Fig 2: miR-143 overexpression affects proliferation of RA-treated NT2/D1 cells, decreases ß-dystrobrevin protein level and increases synapsin I gene expression.(A) Western blot analysis of ß-DB total (left panels) and nuclear (right panels) protein expression level in NT2/D1 cells transiently transfected with miR-143 (miR-143), as compared with untransfected NT2/D1 control cells (+) and NT2/D1 cells transiently transfected with a non-targeting negative control miRNA (miR-C); the degree symbol (°) indicates a concentration of 20 nM, the asterisk (*) represents 10 nM. (B) Cell proliferation analysis of RA-treated miR-143 transiently transfected-NT2/D1 cells compared with miR-C control cells. (C, D) Real-time PCR analysis of MAP2 (C) and NF-L (D) mRNA expression in RA-treated miR-143 transiently transfected-NT2/D1 cells compared with control cells. (E) Real-time PCR analysis of synapsin I (SynI) mRNA expression in RA-treated miR-143 transiently transfected-NT2/D1 cells compared with control cells. (F) Western blot analysis of SynI protein expression level in miR-143 transiently transfected-NT2/D1 cells (miR-143), compared with NT2/D1 control cells (miR-C). (A, F) One representative experiment out of three is shown; actin and nucleolin are shown as internal control of total and nuclear protein extracts, respectively; transfections were performed with 10 nM miRNAs, unless otherwise specified. (B-E) The results of three independent experiments (mean ± SEM values) are shown; * represents p<0.05; the lack of error bars indicates that they are smaller than the symbol. Arrow indicates the start of treatment with RA of the transfected NT2/D1 cells (day 0); (C-E) A.U., arbitrary units.

Fig 3: Delayed expression of synaptic markers after exposure to neonatal hyperoxia. Real-time PCR expression analysis of the synaptic markers: synapsin 1 (Syn1), synapsin 2 (Syn2), synaptotagmin 1 (Syt1), synaptotagmin2 (Syt2), and synaptophysin (Syp) at the age P7, P9, P11, P14, and P30. Gene expression of Syn1 was decreased in cortical samples of animals previously exposed to neonatal hyperoxia (P5-P7) at the ages P7, P9, P11, and P14 and returns to control level at P30 (a). At P7-P14, cortical RNA expression of Syn2 was decreased in hyperoxia animals compared to control litters. At P30, Syn2 expression returns to control level (b). RNA expression of Syt1 was reduced at P9 (c) in the hyperoxia group. Cortical expression of Syt2 was reduced at P11 in mice previously exposed to hyperoxia and returns to control level at P14 (d). At the ages P7, P9, and P11, cortical RNA expression of Syp was decreased in the hyperoxia group (e) (for qPCR: n = 6-8, t-test, *P < 0.05; **P < 0.01; ***P < 0.001). Immunohistochemical staining for parvalbumin (PVALB) and synapsin 1/2 (SYN1/2) of 10 µm coronal brain sections at the age P14 reveals SYN1/2 expression of cortical PVALB+ interneurons (f). Scale bar 20 µm.

Fig 4: Aß-derived fragments differently affect phosphorylation of Syn1 at serine 551 and calcineurin activity. a Representative images of mature cortical neurons (17–22 DIV) treated with vehicle (Ctrl), rat Aß1-40, rat Aß1-42, human Aß1-16, human/rat Aß17-40, human/rat Aß17-42 for 1 h at 37 °C and stained with antibodies recognizing Syn1 phosphorylation at serine 551 (pSyn1S551, green). Syn1,2 (magenta) was used as a synaptic marker. Scale bar 5 µm. b Quantification of the density and c IF intensity of synaptic puncta positive for pSyn1S551. (D) Quantification of calcineurin activity in cortical neurons (21DIV) treated with Aß peptides, BgTx and PNU-120596. Data were normalized to the Ctrl. In plots, the boxes show the interquartile range and median, whiskers minimum and maximum values, and + indicates the mean. Values in brackets are the in b and c number of analysed cells obtained from three independent experiments and in d numbers of independent experiments. One-way ANOVA followed by Dunnett´s post hoc test was applied to assess statistical significance of comparison to Ctrl; *p < 0.05, **p < 0.01, ****p < 0.0001

Fig 5: Hypoxia affects synapsin I expression in NT2/D1 cells.(A) Real-time PCR analysis of SynI mRNA expression in untreated (d0) and RA-treated NT2/D1 cells under normoxic (21% O2) and hypoxic (1% O2) conditions. (B) Lower panels: Western blot analysis of SynI protein expression levels in untreated (d0) and RA-treated NT2/D1 cells, under normoxic (21% O2) and hypoxic (1% O2) conditions. Upper panel: densitometry analysis of SynI protein expression levels compared with actin levels. (A) The results of three independent experiments (mean ± SEM values) are shown; ** represents p<0.01; the lack of error bars indicates that they are smaller than the symbol. A.U., arbitrary units. (B) One representative experiment out of three is shown; actin is shown as internal control of total protein extracts.