Tau inclusions are not immunoreactive for ubiquilin 2.

<p>Double immunostaining for ubiquilin 2 and AT8 in an AD case (A), a G272V MAPT mutation carrier (B) and a sporadic PiD case (C). Detailed images of double labeling show neurons with AT8-positive tau inclusions (D-O). Images were spectrally unmixed, and shown separately and merged in artificial colors (ubiquilin 2: green; AT8: red). AD, Alzheimer’s disease; CA, cornu ammonis FTD, frontotemporal dementia; MAPT, microtubule-associated protein tau, Ub2, ubiquilin 2.</p

Ubiquilin 2 immunoreactivity in tauopathies.

<p>Representative immunohistochemical staining of ubiquilin 2 in the CA1 region of the hippocampus in a control (A) and an AD case (B). Detailed images showing plaque-like structures (C) and perisomatic granules (D) in an AD case. Ubiquilin 2 immunoreactivity in the subiculum of a sporadic PiD case (E), a G272V MAPT mutation carrier (F), and a PSP case (G). AD, Alzheimer’s disease; CA, cornu ammonis, FTD, frontotemporal dementia; MAPT, microtubule associated protein tau.</p

Post-mortem brain material used in this study.

<p>Listed are clinical diagnosis (CON = control, AD = Alzheimer’s disease), Braak stage, gender, age (in years), ApoE genotype, post-mortem interval (PMI, in hours: minutes).</p

QC expression is not upregulated by Aβ or the UPR.

<p>A. Differentiated SK-N-SH cells were treated with 1 µM and 2 µM oligomeric (O) and fibrillary (F) Aβ_1–42 for 24 h. Shown is the average + SD of normalized QC mRNA levels of triplicates from a representative experiment B. Differentiated SK-N-SH cells were treated with different UPR inducers, TM (0.2 µg/µl and 0.5 µg/µl), TG (1 µM and 2 µM) and DD (20 mM) for 16 h. Shown is the average + SD of normalized QC mRNA levels of n = 9 from 3 independent experiments. Normalized BiP mRNA levels are shown as positive control for UPR induction (n = 6 from two independent experiments). The expression levels were normalized to eEF2α mRNA, the levels in untreated cells are set to 1. Asterisks indicate a significant difference compared with control (*p≤0.01, **p≤0.001, ***p≤0.0001).</p

Primers and probes used for qPCR 480 light cycler.

<p>Probe numbers refer to numbers in the Roche universal probe library.</p

Disturbed Ca²⁺ homeostasis increases QC expression and enzyme activity.

<p>A. Differentiated SK-N-SH cells were treated with different concentrations TG as indicated. Shown are the average + SD of normalized QC mRNA levels of n = 6 from two independent experiments. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. B. Differentiated SK-N-SH cells were treated with 1 µM TG for 16 h. The enzymatic activity of QC activity was determined in protein lysates as described in materials and methods, activity in untreated cells is set to 1. Shown is average +SEM of n = 15 from 5 independent experiments. Asterisks indicate a significant difference compared to control (*p≤0.001, **p≤0.0001).</p

Induction of c-fos and c-jun precedes increased QC expression.

<p>Differentiated SK-N-SH cells were treated with 1 µM TG for the times indicated. Shown is the average + SD of normalized QC (A), c-fos (B) and c-jun (C) mRNA levels of triplicates from a representative experiment of three. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p<0.05; **p≤0.001; ***p≤0.0001).</p

Induction of c-fos by ER Ca²⁺ depletion precedes QC expression.

<p>Differentiated SK-N-SH cells were treated with 1 µM TG for 5 h and 16 h with or without pre-incubation with 5 µM BAPTA-AM (B) for 1 h. Shown are the average + SD of triplicates of normalized QC (A) and c-fos (B) mRNA levels from a representative experiment of three. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p<0.05, **p≤0.02 ***p≤0.001).</p

ER Ca²⁺ depletion increases QC levels.

<p>Differentiated SK-N-SH cells were treated with 1 µM TG for 16 h with or without pre-incubation with 5 µM BAPTA-AM (B) for 1 h. Shown are the average + SD of normalized QC mRNA levels of n = 9 from 3 independent experiments. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p≤0.0001).</p

QC mRNA expression is increased in earliest stages of AD pathology.

<p>Expression of QC mRNA was determined in a cohort of patients with varying stages of AD pathology (see materials and methods for details). CON and AD refer to the clinical diagnosis, BS is Braak score for tau pathology, PL is the plaqueload in the hippocampus/entorhinal cortex. Shown is a box-plot of results of the pathological groups as indicated in hippocampus/entorhinal cortex. The expression levels were normalized to eEF2α mRNA. Kruskall-Wallis test was used to evaluate differences between groups followed by the Mann-Whitney U test, to test differences between pairs of groups.</p