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observed for miR146a 17. In mouse macrophages, it was reported that miR147, the murine homolog for miR147b, was up-regulated after Toll-like receptor 4 (TLR4) activation by LPS, acting as a negative regulator of the macrophage inflammatory response 27. In these cultures, miR147b mimic transfection decreased levels of TNF-α and IL-6 under inflam- matory conditions, indicating similar functions of this miRNA in various cell types.
miR146a and miR147b decrease proliferation and promote neuronal differentiation
Other than expressing inflammatory cytokines, severe reactive astrogliosis is also asso- ciated with other classical hallmarks like increased proliferation and aberrant generation of astrocytes 5. This is found in the human epileptogenic brain as well as in experimental epilepsy models. This is illustrated in an astrocyte-specific conditional TSC1 knockout mouse model with spontaneous seizures in which the onset of seizures is concordant with increased astrocytic proliferation 41, 42. Also, in vitro astrocyte proliferation was found to be increased after treatment with IL-1β 7. We found that miR146a and miR147b overexpression decreased the proliferation rate of fetal astrocyte cultures. Our results for miR146a are in line with previous studies showing that infection of murine astrocytes with miR146a-overexpressing lentivirus inhibited proliferation 43, 44. For miR147b, similar effects are observed in colon cancer cells 29 and in a human breast adenocarcinoma cell line, in which overexpression of miR147 led to suppression of proliferation by inhibition of Akt/mTOR signaling 28.
Astrocytes are generated from NSCs, where tight regulation of differentiation is critical for the generation of a balanced number of astrocytes and neurons 45. It was recently reported that neuronal hyperexcitability as seen in epilepsy increases the aber- rant generation of astrocytes from NSCs 6. We found that overexpression of miR147b or miR146a induced neuronal differentiation of NSCs derived from fetal brain. Therefore, miR146a and miR147b might have beneficial effects in astrogliosis and scar formation in several pathologies. In order to find targets concerning NSC cell fate decision, we exam- ined multiple targets in two important signaling pathways that instruct astrogenesis: the
Figure 6. miR147b expression in tuberous sclerosis complex (TSC) cortical tubers and temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), and modulation of inflammation by miR147b in TSC cortical tuber cell cultures. A-H In situ hybridization of miR147b expression in TSC, TLE-HS and control brain tissue. In control cortex, miR147b expression is only visible in neurons in the gray matter (GM, arrows in A), with no expression in the white matter (WM, C). In TSC tubers, besides expression in dysmorphic neurons and giant cells (arrows in B and D), miR147b is also visible in astrocytes (arrow heads, B and D). Inset in D shows co-localization of miR147b with GFAP in the tuber. In control hippocampus, miR147b is expressed by neurons (E, G), whereas in TLE-HS expression is also localized in astrocytes (arrowheads in F and H) besides neurons (arrows in F and H). Inset in H shows co-localization of miR147b with GFAP in the hilus. I-K Quantitative real-time PCR of mRNA expression in TSC cell cultures. TSC cells were transfected for 24 hours with miR147b mimic (50 nM) followed by 24 hours of IL-1β stimulation (10 ng/ml). miR147b mimic decreased the mRNA levels of pro-inflammatory cytokines IL-6 (I) and COX-2 (J), complement component 3 (C3) showed a trend towards decreased expression (K). Scale bar in H: 100 μm, in inset: 25 μm. ***p<0.001 compared to control, Mann Whitney U test. Experiments in I-K were performed in triplicate in cultures from two separate donors and data are mean ± SEM. **p<0.01, Mann Whitney U test.