Increased 14-3-3 levels in neurons treated with Aβ-Reelin.

<p>Primary neurons were treated without Reelin (No Reel) or with Cont-Reelin (C-Reel) or Aβ-Reelin (Aβ-Reel), or Cont-Reelin pre-incubated with the antibody CR50 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072297#pone-0072297-g001" target="_blank">Figure 1</a>; and cell lysates were probed for 14-3-3 and normalized respect to total tau levels (n=3 independent experiments, values are means ± SEM). *Significantly different (<i>p</i> < 0.05) from C-Reel treated cells.</p

Aβ-Reelin fails to induce phosphorylation of Dab1 and GSK3β.

<p>Total cell lysates from primary mouse cortical neuron cultures treated without Reelin (No Reel) or with similar amounts of Reelin obtained from SH-SY5Y cells treated with 0 (control, C-Reel) or 10 µM of Aβ42 (Aβ-Reel) were probed with antibodies specific for total Dab1 and anti-phosphotyrosine Dab1 (P-Dab1), and GSK3β phosphorylated at serine 9 and total GSK3β. Levels of phosphorylated Dab1, GSK3β and tau were normalized to total Dab1, and GSK3β levels and tau respectively (n= 5 independent experiments for Dab1, GSK3β; n= 3 independent experiments for CR50). Data (means ± SEM) were analyzed using ANOVA analysis of variance, followed by Tukey test to compare all groups. *Significantly different (<i>p</i> < 0.05) from C-Reel treated cells.</p

Aβ-Reelin fails to reduce tau phosphorylation.

<p>Levels of phospho-tau (P-tau; antibodies AT8 and PHF13) and total tau were calculated in primary mouse cortical neuron cultures treated with similar amounts of Reelin obtained from SH-SY5Y cells treated with 0 (control, C-Reel), 1 or 10 µM of Aβ42 (Aβ-Reel), or scrambled Aβ42 peptide (Aβsc-Reel; 10 µM) (at least n= 6, from 3 independent experiments). The comparison with non-treated cultures (No Reel) is also shown. In some experiments Cont-Reelin was pre-incubated with the antibody CR50 (15 µg/ml) for 1 h prior to treatment (n=3). Neuronal cultures were also incubated with Reelin glycoforms obtained from SH-SY5Y cells treated with 0 (control, C-Reel) or 100 µM of the mannosidase inhibitor DMJ (DMJ-Reel) (n= 6, from 2 independent experiments). The data represent the percentages of variation (means ± SEM) with respect to values determined for C-Reel treated cells. Data were analyzed using ANOVA analysis of variance, followed by Tukey test to compare all groups. *Significantly different (<i>p</i> < 0.05) from C-Reel treated cells.</p

Elevated plasma microRNA-206 levels predict cognitive decline and progression to dementia from mild cognitive impairment

The need for practical biomarkers for early diagnosis of Alzheimer’s disease (AD) remains largely unmet. Here we investigated the use of blood-based microRNAs as prognostic biomarkers for AD and their application in a novel electrochemical microfluidic device for microRNA detection. MicroRNA transcriptome was profiled in plasma from patients with mild cognitive impairment (MCI) and AD. MicroRNAs Let-7b and microRNA-206 were validated at elevated levels in MCI and AD, respectively. MicroRNA-206 displayed a strong correlation with cognitive decline and memory deficits. Longitudinal follow-ups over five years identified microRNA-206 increases preceding the onset of dementia. MicroRNA-206 was increased in unprocessed plasma of AD and MCI subjects, detected by our microfluidic device. While increased Let-7b levels in plasma may be used to identify patients with MCI, changes in plasma levels of microRNA-206 may be used to predict cognitive decline and progression towards dementia at an MCI stage. MicroRNA quantification via a microfluidic device could provide a practical cost-effective tool for the stratification of patients with MCI according to risk of developing AD