5 research outputs found
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><sup>Q7/7</sup> were compared to ST<i>Hdh</i><sup>Q111/111</sup> 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><sup>Q7/7</sup> 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><sup>Q111/111</sup> cells, PERK-P and eIF2α-P are induced (C,D). Later ER stress responses are increased in ST<i>Hdh</i><sup>Q7/7</sup> 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
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><sup>Q7/7</sup> cells and also in those treated with Tun (5 µg/ml) up to 7h; this is also true in ST<i>Hdh</i><sup>Q111/111</sup> 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><sup>Q111/111</sup> cells from UPR-induced cell death (Tun for 48 h). ***P =  0.0001.</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><sup>Q7/7</sup> cells and even stronger in ST<i>Hdh</i><sup>Q111/111</sup> 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><sup>Q7/7</sup> cells, further aggravated in ST<i>Hdh</i><sup>Q111/111</sup> 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><sup>Q111/111</sup> cells.
<p><b>A</b>) EIF2α phosphorylation in ST<i>Hdh</i><sup>Q111/111</sup> cells is PERK-mediated. ST<i>Hdh</i><sup>Q111/111</sup> 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><sup>Q7/7</sup> 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><sup>Q111/111</sup> 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><sup>Q111/111</sup> 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><sup>Q7/7</sup> levels were quantified from images from 3 experiments ± SE (>20 cells, ***P<0.001).</p