Early and late events induced by polyQ-expanded proteins: identification of a common pathogenic property of polYQ-expanded proteins

Abstract

To find a common pathogenetic trait induced by polyQ-expanded proteins, we have used a conditional expression system in PC12 cells to tune the expression of these proteins and analyze the early and late consequences of their expression. We find that expression for 3 h of a polyQ-expanded protein stimulates cellular reactive oxygen species (ROS) levels and significantly reduces the mitochondrial electrochemical gradient. 24-36 h later, ROS induce DNA damage and activation of the checkpoint kinase, ATM. DNA damage signatures are reversible and persist as long as polyQ-expanded proteins are expressed. Transcription of neural and stress response genes is down-regulated in these cells. Selective inhibition of ATM or histone deacetylase rescues transcription and restores the expression of silenced genes. Eventually, after 1 week, the expression of polyQ-expanded protein also induces endoplasmic reticulum stress. As to the primary mechanism responsible for ROS generation, we find that polyQ-expanded proteins, including native Ataxin-2 and Huntingtin, are selectively sequestered in the lipid raft membrane compartment and interact with gp91, the membrane NADPH-oxidase subunit. Selective inhibition of NADPH oxidase or silencing of H-Ras signaling dissolves the aggregates and eliminates DNA damage. We suggest that targeting of the polyQ-expanded proteins to the lipid rafts activates the resident NADPH oxidase. This triggers a signal linking H-Ras, ROS, and ERK1/2 that maintains and propagates the ROS wave to the nucleus. This mechanism may represent the common pathogenetic signature of all polyQ-expanded proteins independently of the specific context or the function of the native wild type protein.

FIGURE 1.

PolyQ-expanded proteins induce an early mitochondrial dysfunction and a late ER stress. A, rapid induction of ROS and mitochondrial dysfunction by 43polyQ-expanded proteins is shown. Total ROS were measured in PC12 cells in the presence or absence of doxycycline (3, 6, and 18 h). The expression of 17Q or 43Q was monitored by Western blot or immunofluorescence (Ref. and below). PC12 clones carrying polyQ-expressing vectors under the control of tetracycline repressor were maintained in the presence of doxycycline. The induction of the fusion proteins was extensively characterized by immunoblot (HA) and fluorescence (GFP) analysis (5). ROS levels were measured at different times after the removal of doxycycline by fluorescence of the oxidation-sensitive probe, 2,7-dichlorofluorescein diacetate (DCF). The values reported are the means ± S.E. derived from at least 3 independent experiments performed in triplicate (n = 9). Differences between treatment at the given time points were tested for statistical significance using Student's matched t test. *, p < 0.01 compared with the basal (43Q, −Dox, time 0). B, mitochondrial ROS and the mitochondrial membrane gradient were measured with the mitochondrial superoxide indicator, MitoSOX, and with TMRE, respectively, as described under “Experimental Procedures.” Differences between 17Q and 43Q were tested for statistical significance using Student's t test. *, p < 0.01 of t value as compared with the 17Q, −Dox. The experiments were performed in cells grown for 3 h in the absence or presence of doxycycline. The concentration and the time of incubation with MitoSOX and TMRE were optimized by FACS analysis. C, time course of ER stress after polyQ-expanded protein expression is shown. ER stress was assayed by analyzing XBP-1 accumulation by RT-PCR. RNA was isolated from PC12 expressing 17Q or 43Q from 3 h to 6 weeks as indicated in the upper part of the figure on each lane. The two arrows on the right indicate the bands corresponding to the unspliced and spliced XBP-1 RNA. RT-PCR was performed using total RNA extracted from cells treated as described under “Experimental Procedures.”

FIGURE 2.

Inhibition of NADPH oxidase reverses the early mitochondrial dysfunction induced by 43Q protein. A, mitochondrial gradient in cells expressing 17Q- or 43Q-expanded proteins is shown. 17Q (left) or 43Q (right) cells were treated for 3 (3h) or 72 h (3dd) with AEBSF (40 μm) and subjected to TMRE fluorescent analysis. The quantitative data, expressed as the mean ± S.E. of TMRE fluorescence of three experiments in triplicate (n = 9), are represented as -fold increase relative to the untreated controls (+Dox, −AEBSF). The intensity of fluorescence was evaluated in each positive cell by Meta-Morph software analysis (Universal Imaging, West Chester, PA). Differences between groups were tested for statistical significance using Student's matched pairs t test. *, p < 0.01 as compared with the basal (untreated, open bars); **, p < 0.01 relative to the untreated control (+Dox, untreated). B, cytofluorimetric analysis of the mitochondrial gradient in cells expressing 17Q- or 43Q-expanded proteins for 3 h challenged with apocynin is shown. The left panel shows cells grown in the absence of doxycycline (−D) for 3 h and treated with apocynin (Apo) or AEBSF as indicated under “Experimental Procedures” and analyzed by FACS. The right panel shows a representative FACS analysis. All data derive from at least three independent experiments, performed in triplicate (n = 9). Differences between groups were tested for statistical significance using Student's t test. *, p < 0.01 of t value (matched pairs test) as compared with the untreated 43Q (basal); ***, p < 0.01 of t value as compared with the untreated 17Q.

FIGURE 3.

Recombinant expanded polyQ protein or Huntingtin or Ataxin-2 induces oxidative DNA damage in neurons or fibroblasts. A, expression of 43Q protein activates ATM in primary neurons. Primary rat neurons (from embryonic rat cortex) were transiently transfected with the vectors encoding HA-17-GFP or HA-43Q-GFP and stained for p-H2AX, as described under “Experimental Procedures.” The efficiency of transfection was 1–3% + 1, and at least 20 cells were counted/sample in duplicate. The p-H2AX signal was quantified by ImageJ 1.43 (NIH), and positive cells, containing a signal 2 S.D. higher than controls (cells transfected with an empty vector) were 75% in 43Q and 10% in 17Q transfected cells (right panel). A representative micrograph of cells expressing 17 or HA-43Q-GFP proteins is shown (left panel). Differences between groups were tested for statistical significance using Student's t test. **, p < 0.01 as compared with the cells expressing the HA-17Q-GFP protein. All data derive from at least three independent experiments performed in triplicate (n = 9). B, FACS analysis of phosphorylated H2AX in cells expressing 17Q or 43Q proteins and treated with the anti-oxidant NAC are shown. Cells were grown for 6 h in the absence of doxycycline, treated with NAC (25 mm), and analyzed by FACS (right panel) with anti-phosphorylated H2AX. Differences between treatments were tested for statistical significance using Student's matched pairs t test. **, p < 0.02 for t value as compared with the cells grown in the presence of doxycycline. ***, p < 0.01 for t value relative to the cells grown in the absence of NAC. The left panel shows the analysis of cumulative data derived from three experiments performed in triplicate (n = 9). C, oxidative DNA damage in fibroblasts derived from HD and SCA-2 patients. Human primary fibroblasts derived from control, HD, and SCA-2 patients were treated with 25 mm NAC. After 15 min the cells were fixed and analyzed as described under “Experimental Procedures” for the accumulation of 8-oxodG (right panel). The 8-oxodG signal was quantified by ImageJ 1.43 (NIH). Positive cells, containing a signal 2 S.D. higher than controls (CTRL), were 85% in fibroblasts derived from HD and SCA-2 patients (left panel). All data derive from three independent patients; all experiments were performed in triplicate. Differences between groups were tested for statistical significance using Wilcoxon matched test. *, p < 0.01 as compared with the untreated fibroblast derived from controls subjects (open bars); **, p < 0.02 as compared with the basal (untreated) of the same patient. D, reversible activation of DNA damage checkpoint by 43 polyQ-expanded protein is shown. ATM-substrates, p-H2AX, and 17Q or 43Q GFP proteins were assayed by immunoblot on extract derived from PC12 expressing 17 or 43Q for 1–6 days. The values reported are the means ±S.D. of experiments performed in triplicate and represent the levels of the proteins indicated above relative to β-tubulin.

FIGURE 4.

Inhibition of transcription by polyQ-expanded proteins is dependent on the DNA damage checkpoint. A, VGF8 expression is silenced in 43Q expressing cells. 17Q- and 43Q-expressing cells were stimulated with NGF in the presence or absence of doxycycline. Kudos, the ATM inhibitor, was added to the cultures for 15 h. VGF8 mRNA was determined by real time PCR as described under “Experimental Procedures.” The absolute levels of VGF8 and the induction by NGF are less efficient in 43Q + Dox-expressing cells relative to 17Q + Dox. The partial silencing of the gene depends on the time in culture of the clones. The same experiment has been carried out on the pool of clones with similar results. Differences between treatments were tested for statistical significance using Student's matched pairs t test. *, p < 0.01 as compared with the control (+Dox, NGF/Kudos untreated); **, p < 0.01 as compared with NGF treated control (+Dox, +NGF, or +NGF/Kudos). B, reactivation of VGF8 expression by ATM inhibition. The cells were grown as indicated in A for 6 or 15 days in the absence of doxycycline and treated 15 h with Kudos (5 μm). Differences between treatments were tested for statistical significance using Student's matched pairs t test. *, p < 0.01 as compared with the each control (+Dox, NGF/Kudos untreated); **, p < 0.01 as compared with each NGF treated control (−Dox, +NGF, −Kudos) C, reactivation of VGF8 expression by histone deacetylase inhibitor, trichostatin A (TSA). The cells were grown as indicated in A for 6 h in the absence of doxycycline and treated 15 h with trichostatin A, as described under “Experimental Procedures.” *, p < 0.01 as compared with the each control (+Dox, NGF/Kudos untreated); **, p < 0.01 as compared with NGF treated control (−Dox, +NGF, −TSA). Trichostatin A and Kudos did not modify ROS levels in cells expressing 43Q.

FIGURE 5.

PolyQ-expanded proteins segregate in the lipid rafts and interact with the resident NADPH oxidase subunit gp91. A, neuroblastoma cells expressing 17Q and 43Q were lysed for 20 min in ice-cold Triton X-100 buffer (see “Experimental Procedures”) and separated by centrifugation as supernatant (S) and pellet (P) fractions. The pellet fraction was dissolved in SDS buffer (indicated under “Experimental Procedures”). Lysates were immunoblotted with anti-HA, anti-ERK1/2 and anti-actinin antibodies (Ac) (upper panel). The histogram is derived from the analysis of cumulative data, derived from three experiments performed in triplicate (n = 9) and shows the fraction of soluble (on the left) and insoluble (on the right) polyQ proteins. Differences between groups were tested for statistical significance using Student's t test. *, p < 0.01 as compared with the HA-17Q-GFP expressing cells. B and C, segregation of wild type and mutant Ataxin-2 (B) and Huntingtin (C) in the lipid rafts is shown. Fibroblasts derived from controls and patients HD and SCA-2 were processed as in A except that in this case the cells were challenged for 30 min with 10 mm methyl-β-cyclodextrin (+mβC) (as described under “Experimental Procedures”). Cell lysates were immunoblotted with antibodies against Huntingtin (HTT), Ataxin-2 (ATX-2), Nox subunit gp91, flotillin, ERK1/2 (right panel S) and actinin (left panel). The histograms are derived from the analysis of cumulative data derived from three experiments performed in triplicate (n = 9) and show the fraction of supernatant (left panel) and pellet (right panel) Ataxin-2 (B) or Huntingtin (C). D, interaction of gp91 with polyQ-expanded proteins is shown. Extracts derived from neuroblastoma cells stably transfected with either pCMV-HA-17Q-GFP or pCMV-HA-43Q-GFP were subjected to immunoprecipitation (IP) with HA antibody. The immunoprecipitates (1 mg) and total proteins were resolved on an SDS-10% PAGE and immunoblotted with anti-gp91 (upper panel) and anti-HA (lower panel) antibodies. Differences between groups were tested for statistical significance using Wilcoxon matched pairs test. *, p < 0.01 as compared with the controls; **, p < 0.01 as compared with methyl-β-cyclodextrin (−mβC) untreated samples.

FIGURE 6.

Inhibition of NADPH oxidase reduces Ras levels and DNA damage induced by ROS in cells of SCA-2 and HD patients. A, HD or SCA-2 fibroblasts contain high levels of H-Ras, and inhibition of NADPH oxidase reduces H-Ras levels. Primary fibroblasts derived from control, HD, and SCA-2 patients were immuno-stained with anti-H-Ras antibody (as described under “Experimental Procedures”) and analyzed by fluorescence microscopy. Where indicated, the cells were treated with 0.5 mm H₂O₂ for 15 min, 10 μm gp91-TaT or control (CTRL) peptides for 15 h, and 25 μm MG132 15 h (upper panel). The H-Ras signal was quantified by ImageJ 1.43 (NIH), and positive cells containing a signal 2 S.D. higher than controls were 90% in fibroblasts derived from HD and SCA-2 patients (lower panel). All data derive from at least three independent experiments performed in triplicate (n = 9). Differences between groups were tested for statistical significance using Wilcoxon matched pairs test. *, p < 0.02 compared with the fibroblast derived from controls subjects; **, p < 0.01 compared with the untreated basal HD and SCA cells. B, inhibition of NADPH oxidase reduces DNA damage in HD and SCA-2 cells is shown. Human primary fibroblasts derived from normal, HD, and SCA-2 patients were seeded onto glass slides, fixed, and analyzed as described under “Experimental Procedures” for the presence of 8-oxodG. The cells were analyzed by fluorescence microscopy. Where indicated, the cells were treated with 0.5 mm H₂O₂, the NADPH oxidase inhibitors (20 μm AEBSF for 15 h, 10 μm gp91-TaT and control peptides for 15 h, 20 μm apocynin for 15 h, and 20 μm DMSO for 15 h). The 8-oxodG signal was quantified by ImageJ 1.43 (NIH), and positive cells containing a signal 2 S.D. higher than controls were 85% in fibroblasts derived from HD and SCA-2 patients (left panel). All data derive from at least 3 independent experiments performed in triplicate (n = 9). Wilcoxon matched pairs test: *, p < 0.02 compared with the basal, normal cells; **, p < 0.01 compared with the untreated basal HD and SCA cells.

FIGURE 7.

Silencing gp91 NADPH oxidase subunit inhibits selectively DNA damage induced by expression of 43Q in neuroblastoma cells. A, SK-N-BE neuroblastoma cells were transiently cotransfected with the vectors encoding HA-17Q-GFP or HA-43Q-GFP and several pRS shRNA expression vectors encoding a 29-mer shRNA against CYBB (gp91 phox) as described under “Experimental Procedures.” After 48 h, 10⁶ cells was used for mRNA quantitation, and 2 × 10⁵ cells in triplicate were fixed with 4% paraformaldehyde and stained for p-H2AX, as described under “Experimental Procedures.” CTRL, cells transfected only with HA-17-GFP or HA-43Q-GFP; SCR, cells cotransfected with scrambled sh; shRNA-1 and shRNA-2, cells cotransfected with two different shRNA against CYBB. B, the p-H2AX fluorescence intensity was determined on the polyQ expressing cells (GFP+) by ImageJ 1.43 (NIH) software. Fluorescence intensity differences between groups were tested for statistical significance using Student's t test (n = 20). *, p < 0.01 compared with untreated C1 (basal control); **, p < 0.01 compared with the basal 17Q; ***, p < 0.01 compared with the 43Q treated with scrambled shRNA.

FIGURE 8.

H-Ras inhibition reverses DNA damage in HD and SCA-2 cells. Primary fibroblasts derived from control (CTRL), HD, and SCA-2 patients were challenged with siRNAs targeting H-Ras or scrambled H-Ras human sequences as indicated under “Experimental Procedures.” Immunofluorescence for 8-oxodG was carried out as described under “Experimental Procedures.” Where indicated, the cells were treated with 15 μm PDGF for 15 min. The 8-oxodG signal was quantified by ImageJ 1.43 (NIH), and positive cells containing a signal 2 S.D. higher than controls were 85% in fibroblasts derived from HD and SCA-2 patients (left panel). All data derive from at least 3 independent experiments performed in triplicate (n = 9). Wilcoxon matched pairs test: *, p < 0.02 compared with the basal, normal cells; **, p < 0.01 compared with the untreated basal HD and SCA cells.

FIGURE 9.

Inhibition of NADPH oxidase dissolves nuclear aggregates. A, time-dependent expression of polyglutamine-GFP fusion proteins in inducible PC12 cell lines is shown. Phase contrast (lower) and fluorescence images (upper) of PC12 cells grown in the absence of doxycycline for the indicated times. The localization of GFP-fusion proteins was examined by fluorescence microscopy. Where indicated the cells were treated with 10 μm gp91-TAT and control peptides 15 h and 10 μm MG132 15 h. d, days. B, Western blot analysis was performed by using total extracts derived from PC12 cells expressing 17Q and 43Q (Dox−) and not expressing (Dox+). The extracts were immunoblotted with anti-HA antibody. The arrows indicate the apparent molecular weight of fusion proteins. The high molecular weight bands visible in the stacking gel appear only in extracts of HA-43Q-GFP cells induced for 6 days (open arrow in the right panel). C, the histogram shows the statistical analysis of the aggregates derived from at least three independent experiments performed in triplicate (n = 9), expressed as arbitrary units relative to the concentration of the 43Q protein. Wilcoxon matched pairs test: *, p < 0.05 compared with -Dox +gp91-TAT-expressing cells.