GADD34 mediates cytoprotective autophagy in mutant huntingtin expressing cells via the mTOR pathway

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Research Article

GADD34 mediates cytoprotective autophagy in mutant huntingtin expressing cells via the mTOR pathway Alise Hyrskyluoto a, b , Sami Reijonen c , Jenny Kivinen a, b , Dan Lindholm a, b, 1 , Laura Korhonen a, b, d,⁎, 1 a

Institute of Biomedicine/Biochemistry, University of Helsinki, Biomedicum Helsinki, Haartmaninkatu 8, FIN-00014, Finland Minerva Medical Research Institute, Biomedicum Helsinki, Tukholmankatu 8, FIN-00290 Helsinki, Finland c Department of Veterinary Biosciences, Unit of Biochemistry and Cell Biology, University of Helsinki, P.O. Box 66, FIN-00014 University of Helsinki, Finland d Division of Child Psychiatry, Helsinki University Central Hospital, FIN-00029 HUS, Finland b

A R T I C L E I N F O R M A T I O N

A B S T R A C T

Article Chronology:

Increased protein aggregation and altered cell signaling accompany many neurodegenerative

Received 17 June 2011

diseases including Huntington's disease (HD). Cell stress is counterbalanced by signals

Revised version received

mediating cell repair but the identity of these are not fully understood. We show here that the

28 August 2011

mammalian target of rapamycin (mTOR) pathway is inhibited and cytoprotective autophagy is

Accepted 30 August 2011

activated in neuronal PC6.3 cells at 24 h after expression of mutant huntingtin proteins.

Available online 7 September 2011

Tuberous sclerosis complex (TSC) 1/2 interacted with growth arrest and DNA damage protein 34 (GADD34), which caused TSC2 dephosphorylation and induction of autophagy in mutant

Keywords:

huntingtin expressing cells. However, GADD34 and autophagy decreased at later time points,

Autophagy

after 48 h of transfection with the concomitant increase in mTOR activity. Overexpression of

GADD34

GADD34 counteracted these effects and increased cytoprotective autophagy and cell survival.

TSC

These results show that GADD34 plays an important role in cell protection in mutant huntingtin

mTOR

expressing cells. Modulation of GADD34 and the TSC pathway may prove useful in

Cell death

counteracting cell degeneration accompanying HD and other neurodegenerative diseases.

HD

© 2011 Elsevier Inc. All rights reserved.

⁎ Corresponding author at: Institute of Biomedicine/Biochemistry, Biomedicum Helsinki, Haartmaninkatu 8, FIN-00014, Finland. Fax: + 358 9 191 25 701. E-mail address: laura.t.korhonen@helsinki.fi (L. Korhonen). Abbreviations: ACC, Acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; ATF4, activating transcription factor 4; Bcl-2, B-cell CLL/lymphoma 2; CHOP, CCAAT/enhancer binding protein (C/EBP) homologous protein also called for growth arrest and DNA-damage-inducible protein GADD153; eIF2α, elongation initiator factor 2 alpha; ER, endoplasmic reticulum; ERAD, ER associated degradation; GADD34, Growth arrest and DNA damage-inducible protein 34; CAG, glutamine; FL, full-length; GFP, green fluorescent protein; HD, Huntington's disease; IRE1α, inositol requiring enzyme 1 alpha; JNK, jun-N-terminal kinase; kDa, kilodalton; LAMP1, Lysosomal-associated membrane protein 1; LC3, light chain 3 protein; PC6.3, pheochromocytoma cell line subline 6.3; PCR, polymerase chain reaction; PERK, PKR-like ER-localized eIF2α kinase; polyQ, polyglutamine; PP1, protein phosphatase 1; RFP, red fluorescence protein; S6K, RPS6-p70-protein kinase; TBS, Tris buffered saline; mTOR, mammalian target of rapamycin; TSC1, tuberous sclerosis 1; TSC2, tuberous sclerosis 2; UPR, unfolded protein response; Q, glutamine 1 These authors made equal contribution to the work. 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.08.020

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Introduction

Materials and methods

Huntington's disease (HD) is an autosomally dominantly inherited disease characterized by severe motor and cognitive symptoms due to neurodegeneration in striatum and other parts of the brain [1]. HD is caused by a CAG-repeat expansion in exon I of the IT15 gene that encodes huntingtin [2]. Increased CAG-repeats in huntingtin cause accumulation of intracellular protein aggregates in cells, including neurons [3,4]. Inhibition of the autophagy–lysosome pathway has been shown to impair the degradation of the huntingtin aggregates [5]. As shown in HD models in mice and Drosophila, blocking of the mammalian target of rapamycin (mTOR) pathway by rapamycin induces autophagy and reduces mutant huntingtin toxicity [6,7]. This suggests a role for the mTOR pathway and autophagy in cytoprotection in HD that warrants further studies. mTOR is a serine/threonine kinase that is important in the control of cell growth, proliferation and survival [8]. The activity of mTOR is regulated by various growth factors and cell signals, as well as by the supply of amino acids and nutrients sensing the general energy state of the cell. mTOR inhibits autophagy by phosphorylating proteins encoded by the autophagy-related genes (Atgs), whereas phosphorylation of the ribosomal protein S6 kinases (S6K1 and S6K2) and the eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP) stimulates translation of specific proteins involved in cell repair after stress [9–11]. The precise links between altered protein synthesis and the process of autophagy under different cell conditions are not fully understood. Growth arrest and DNA damage-inducible gene 34 (GADD34) is a protein induced by cell damage and it is a major regulator of translation during cell stress [12–16]. GADD34 forms a complex with the protein phosphatase 1 (PP1) to dephosphorylate the eukaryotic initiator factor-2α (eIF2α) [15,17]. Phosphorylation of eIF2α decreases global protein synthesis but is required for translation of a subset of specific mRNAs encoding proteins that are involved in cell repair [17,18]. Studies of GADD34 gene deficient mice have shown that GADD34 promotes cell survival and the recovery from protein synthesis inhibition induced by endoplasmic reticulum (ER) stress [19]. However, the possible roles played by GADD34 in human neurodegenerative diseases and in the regulation of autophagy in cells are so far less understood. We have recently shown that mutant N-terminal huntingtin fragment proteins as well as disease-causing full-length (FL) huntingtin proteins trigger ER stress in neuronal PC6.3 cells with an increase in the phosphorylation of eIF2α [20,21]. eIF2α is a target for GADD34 in regulation of protein synthesis but GADD34 may have additional functions during cell stress [22]. In the present study, we showed that GADD34 induced cytoprotective autophagy in mutant huntingtin expressing cells via influencing the mTOR pathway. GADD34 interacted with Tuberous sclerosis complex 1 (TSC1), which caused dephosphorylation of TSC2. However, at later time points the mTOR inhibition was reversed and correlated with decreased levels of GADD34 and an inhibition of autophagy. Overexpression of GADD34 induced cytoprotective autophagy and increased cell viability in mutant huntingtin expressing cells. Interfering with the levels or the function of the GADD34 may represent novel targets to combat cell stress and the deleterious effects of mutant huntingtin proteins.

Cell culture and transfections Rat pheocromocytoma cells (PC6.3) were cultured in RPMI 1640 (Biochrom AG) medium supplemented with 5% fetal calf serum and 10% horse serum. Cells were transfected with expression vectors encoding for different CAG-repeat lengths of huntingtin exon1 fused to EGFP, as well as full-length (FL) huntingtin with 17- and 75 polyglutamine-repeats as described [20,21]. LC3-RFP (light chain 3 protein-red fluorescent protein), LC3-EGFP (LC3-enhanced green fluorescent protein), LAMP1-RFP (Lysosomal-associated membrane protein 1-RFP) and GADD34 expression plasmids were obtained from Addgene. Cells were transfected using the Transfectin reagent (BioRad) with the above plasmids or with the control EGFP plasmid (Clontech). In some experiments, 1 mM 3methyladenine (3-MA, Sigma), 200 nM Rapamycin (Calbiochem), 2 μM thapsigargin (Sigma) or 1 μg/ml tunicamycin (Sigma) was added. Rapamycin was added 4 h after transfection, 3methyladenine for 6 h, thapsigargin and tunicamycin for 24 h. LC3 immunoblotting was done also in the presence of lysosomal proteasome inhibitors: 5 μM E64d (Sigma) and 10 μM pepstatin (Sigma). Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Calbiochem) assay as described previously [21,23].

Silencing of GADD34 expression using siRNAs GADD34 expression was specifically silenced in neuronal PC6.3 cells using siRNA (small interfering RNAs) duplexes (Silencer®, Applied biosystems). Ppp1R15a (GADD34) siRNAs (P/N:s 4390815 and 4390815) and siRNA universal negative control (Mission®, Sigma) were transfected by using the Transfectin reagent (BioRad) following the manufacturer's instructions.

Immunocytochemistry PC6.3 cells plated on polylysine-laminin-coated coverslips were fixed for 20 min using 4% paraformaldehyde or methanol (staining of endogeneous LC3). Cells were incubated overnight with primary GADD34 (1:1000, Santa Cruz Biotechnology) or LC3 antibody (LC3 (1:100, Cell Signaling) followed by 1 h incubation with Alexa594-conjugated secondary antibodies (1:500, Molecular Probes). Stainings were analyzed with Zeiss LSM 5 duo confocal microscope at Molecular Imaging unit, Biomedicum Helsinki.

Immunoblots Cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris–HCl and 1% SDS, pH 8.0) containing phosphatase inhibitor cocktail (Roche), or in Poly (ADP-ribose) polymerase (PARP) lysis buffer (62.5 mM Tris, pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.003% bromophenol blue and 5% 2-mercaptoethanol). Equal amounts of protein (40 μg) were separated by SDS-PAGE, and transferred to a nitrocellulose filters (Amersham Biosciences, Helsinki, Finland). Filters were blocked for 1 h in 5% milk-TBS or 5% BSA-TBS, followed by an overnight incubation at + 4 °C using primary

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antibodies. These included antibodies against active-caspase-3 (diluted 1:350, Cell Signaling), Acetyl-CoA carboxylase (ACC, 1:1000, Cell Signaling), p-ACC Ser79 (1:3000, Cell Signaling), ATF4 (1:500, Abnova), Chop (1:200, Santa Cruz), GFP (1:4000, Sigma), GADD34 (1:2000, Santa Cruz), LC3 (1:400, Cell Signaling), Akt (1:1000, Cell Signaling), p-Akt Ser473 (1:1000, Cell Signaling), PARP (1:1000, Cell Signaling), eEF2 (1:1000, Cell Signaling), p-eEF2 Thr56 (1:8000, Cell Signaling), eIF2α (1:1000, Cell Signaling), p-eIF2α Ser51 (1:3000, Cell Signaling), mTOR (1:1000, Cell Signaling), p-mTOR Ser2448 (1:1000; Cell Signaling), p70 S6K (1:1000, Cell Signaling), p-p70-S6K Thr389 (1:1000, Cell Signaling), TSC2 (1:1500, Cell Signaling), p-TSC2 Thr1462 (1:2000, Cell Signaling), p62 (1:10,000, Cell Signaling), TSC1 (1:5000, Cell Signaling). After washing, the filters were incubated with horseradish peroxidase conjugated secondary antibodies (1:2500, Pierce), followed by a detection using the enhanced chemiluminescent method (Thermo Scientific). Filters were stripped for 30 min at 60 °C (62.5 mM Tris–HCl, pH 6.8, 100 mM β-mercaptoethanol, 2% SDS), and reprobed with antibody for β-actin (1:5000, Sigma).

Immunoprecipitation Cells were lysed in RIPA buffer supplemented with protease inhibitors (Roche). Lysates were incubated with anti-GADD34 antibody (1 μg antibody per 500 μl lysate) overnight at +4 °C. Immunocomplexes were precipitated using protein A-Agarose (Roche) for 2 h at + 4 °C and washed three times with RIPA buffer. The beads were boiled in SDS-PAGE sample buffer and samples separated using a 10% SDS-PAGE gel followed by transfer to nitrocellulose membranes. Membranes were probed with GADD34 and TSC1 antibodies.

Polymerase chain reaction RNA was prepared using the mammalian RNA purification kit (Sigma), and cDNA made by reverse transcription using 50 U SuperScript II reverse transcriptase. PCR reactions were started with a 5 min denaturation step at 95 °C followed by 30 cycles carried out at 95 °C for 60 s, 63° for 60 s and 72 °C for 90 s. The following primers were used: GADD34: 5′-GTCCATTTCCTTGCTGTCTG-3′ (forward), 5′-AAGGCGTGCCCATGCTCTGG-3′ (reverse). ß-actin: 5′CTTCAACACCCCAGCCATG-3′ (forward); 5′-GTGGTACGACCAGAGGCATAC-3′ (reverse). Fig. 1 – Expression of N-terminal huntingtin fragment proteins differentially affects the phosphorylation of translational regulators in neuronal PC6.3 cells. (A–B) Immunoblots. eEF2 (Thr56) and eIF2α (Ser51) phosphorylation was studied in EGFP (control), and 18Q- and 120Q-huntingtin expressing PC6.3 neuronal cells at 24 h after transfection as described in Materials and methods. Also the total protein levels of eIF2α and eEF2 were studied. ß-actin was used as control. Quantification was done using NIH Image. Values are means ± SD, n = 3. *p < 0.05 for 18Q vs 120Q-expressing cells. (C–D) Levels of ATF4 and CHOP increase despite decreased translation in 120Q-huntingtin expressing cells. Quantification was as above. *p < 0.05 for 18Q vs 120Q. (E–F) Phosphorylations of the p70 S6 kinase (Thr389) and the Akt kinase (Ser473) were inhibited in 120Q-huntingtin expressing cells at 24 h. Values are means ± SD, n = 3. *p < 0.05 for 18Q vs 120Q.

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Autophagy detection PC6.3 cells were transfected with different huntingtin constructs together with LC3-RFP and fixed after 24 h or 48 h using 4% paraformaldehyde. The number of cells with LC3 positive vesicles was analyzed. Cells with more than five dots were considered positive. Conversion of LC3 was also studied using immunoblotting and the appearance of the lower LC3-II band was used to indication of ongoing autophagy.

Quantifications and statistic Immunoblots were quantified using the NIH Image quantification software. Results are expressed as percentage of controls

(mean± SD). Statistical analyses were done using Students t-test with Bonferroni post hoc test. p < 0.05 was considered as significant.

Results Expression of mutant huntingtin fragment proteins inhibits protein synthesis Expression of N-terminal mutant huntingtin fragment proteins containing 120 polyglutamine repeats (120Q) for 24 h increased the phosphorylation of eukaryotic elongation factor 2 (eEF2 Thr56) and eIF2 (Ser51) in neuronal PC6.3 cells reflecting an inhibition of global protein synthesis (Figs. 1A–B). This translational

Fig. 2 – Induction of autophagy by mutant huntingtin proteins. (A) Cells were transfected for 24 h with the N-terminal huntingtin fragment constructs (green fluorescence) together with the autophagy marker, RFP-LC3 (red). Note the presence of autophagosomal vacuoles. 200 nM rapamycin was used as a positive control to inhibit mTOR. Scale bar, 10 μm. (B) Cells were transfected for 24 h using RFP-LC3 and control huntingtin (17Q-full-length or 18Q N-terminal fragment) or with the disease-causing 75Q-full-length huntingtin or the 120Q N-terminal fragment encoding plasmids. The number of green fluorescent cells containing more than five autophagosomal vacuoles per cross section were counted and quantified. Values are means ± SD, n = 3. **p < 0.01 120Q vs 18Q and 75Q-FL vs 17Q-FL. (C) Immunoblot. Cells were transfected for 24 h as above, with or without lysosomal protease inhibitors E64d and pepstatin. Immunoblot was made using anti-LC3 antibody. The presence of the converted LC3-II band showed on-going autophagy. Typical experiments are shown and were repeated three times. (D) Staining for endogenous LC3 in the Htt-expressing cells. Cell were transfected for 24 h with 18Q or 120Q N-terminal huntingtin fragments (green fluorescence) and stained for LC3 (red fluorescence). Note the presence of autophagosomal vacuoles in 120Q huntingtin expressing cells and in rapamycin (200 nM) treated cells. Scale bar, 10 μm.

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block is not absolute and the proteins activating transcription factor 4 (ATF4) and CHOP involved in cell protection and ER stress were still synthesized under these conditions (Figs. 1C–D). In contrast to eEF2 and eIF2α, the phosphorylation of the ribosomal p70 S6 kinase (p70 S6K Thr389) was decreased in cells expressing the 120Q huntingtin proteins (Figs. 1E–F). p70 S6K is a target for the mTOR kinase in cells and the decreased phosphorylation correlates with mTOR inhibition [8]. The reduced p70 S6K phosphorylation cause a further reduction in protein synthesis in mutant huntingtin expressing cells. We observed that the phosphorylation of Akt (Ser473) was decreased in mutant huntingtin expressing cells (Figs. 1E–F), and decreased p-Akt (Ser473) levels have been correlated with inhibition of mTOR activity during ER stress [24,25].

Inhibition of the mTOR pathway induces autophagy in mutant huntingtin expressing cells Apart from affecting protein synthesis, the activity of mTOR is known inhibit the process of autophagy on cells. To study whether the inhibition of mTOR observed in 120Q huntingtin expressing cells affects autophagy, we employed the marker LC3-RFP. Data

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showed that the number of LC3 positive autophagosomes was increased in these cells compared with control and 18Q huntingtin expressing cells (Fig. 2A). In these studies, rapamycin was used as a positive control to inhibit mTOR (Fig. 2A). Quantification showed that the number of autophagosome positive cells increased significantly in 120Q huntingtin expressing cells as well as in cells expressing the disease-causing full-length 75Q huntingtin proteins (p < 0.01 control vs mutant huntingtin) (Fig. 2B). Immunoblotting and staining for endogeneous LC3 showed a conversion of LC3 (LC3-I to LC3-II) in 120Q-expressing cells (Fig. 2C and E; p < 0.01 for C vs 120Q in case of LC3-II). In these cells, the mutant huntingtin proteins co-localized with the lysosomal marker LAMP-1, in line with their localization in lysosomes (data not shown).

GADD34 interacts with tuberous sclerosis complex (TSC) proteins in mutant huntingtin expressing cells mTOR is regulated by the TSC complex, consisting of the TSC1 and the TSC2 proteins [26]. Phosphorylation of TSC2 by upstream kinases inhibits the activity of this complex and reduces mTOR

Fig. 3 – TSC1 interacts with GADD34. (A) GADD34 expression was analyzed 24 h after transfection with huntingtin fragment proteins. Upper panel; semi-quantitative PCR was done as described in Materials and methods. ß-actin was used as control. Lower panel: immunoblot. No significant change in GADD34 expression. (B) Immunostaining of GADD34 in control and transfected cells. Scale bar, 10 μm. (C) Immunoprecipitation was done as described in Materials and methods using anti-GADD34 antibodies followed by immunoblotting using anti-TSC1 antibodies. Note interaction of GADD34 with TSC1 in 120Q-huntingtin expressing cells. Two bands of TSC1 (170 and 150 kDa) were enriched in the immunoprecipitate. These bands were present also in cell lysates after longer exposure of the film. Typical experiment is shown and was repeated three times. (D–E) Immunoblots. mTOR phosphorylation (Ser2448) and total protein levels of mTOR were analyzed 24 h and 48 h after transfection with huntingtin fragment proteins. Phosphorylation of mTOR and total protein levels of mTOR were decreased in 120Q-huntingtin expressing cells at 24 h. However phosphorylation of mTOR and total protein levels of mTOR were recovered 48 h after transfection. Quantification was done using ImageJ. Values are means ± SD, n = 3. *p < 0.05 for 18Q vs 120Q-expressing cells.

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signaling [27], whereas dephosphorylation of TSC2 by various phosphatases inhibits mTOR and activates autophagy. This promoted us to study GADD34, which is engaged in dephosporylation reactions [14,15] and which was previously shown to interact with the TSC complex [22]. GADD34 levels may increase during cell stress, but little is known about the precise temporal changes of GADD34 after various insults. Data obtained showed that the expression of GADD34 was not significantly changed at 24 h after transfection with mutant huntingtin constructs as determined by semi-quantitative PCR or immunoblotting (Fig. 3A), neither was the subcellular localization of GADD34 altered in mutant huntingtin expressing cells (Fig. 3B). However, using immunoprecipitations to study protein interactions showed that GADD34 specifically interacted with TSC1 in 120Q huntingtin proteins expressing cells but not in controls (Fig. 3C). The binding of GADD34 to TSC1 may stabilize the TSC-complex, as shown previously for GADD34 in non-neuronal cells [27], and cause inhibition of mTOR signaling and thus activate cytoprotective autophagy in PC6.3 neuronal cells at 24 h. In line with this the phosphorylation of mTOR (Ser2448) was decreased in 120Q mutant huntingtin expressing cells at 24 h (Fig. 3D).

Autophagy is inhibited at 48 h in mutant huntingtin expressing cells—relationship to changes in GADD34 levels and AMP-activated protein kinase (AMPK) activity In contrast to the situation at 24 h, we observed that GADD34 was downregulated in mutant 120Q huntingtin proteins expressing cells 48 h after transfection (Figs. 4A–B). This effect is dependent on mutant huntingtin since treatment with two well-known ER stress inducer tunicamycin and thapsigargin did decrease GADD34 levels at 24 h (Fig. 4C). The decrease in GADD34 at 48 h was accompanied by an increase in the phosphorylation of TSC2 (Thr1462; Figs. 4D–E) that in turn leads to activation of mTOR and the inhibition of cytoprotective autophagy. In line with this, we observed that there was a restoration in the phosphorylation

of p70S6K (Thr389) and Akt (Ser473) in 120Q mutant huntingtin expressing cells at 48 h compared with the situation at 24 h (Figs. 4F–H). We also studied the activity of AMPK in huntingtin expressing cells since AMPK can negatively influence mTOR and stimulate autophagy [8,28]. Data showed that AMPK is activated in 120Q mutant huntingtin expressing cells at 24 h as shown by phosphorylation of its substrate ACC (Ser79; Fig. 4I). This indicates that AMPK may contribute to cytoprotective autophagy at early time points in mutant huntingtin expressing cells. However at 48 h the AMPK activity decreased that may lead to an increase in mTOR and an inhibition of autophagy in these cells. To study this more directly, we stained for LC3 positive autophagosomes in mutant huntingtin expressing cells at 48 h (Fig. 4J). Quantification showed that the number of autophagosome positive cells was low in both wild type cells and in those expressing either the mutant 120Q fragment protein or the disease-causing full-length 75Q huntingtin protein (Fig. 4K). This data is in contrast to results obtained at 24 h, indicating that the lack of cytoprotective autophagy at 48 h may contribute to the deleterious effects of the mutant huntingtin protein at longer time points.

Overexpression of GADD34 elevates autophagy and protects cells against mutant huntingtin-induced cell degeneration Results above indicate that GADD34 is an important regulator of mTOR and autophagy in mutant huntingtin expressing cells. To study this further, we overexpressed and downregulated GADD34 in PC6.3 cells (Figs. 5A, C) and analyzed their effects on the activity of the TSC/mTOR pathway and on autophagy. Data showed that overexpression of GADD34 decreased the phosphorylation of TSC2 Thr1462 at 48 h (Fig. 5B) thereby activating autophagy in wild type cells and in those expressing the disease-causing full-length 75Q huntingtin protein (Fig. 5D). The increase in autophagy brought about by GADD34 was accompanied by an increase in cell viability in mutant 120Q-huntingtin expressing cells (Fig. 5E), and a reduction in the cleavage of PARP (Fig. 5F). PARP is a substrate for caspase-3, and we reasoned that silencing of

Fig. 4 – GADD34 was decreased and autophagy inhibited at 48 h in mutant huntingtin expressing cells. (A–B) Immunoblot. GADD34 expression was analyzed 24 h and 48 h after transfection with huntingtin fragment proteins. GADD34 is downregulated at 48 h in cells expressing 120Q huntingtin proteins. Values are means ± SD, n = 3. *p < 0.05 for 18Q vs 120Q-expressing cells. (C) Immunoblot. Cells were treated for 48 h with ER stress inducers tunicamycin (1 μ/ml) or thapsigargin (2 μM) and GADD34 expression was analyzed. (D–E) Immunoblots. Total protein levels of TSC1, TSC2 phosphorylation (Thr1462) and total protein levels of TSC2 were analyzed 24 h and 48 h after transfection with huntingtin fragment proteins. TSC2 was dephosphorylated in 120Q-huntingtin expressing cells at 24 h and phosphorylated at 48 h. Values are means ± SD, n = 3. *p < 0.05 for 18Q vs 120Q. (F–H) Immunoblots. P70 S6kinase (Thr389) and Akt kinase (Ser473) phosphorylation was analyzed 24 h and 48 h after transfection with huntingtin fragment proteins. Also the total protein levels of p70 S6K and Akt kinase were studied. Phosphorylation of the p70 S6K and Akt kinase was inhibited in 120Q-huntingtin expressing cells at 24 h. However phosphorylation of p70 S6K and Akt were increased in 120Q huntingtin expressing cells 48 h after transfection, reflecting an increased mTOR activity. Typical experiment is shown and was repeated three times. Quantification was done using ImageJ. Values are means ± SD, n = 3. *p < 0.05 for 18Q vs 120Q. (I) Immunoblots. Phosphorylation of ACC (Ser79) reflects an increased AMPK activity in 120Q-huntingtin expressing cells at 24 h, and a decreased activity at 48 h. Typical experiment is shown and was repeated. (J) Left panel: Cells were transfected for 48 h with the N-terminal huntingtin fragment constructs (green fluorescence) together with the autophagy marker RFP-LC3 (red). Note a lack of autophagosomal vacuoles. Right panel: Cells expressing N-terminal huntingtin fragment constructs (green fluorescence) were stained for LC3 at 48 h. Scale bar, 10 μm. (K) Quantification. Cells were transfected with control or mutant huntingtin expressing plasmids together with RFP-LC3 as indicated. The number of cells with LC3-autophagosomes was counted as described in Materials and methods.

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endogenous GADD34 could affect caspase-3 activation and thereby cell viability. Data obtained showed indeed that 120Qhuntingtin expression induced caspase-3 cleavage (17 kDa fragment) was increased by GADD34 silencing (Fig. 5G). These effects on TSC2 phosphorylation, autophagy and cell death were abolished by GADD34 silencing (Figs. 5C, E–G). This data link the expression levels of GADD34 to cell death and to the susceptibility of neuronal PC6.3 cells to undergo cell degeneration after expression of mutant huntingtin proteins. To further elucidate the role of autophagy in cytoprotection, we employed the autophagy inhibitor 3-MA [29], which significantly decreased the viability of mutant huntingtin expressing cells (p < 0.05 for 120Q vs 120Q + 3-MA) (Fig. 5H). This indicates that an unperturbed autophagy mediates protection against cell

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degeneration induced by mutant huntingtin proteins in the PC6.3 cells.

Discussion The pathophysiological changes accompanying HD are complex and include alterations in gene transcription, cell signaling, energy metabolism, protein trafficking, transport and synthesis, as well as an impaired degradation of misfolded and aggregated proteins. The precise signals regulating these events are not fully understood but ER stress and changes in autophagy [30,31] may contribute to cell death observed in HD. We show here that the mutant huntingtin proteins, but not general ER stress inductors

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Fig. 5 – Overexpression of GADD34 activates autophagy and protects cells against mutant huntingtin-induced cell degeneration. (A) Levels of GADD34 and huntingtin-fusion protein were determined by immunoblots using anti-GADD34 and anti-GFP antibodies. Note equal levels of huntingtin in co-transfected cells. Cells were transfected with GADD34 together with pcDNA3 plasmid as control and mutant huntingtin plasmids as indicated. (B) Immunoblot. Expression of GADD34 decreases the phosphorylation of TSC2 (Thr1462) and inhibits mTOR. (C) Immunoblot. Effect of GADD34 siRNA reduced GADD34 counteracted the decrease in phosphorylation of TSC2 (Thr1462) by mutant huntingtin protein. (D) Cells were transfected for 48 h and immunoblot was made using GFP antibodies. The presence of the converted LC3-II band shows on-going autophagy in GADD34 overexpressing cells. The positions of GFP-LC3-I, GFP-LC3-II and cleaved GFP are indicated. Typical experiments are shown and were repeated three times. (E) GADD34 overexpression promoted and GADD34 siRNA reduced cell viability in 120Q-huntingtin expressing cells at 48 h. MTT assay was done as described in Materials and methods. Values are means ± SD, n = 3. *p < 0.05 for 18Q vs 120Q-expressing cells, for 120Q vs GADD34 + 120Q and for 120Q vs GADD34 siRNA + 120Q. (F) Immunoblot. PARP is a downstream substrate of caspase-3. GADD34 reduced and GADD34 siRNA increased PARP cleavage (89 kDa band) that was induced by the 120Q-huntingtin proteins at 48 h. (G) Immunoblot. GADD34 overexpression decreased and GADD34 siRNA enhanced caspase-3 activation, as evident from the increased levels of cleaved caspase-3 (17 kDa band). Typical experiments are shown and were repeated three times. (H) Treatment with the autophagy inhibitor 3-methyladenine for 6 h increased mutant huntingtin-induced cell death. *p < 0.05 for 75Q-FL vs 75Q-FL + 3-MA.

thapsigargin and tunicamycin, regulate the mTOR pathway in neuronal PC6.3 cells with a decrease in protein synthesis and time-dependent change in the process of autophagy. Particularly,

GADD34 was shown to play a hitherto unrecognized role in the regulation of mTOR pathway and autophagy in HD. Thus, GADD34 via binding TSC1 inhibited mTOR activity and induced

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cytoprotective autophagy at 24 h in the mutant huntingtin expressing cells. This, however, was followed by an inhibition of autophagy at later time points. Overexpression of GADD34 prolonged the inhibition of mTOR and stimulated autophagy, thereby counteracting cell death of mutant huntingtin expressing cells. These data underscore the importance of autophagy to counteract neurodegeneration in HD [6,7,32], and address an important function of GADD34 and the TSC–mTOR pathway in the regulation of this process. Previously, GADD34 has been shown to play an important function in the control of protein synthesis after global translation block [14]. GADD34 interacts with PP1 and dephosphorylates eIF2α, which stimulates protein synthesis during cell injury [15,17]. The levels of GADD34 in the brain correlate with increased cell survival after brain ischemia [33,34]. Apart from PP1, few interactors for GADD34 have so far been found. Recently, it was shown that GADD34 inhibits protein synthesis via the TSC protein complex [22] and this complex is also involved in the control of neuronal responses to stress [35]. We observed here that GADD34 interacts with TSC1 in mutant huntingtin expressing neuronal cells at 24 h. TSC2 was dephosphorylated at 24 h, whilst the levels of GADD34 did not significantly change. However, the levels of GADD34 decreased at later time points, which increased TSC2 phosphorylation (Thr1462) and inhibited cytoprotective autophagy. The decrease in GADD34 at 48 h contrasts to the situation at 24 h and is probably due to an inhibition of protein synthesis that ultimately reduces GADD34 levels in mutant huntingtin expressing cells. Recently it was shown that the loss of TSC1 or TSC2 triggers the unfolded protein response (UPR) in the ER [36]. Interestingly, we observed largely intact levels of TSC1 or TSC2 in mutant huntingtin expressing cells at 24 h, when the ER stress pathways are already activated [20]. This probably reflects the nature of the upstream signals that activate UPR and ER stress in different cell types. It was recently shown that the expression of mutant huntingtin in yeast and in PC12 cells leads to an impairment of the ER-associated degradation (ERAD) pathway that contributes to cell toxicity mediated by the misfolded proteins [30]. It is reasonable to assume that the inhibition of the ERAD pathways together with a reduction in autophagy may enhance the toxicity of mutant huntingtin proteins in neuronal cells. Previously it has been shown that the inhibition of mTOR pathway by using rapamycin can reduce cell toxicity induced by mutant huntingtin proteins [6,7]. However, little is known about the upstream mechanisms controlling mTOR in neurodegenerative diseases, including HD. We show here that GADD34 binds TSC1 in mutant huntingtin expressing cells causing inhibition of the mTOR pathway. A similar mechanism for mTOR inhibition has previously been reported in non-neuronal cells after cell stress induction by energy failure [22]. Extending these results, we show here that the GADD34–TSC complex inhibits mTOR and enhances cytoprotective autophagy in a time-dependent manner that probably is of physiological importance. Autophagy has been linked to increased cell survival after ER stress [37], and the cytoprotective autophagy observed here probably serves as a protective measure against the deleterious effects of mutant huntingtin protein and thus is a cell protective signal. Results obtained using the inhibitor 3-methyladenine demonstrate that inhibition of autophagy cause cell degeneration in neuronal PC6.3 cells. Previous data also support the view that

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active autophagy may facilitate the removal of misfolded proteins, and other toxic products and damaged organelles during cell stress [31,32,37]. Apart from GADD34, we report here that AMPK could contribute to the time-dependent changes in TOR signaling and autophagy observed in mutant huntingtin expressing cells at 24 h and 48 h of transfection. The activity of AMPK was measured by changes in the phosphorylation of its downstream substrate ACC (Ser79), which was increased at 24 h and decreased at 48 h. AMPK has recently gained an increased attention as a potentially important protein in neuronal survival and neuroprotection [38]. In view of our data presented here on AMPK in mutant huntingtin expression cells, it is important to study the role of AMPK in cytoprotective autophagy in neurons in more details in the future. Taken together, the present results demonstrate that GADD34 has a protective function in neuronal cells by inducing cytoprotective autophagy via the TSC/mTOR pathway and by enhancing cell viability in mutant huntingtin expressing cells. Previous studies have shown that GADD34 stimulates protein synthesis via the eIF2. Interestingly, mTOR itself is known to regulate both the process of translation and the induction of autophagy. These data show a functional overlap between GADD34 and mTOR in regulation of autophagy and protein synthesis. It remains to be studied whether GADD34 may specifically interact with mutant huntingtin and other polyQ proteins. It is tempting to speculate that GADD34 may have other targets as well that may be active during cell stress and neurodegeneration. In view of its neuroprotective effects shown here, GADD34 may be a useful target to consider in defining novel strategies for counteracting some of the adverse cellular changes observed in HD.

Acknowledgments We thank A. Norremolle and L. Hasholt for the N-terminal huntingtin plasmids, F. Saudou for the full-length huntingtin constructs, and E. Lehto and J. Mäkelä for skillful technical assistance. LC3-RFP, LAMP1-RFP and GADD34 plasmids were obtained from Addgene as courtesy by T.Yoshimori, W. Mothes and D. Ron respectively. Confocal imaging was done at Molecular Imaging Unit, Biomedicum Helsinki. This work was supported by Academy of Finland, Sigrid Juselius Foundation, Arvo and Lea Ylppö Foundation, Liv and Hälsa Foundation, Finska Läkaresällskapet, Emil Aaltonen Foundation, von Frenckell Foundation, Oskar Öflund and Minerva Foundation. AH is a PhD student in Finnish Graduate School of Neuroscience.

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