Regulation of phosphorylated eIF2α levels by inhibition of its dephosphorylation.

<p><b>A</b>) Guanabenz (Gz), at a relatively high concentration (100 µM), inhibits eIF2α dephosphorylation in untreated ST<i>Hdh</i>^Q7/7 cells and also in those treated with Tun (5 µg/ml) up to 7h; this is also true in ST<i>Hdh</i>^Q111/111 cells but only after very short treatments. *P = 0.02, **P = 0.01. EIF2α-P levels were normalized by total eIF2α. <b>B</b>) Similar to (A), but for cells treated for 24 h. After these long treatments Gz did not inhibit ER stress-induced eIF2α dephosphorylation, it increased CHOP levels. The values in the graphs are averages from 3-4 independent experiments±SE. *P<0.05, **P = 0.002. <b>C</b>) Gz showed a minor effect in rescuing ST<i>Hdh</i>^Q111/111 cells from UPR-induced cell death (Tun for 48 h). ***P =  0.0001.</p

Striatal neurons show a low induction of early UPR markers, whereas later ER stress responses are upregulated.

<p>Early responses and in some cases late ones are increased by expression of Htt111Q. A,B) Levels of UPR markers after short term ER stress. ST<i>Hdh</i>^Q7/7 were compared to ST<i>Hdh</i>^Q111/111 and NIH 3T3 cells. Immunoblots show results of a representative experiment of 3. Vertical lines indicate removal of irrelevant lanes. C-H) Quantification of A,B. ST<i>Hdh</i>^Q7/7 cells do not activate properly an early stress response, mediated by PERK-P and its target eIF2α-P, induced by Tun (C, 10 µg/ml) or by MG-132 (D, 40 µM). In ST<i>Hdh</i>^Q111/111 cells, PERK-P and eIF2α-P are induced (C,D). Later ER stress responses are increased in ST<i>Hdh</i>^Q7/7 compared to NIH 3T3 cells (E-H). Htt111Q expression causes even more enhanced upregulation of the UPR markers in some cases. Values were normalized to β-actin levels as a loading control.</p

High sensitivity of striatal neurons to ER stress, further aggravated by expression of pathogenic huntingtin.

<p><b>A-C</b>) Strong induction of GADD34 and CHOP upon prolonged ER stress in ST<i>Hdh</i>^Q7/7 cells and even stronger in ST<i>Hdh</i>^Q111/111 cells; (3 independent experiments ±SE). *P = 0.02, <b>*</b>*P = 0.01, ***P = 0.0002. Immunoblots of a representative experiment are shown in A. GAPDH levels served here as a loading control. <b>D</b>) Prolonged ER stress induced with Tun or MG-132 leads to extensive death of ST<i>Hdh</i>^Q7/7 cells, further aggravated in ST<i>Hdh</i>^Q111/111 cells, as measured by FACS analysis of cell cycle progression with propidium iodide (PI) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090803#pone.0090803.s002" target="_blank">Fig. S2</a>); (6 independent experiments ± SE). *P<0.05, **P = 0.01, ***P = 0.001.</p

Regulation of phosphorylated eIF2α levels by inhibition of its phosphorylation and rescue of ST<i>Hdh</i>^Q111/111 cells.

<p><b>A</b>) EIF2α phosphorylation in ST<i>Hdh</i>^Q111/111 cells is PERK-mediated. ST<i>Hdh</i>^Q111/111 cells left untreated or treated with the PERK inhibitor A4 (50 µM) or the PKR inhibitor PKRi (1 µM) for the indicated times. **P = 0.009. <b>B</b>) ER stress-mediated eIF2α phosphorylation is inhibited by A4 and not by PKRi. As in (A), but with ST<i>Hdh</i>^Q7/7 cells treated for different times with Tun. <b>C</b>) PKR-mediated eIF2α phosphorylation is inhibited by PKRi and not by A4. As in (B), but with cells treated for 7h with the PKR inducer poly-I:C (200 µg/ml). <b>D-E</b>) A4 rescued ST<i>Hdh</i>^Q111/111 cells from UPR-induced cell death (Tun for 48 h, D), whereas PKRi had no effect (E). ***P =  0.0001. <b>F</b>) Total protein synthesis levels are much increased in ST<i>Hdh</i>^Q111/111 cells after prolonged ER stress (Tun for 24h) and reduced by A4 (50 µM). **P<0.002 (3 repeat experiments).</p

Very low eIF2α-P levels in striatal cells, much increased by expression of Htt111Q.

<p><b>A</b>) Basal level of eIF2α-P in murine cell lines normalized by total eIF2α. Graph: average of 3 experiments ± SE<b>.</b> **P = 0.004, ***P  = 0.001. <b>B</b>) Immunofluorescence images of cells fixed, permeabilized and stained with rabbit anti-eIF2α-P and mouse anti-eIF2α followed by secondary antibodies. Bar = 10 µm. Image exposure time was kept constant to be able to compare protein levels in the different cell types. Levels relative to ST<i>Hdh</i>^Q7/7 levels were quantified from images from 3 experiments ± SE (>20 cells, ***P<0.001).</p

HCV patients.

1<p>Median of absolute values divided by upper limit of normal range.</p

ROC plots of sH2a and of the combination score of sH2a and ALT.

<p><b>A</b>) Receiver operating characteristic (ROC) plot of sH2a values for advanced fibrosis and cirrhosis (METAVIR 0–1 vs 3–4) for the group of 44 HCV patients. <b>B–C</b>) ROC plots of the combined scores of sH2a and ALT were made for advanced fibrosis and cirrhosis (METAVIR 0–1 vs 3–4) (B) or for intermediate fibrosis (METAVIR 0–1 vs 2–3) (C) of the same group of patients.</p

Healthy individuals.

<p>Healthy individuals.</p

Model depicting the sequence of events in the fibrogenic process and changes in sH2a secretion.

<p>The membrane-bound precursor of sH2a is cleaved in the endoplasmic reticulum of healthy hepatocytes, traverses the secretory pathway and is secreted to the plasma (step 1). As a consequence of HCV infection and possibly also upon other insults, hepatocytes are damaged, their overall function is compromised and sH2a secretion is reduced, while intracellular enzymes like ALT are released to the plasma (step 2). This starts a process of inflammation (step 3), activation of hepatic stellate cells leading to fibrosis and then cirrhosis, which involve secretion and progressive accumulation of ECM components (step 4).</p

Univariate analysis of correlation of characteristics with advanced fibrosis.

1<p>Data is presented as n (%) except for age, which is presented as median (IQR); odds ratios are from simple logistic regression, using the forced entry method, on each variable. The dependent variable in the regression is state and the outcome is METAVIR score (1 if state is 3–4); CI, confidence interval presented for each explanatory variable.</p