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. 2014 Mar 3;9(3):e90803.
doi: 10.1371/journal.pone.0090803. eCollection 2014.

ER stress-induced eIF2-alpha phosphorylation underlies sensitivity of striatal neurons to pathogenic huntingtin

Julia Leitman  1 , Boaz Barak  2 , Ron Benyair  1 , Marina Shenkman  1 , Uri Ashery  2 , F Ulrich Hartl  3 , Gerardo Z Lederkremer  1
Affiliations

ER stress-induced eIF2-alpha phosphorylation underlies sensitivity of striatal neurons to pathogenic huntingtin

Julia Leitman et al. PLoS One. .

Abstract

A hallmark of Huntington's disease is the pronounced sensitivity of striatal neurons to polyglutamine-expanded huntingtin expression. Here we show that cultured striatal cells and murine brain striatum have remarkably low levels of phosphorylation of translation initiation factor eIF2α, a stress-induced process that interferes with general protein synthesis and also induces differential translation of pro-apoptotic factors. EIF2α phosphorylation was elevated in a striatal cell line stably expressing pathogenic huntingtin, as well as in brain sections of Huntington's disease model mice. Pathogenic huntingtin caused endoplasmic reticulum (ER) stress and increased eIF2α phosphorylation by increasing the activity of PKR-like ER-localized eIF2α kinase (PERK). Importantly, striatal neurons exhibited special sensitivity to ER stress-inducing agents, which was potentiated by pathogenic huntingtin. We could strongly reduce huntingtin toxicity by inhibiting PERK. Therefore, alteration of protein homeostasis and eIF2α phosphorylation status by pathogenic huntingtin appears to be an important cause of striatal cell death. A dephosphorylated state of eIF2α has been linked to cognition, which suggests that the effect of pathogenic huntingtin might also be a source of the early cognitive impairment seen in patients.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Striatal neurons show a low induction of early UPR markers, whereas later ER stress responses are upregulated.
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. STHdh Q7/7 were compared to STHdh 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. STHdh 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 STHdh Q111/111 cells, PERK-P and eIF2α-P are induced (C,D). Later ER stress responses are increased in STHdh 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.
Figure 2
Figure 2. Very low eIF2α-P levels in striatal cells, much increased by expression of Htt111Q.
A) Basal level of eIF2α-P in murine cell lines normalized by total eIF2α. Graph: average of 3 experiments ± SE. **P = 0.004, ***P  = 0.001. 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 STHdh Q7/7 levels were quantified from images from 3 experiments ± SE (>20 cells, ***P<0.001).
Figure 3
Figure 3. Low eIF2α-P levels in striatal cells are due to reduced phosphorylation (PERK activity), not increased de-phosphorylation.
A) Striatal cells have very low eIF2α-P dephosphorylating activity, measured in vitro at 37°C with eIF2αGFP-P (Materials and Methods). **P  = 0.01, ***P = 0.001. B) Striatal cells have a very low basal level of CreP (RT-PCR of CreP mRNA). NIH 3T3 cells treated overnight with Tun, compared to the untreated cells, served as a control of UPR-independent constitutive CreP expression. **P  = 0.01. C) Striatal cells have a very low basal level of PERK-P, which is increased in STHdh Q111/111 cells, and a low basal level of GADD34. *P <0.04, ** P (PERK-P) = 0.01, P (GADD34) = 0.002.
Figure 4
Figure 4. Mouse brain striatum presents a low level of eIF2α-P compared to other brain areas, which is increased in an HD mouse model.
A-C) EIF2α-P levels in the striatum of WT mouse brains are significantly lower than those in other brain regions (A, N = 10) and are increased in HD model mouse (N171-82Q) brains (B, N = 6) compared to WT. Bar = 100 µm. Levels were quantified as explained in Materials and Methods (C). *P = 0.015, **P<0.003, ***P = 0.0001. D-E) The striatum marker DARPP-32 was stained in brain sections of the same WT (D) and HD model mice (E), labeling the striatum and not the cortex.
Figure 5
Figure 5. High sensitivity of striatal neurons to ER stress, further aggravated by expression of pathogenic huntingtin.
A-C) Strong induction of GADD34 and CHOP upon prolonged ER stress in STHdh Q7/7 cells and even stronger in STHdh Q111/111 cells; (3 independent experiments ±SE). *P = 0.02, **P = 0.01, ***P = 0.0002. Immunoblots of a representative experiment are shown in A. GAPDH levels served here as a loading control. D) Prolonged ER stress induced with Tun or MG-132 leads to extensive death of STHdh Q7/7 cells, further aggravated in STHdh Q111/111 cells, as measured by FACS analysis of cell cycle progression with propidium iodide (PI) (see Fig. S2); (6 independent experiments ± SE). *P<0.05, **P = 0.01, ***P = 0.001.
Figure 6
Figure 6. Regulation of phosphorylated eIF2α levels by inhibition of its dephosphorylation.
A) Guanabenz (Gz), at a relatively high concentration (100 µM), inhibits eIF2α dephosphorylation in untreated STHdh Q7/7 cells and also in those treated with Tun (5 µg/ml) up to 7h; this is also true in STHdh Q111/111 cells but only after very short treatments. *P = 0.02, **P = 0.01. EIF2α-P levels were normalized by total eIF2α. 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. C) Gz showed a minor effect in rescuing STHdh Q111/111 cells from UPR-induced cell death (Tun for 48 h). ***P =  0.0001.
Figure 7
Figure 7. Regulation of phosphorylated eIF2α levels by inhibition of its phosphorylation and rescue of STHdh Q111/111 cells.
A) EIF2α phosphorylation in STHdh Q111/111 cells is PERK-mediated. STHdh 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) ER stress-mediated eIF2α phosphorylation is inhibited by A4 and not by PKRi. As in (A), but with STHdh Q7/7 cells treated for different times with Tun. C) 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). D-E) A4 rescued STHdh Q111/111 cells from UPR-induced cell death (Tun for 48 h, D), whereas PKRi had no effect (E). ***P =  0.0001. F) Total protein synthesis levels are much increased in STHdh Q111/111 cells after prolonged ER stress (Tun for 24h) and reduced by A4 (50 µM). **P<0.002 (3 repeat experiments).
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