CSF from patients with MDE, but not with FTLD, contains elevated levels of let-7b and let-7e copies compared to healthy controls.

<p>(A) CSF from healthy controls (<i>n</i> = 10, Co-1 –Co-10) and from patients with FTLD (<i>n</i> = 8, FTLD-1 –FTLD-8) or MDE (<i>n</i> = 8, D-1 –D-8) were assayed by qPCR using primers specific for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, or miR-124. Data are presented as box-and-whisker-plots of mean CT-values performed in triplicates with median for each miRNA. (B) CSF from samples described in (A) were assayed by qPCR using primers specific for let-7b or let-7e and were normalized to the standard of the respective synthetic miRNA. Data are presented as mean ± SD. Each patient resembles one dot. Statistical analysis was performed using one-way ANOVA followed by Sidak’s multiple comparison post hoc test of the respective patient group <i>vs</i>. control group. Global <i>p</i>-values, as indicated in the figure. ns, not significant. <i>p</i>-values were adjusted (adj.) for age and gender by ANCOVA, as indicated.</p

Median CT-values from data depicted in Fig 1 and Fig 3A.

<p>Median CT-values from data depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200602#pone.0200602.g001" target="_blank">Fig 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200602#pone.0200602.g003" target="_blank">Fig 3A</a>.</p

CSF from patients with AD contains elevated levels of let-7b and let-7e copies compared to healthy controls.

<p>CSF from patients with AD (<i>n</i> = 10–11, AD-1 –AD-6; AD-8 –AD-12) and healthy controls (<i>n</i> = 10, Co-1 –Co-10) were assayed by qPCR using primers specific for let-7a, let-7b, let-7c, let-7e, or let-7g and were normalized to the standard of the respective synthetic miRNA. Data are presented as mean ± SD. Each patient resembles one dot. Statistical analysis was performed using unpaired <i>t</i>-test, and <i>p</i>-values were adjusted (adj.) for age and gender by ANCOVA, as indicated.</p

Demographic data, neuropsychometric data, and biomarkers of patients analyzed in Fig 1, Fig 2, and Fig 3.

<p>Demographic data, neuropsychometric data, and biomarkers of patients analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200602#pone.0200602.g001" target="_blank">Fig 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200602#pone.0200602.g002" target="_blank">Fig 2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200602#pone.0200602.g003" target="_blank">Fig 3</a>.</p

Neurotoxic potential of let-7b and let-7e <i>in vitro</i>.

<p>(A) Cortical neurons from C57BL/6 mice were incubated with 5 μg/ml of the respective native oligoribonucleotide, as indicated, for 5 days. Treatment with imiquimod (10 μg/ml) served as a positive control. Subsequently, cells were fixed and immunostained with neurofilament Ab (red), MAP-2 Ab (green), and stained with DAPI (blue). Scale bar 100 μm. (B, C) C57BL/6 neurons or (D, E) SH-SY5Y cells were incubated with indicated doses of native let-7b, let-7e, or a mutated control oligoribonucleotide for 4 days or with 5 μg/ml of native let-7b, let-7e, or a mutated control oligoribonucleotide for indicated durations. Treatment with imiquimod (10 μg/ml) served as a positive control. Cells were immunostained with NeuN Ab, stained by TUNEL assay, or with DAPI. Each condition was performed in duplicate and averaged. NeuN-positive cells were quantified and were expressed as relative neuronal viability. TUNEL-positive cells were quantified, set in relation to NeuN-positive cells, and were expressed as fold-change. Mean ± SD from 3–4 individual experiments, Kruskal-Wallis test followed by Dunn´s Multiple comparison post hoc test of negative control <i>vs</i>. treatment. <i>P</i> values in (B) depicting the dose response: let-7b **<i>p</i> = 0.0036; time response: let-7b ***<i>p</i> = 0.0002, let-7e *<i>p</i> = 0.0107. <i>P</i> values in (C) depicting the dose response: let-7b *<i>p</i> = 0.0136; time response: let-7b ***<i>p</i> = 0.0005. (D, E) <i>P</i>-values for relative neuronal viability: let-7b **<i>p</i> = 0.0047; TUNEL assay: let-7b ***<i>p</i> = 0.0001. #<i>p</i><0.05, ##<i>p</i><0.01, ###<i>p</i><0.005. mut. oligo, mutated oligoribonucleotide.</p

Members of the <i>let-7</i> miRNA family are present in CSF from healthy controls and patients with AD.

<p>CSF from healthy controls (<i>n</i> = 10, Co-1 –Co-10) or patients with AD (<i>n</i> = 12, patient AD-1 –AD-12) were assayed by qPCR using primers specific for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, or miR-124. Data are presented as box-and-whisker-plots of mean CT-values performed in triplicates with median for each miRNA.</p