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DR (microglial marker, mouse clone CR3/43; Dako; 1:100). Signal was detected using the chromogen 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO, USA).
Quantitative reverse-transcription PCR analysis (RT-qPCR)
miRNAs (miR34a-5p, miR34b-5p, miR34c-5p, miR302a-3p, miR21-5p and the reference small nuclear RNAs, Rnu6B and Rnu44) expression was analyzed using Taqman micro RNA assays (Applied Biosystems, Foster City, CA). cDNA was generated using Taqman MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions and the PCRs were run on a Roche Lightcycler 480 thermo- cycler (Roche Applied Science, Basel, Switzerland). Quantification of data was performed using the computer program LinRegPCR in which linear regression on the Log (fluores- cence) per cycle number data is applied to determine the amplification efficiency per sample43, 44. The starting concentration of each specific product was divided by the start- ing concentration of reference genes (geometric mean of Rnu6B and Rnu44 values) and this ratio was compared between groups.
To evaluate expression of miRNA targets and inflammation-related genes, 2.5μg of total RNA was reverse-transcribed into cDNA using oligodT primers. PCR primers (Eurogentec, Belgium) were designed using the Universal ProbeLibrary of Roche (https:// www.roche-applied-science.com) on the basis of the reported cDNA sequences. For each PCR, a mastermix was prepared on ice, containing per sample: 1 μl cDNA, 2.5 μl of 2x SensiFASTTM SYBR Green Reaction Mix (Bioline Inc, Taunton, MA, USA), 0.4 μM of both reverse and forward primers and the PCRs were run on a Roche Lightcycler 480 thermocycler (Roche Applied Science, Basel, Switzerland). Quantification of data was performed as described for the Taqman PCR and the starting concentration of each specific product was divided by the geometric mean of the starting concentration of ref- erence genes (EF1A, C1orf43 and SNRPD3) and this ratio was compared between patient/ control groups.
Human astrocyte-enriched cell cultures
Primary fetal astrocyte-enriched cell cultures were prepared from tissue collected from donors from whom a written informed consent for the use of the material for research purposes has been obtained by the Bloemenhove Clinic (Heemstede, The Netherlands). Cell isolation was performed as described elsewhere 42. Cell cultures were stimulated with human recombinant (r)IL-1β (Peprotech, NJ, USA; 10 ng/ml) for 24 hours. For transfection Fetal astrocyte cultures were transfected with either mirVanaTM miR34b-5p miRNA mimic or mirVanaTM miRNA Mimic, Negative Control #1 (both from ThermoFisher Scientific, Landsmeer, Netherlands) at a final concentration of 50 nM using Lipofectamine® 2000 transfection reagent (ThermoFisher Scientific, Landsmeer, Netherlands). Cells were harvested after 24 hours of stimulation/transfection for RNA isolation and RT-qPCR (miR34a5p, miR34b-5p, IL-1β, IL-6, COX-2).
Primary mouse hippocampal neuron cultures, immunofluorescent staining and neurite growth analysis
Primary mouse hippocampal neuron cultures were prepared from postnatal day 0 (P0) C57BL/6 mouse brains. Cells were plated on 15 mm coverslips coated with poly-D-ly-