Oligodendrocyte-specific Argonaute profiling identifies microRNAs associated with experimental autoimmune encephalomyelitis

摘要

Conditional tAGO2 mice express epitope-tagged Argonaute 2 in OLs. a Cre-Lox system adopted to generate a conditional tAGO2 line specific for OLs. b Brain and spinal cord lysates from conditional tAGO2 and floxed AGO2 mice probed by immunoprecipitation (IP) with antibodies specific for AGO2, Myc and GFP. Normal IgGs were used as negative controls in mock IPs. All IP samples were analyzed by Western blotting using an antibody for AGO2. A signal corresponding to the epitope-tagged form of AGO2 was observed at around 120–130 kDa only in conditional tAGO2 mice. c Brain sections from both mouse lines were stained with an antibody for GFP (in green) and one for OLIG1 (in red) and inspected by confocal microscopy. Overlapping staining patters were observed exclusively in conditional tAGO2 mice while floxed controls were positive only to OLIG1 stain. Scale bar: 50 μm. d Brain samples from conditional tAGO2 mice were subjected to miRAP using antibodies for Myc and IgG as negative control. Purified samples were probed for miR-219 and miR-124 levels by qRT-PCR. As miR-219 is more enriched in OLs, its levels are higher in miRAP samples from Myc IPs in comparison to miR-124, which is more enriched in neurons. MiRNA levels are shown as fold differences (mean ± SEM) compared to IgG immunoprecipitated samples, coming from at least two independent miRAP experiments. **P ≤ 0.01