predicted to be targets of miR-146a using the TargetScan 5.1 program and IPA software.</p

A summary showing key inflammatory response-related genes whose expression is modulated upon the over-expression of miR-146a.

<p>A summary showing key inflammatory response-related genes whose expression is modulated upon the over-expression of miR-146a.</p

Analysis of genes dysregulated by over-expression, or knock-down of miR-146a in resting EOC 13.31 cells using either miRNA mimics or anti-miRs.

<p><b>A.</b> Venn diagram to show the intersection between genes down-regulated by miR-146a over-expression, up-regulated on miR-146a knock-down and those targets bioinformatically predicted using the TargetScan 5.1 program and IPA software. <b>B.</b> Networks showing the interactions between several predicted miR-146a target genes. Shaded grey are those genes also bioinformatically predicted using the TargetScan 5.1 program and IPA software.</p

Altering the expression of miR-146a in microglia.

<p><b>A.</b> MiR-146a over-expression as measured by TaqMan® qRT-PCR in microglia cells after 8, 24, and 48 hrs of treatment with 30 nM (previously optimized, data not shown) of transfected pre-miR-146a in comparison to 30 nM of transfected scrambled negative control miRNA. An average fold change and standard deviation were measured from triplicate experiments. <b>B.</b> MiR-146a knock-down as measured by TaqMan® qRT-PCR in microglia cells after 8, 24, and 48 hrs of treatment with 50 nM (previously optimized, data not shown) of transfected anti-miR-146a in comparison to 50 nM of transfected scrambled negative control miRNA. An average fold change and standard deviation were measured from triplicate experiments.</p

Analysis of genes dysregulated upon over-expression, or knock-down, of miR-146a in stimulated EOC 13.31 cells using either miRNA mimics or anti-miRs.

<p><b>A.</b> Venn diagram to show the intersection between genes down-regulated in LPS stimulated EOC 13.31 cells by miR-146a over-expression, and those up-regulated upon miR-146a knock-down. Also shown are those targets bioinformatically predicted using the TargetScan 5.1 program and IPA software. <b>B.</b> Schematic showing alterations in expression in key inflammatory response-related genes in LPS stimulated EOC 13.31 cells following miR-146a over-expression. Colored green are those down-regulated genes, colored red are up-regulated genes, while those highlighted in blue are bioinformatically predicted targets of miR-146a using the TargetScan 5.1 program and IPA software.</p

Expression levels of microglial cell surface receptors in hippocampal tissue of RML infected, symptomatic mice as determined by microarray analysis.

<p>Expression levels of microglial cell surface receptors in hippocampal tissue of RML infected, symptomatic mice as determined by microarray analysis.</p

Inflammation-related genes that are altered in response to increased expression of miR-146a in LPS stimulated EOC 13.31 cells.

<p>Down-regulated genes are in bold italics, up-regulated genes are in plain type.</p