Background
Macroautophagy (simply referred to as autophagy) is a lysosome degradation pathway involving the synthesis, trafficking, and degradation of autophagic vacuoles, or autophagosomes. Basal autophagy is responsible for the turnover of long-lived proteins, protein aggregates, and damaged organelles, but can also be upregulated to cope with various cellular stressors. In fact, autophagy disregulation has been implicated in many disease states. Several studies of postmortem Huntington’s disease (HD) brains and animal models have indicated altered autophagic activity [
1‐
4]. As a bulk cellular degradation pathway, autophagy has also been extensively studied for its neuroprotective potential through the removal of mutant huntingtin (Htt) [
5]. However, the precise pathways involved in autophagy during Huntington’s disease (HD) are still being clarified.
Autophagy is tightly regulated by multiple signaling pathways related to nutrient sensing and cellular stress. ULK1 is a serine/threonine kinase that initiates the autophagy cascade [
6]. ULK1 is regulated in part by mTOR and AMPK, which inhibit and activate ULK1, respectively [
7]. Immediately downstream of ULK1 is the class III PI 3-kinase, Vps34. Vps34 phosphorylates phosphatidylinositol at the 3′ position to form PI(3)P [
8], which serves as a second messenger to facilitate the recruitment of later stage, autophagy-related proteins to the site of autophagosome formation. However, Vps34 activity is not limited to autophagy; it is also involved in endosomal sorting and cytokinesis [
9,
10]. Vps34 exists in multiple distinct complexes with Beclin 1 and VPS15 [
11], but Vps34 in complex with ATG14 is exclusive to autophagy initiation. Upon autophagy induction, ATG14-Vps34 is recruited to the site of autophagosome biogenesis in an ULK1-dependent manner [
12].
HD is a fatal neurodegenerative disease caused by mutations in the Htt gene that code for expanded polyglutamine tracts (polyQ) in the first exon, which causes protein aggregation and neuronal loss throughout the brain, most notably in the striatum and cortex. The precise nature of autophagy alterations in HD is not completely understood. However, increasing autophagy has been shown to facilitate the clearance of mutant Htt aggregates [
1,
13]. Therefore, understanding the status of autophagy in the context of HD is crucial for the rational design of autophagy-based therapeutics.
Recent work has aimed at understanding the autophagy pathway in finer detail, during the HD pathogenesis. As part of this, a link between the ULK1 kinase and the autophagy receptor, p62/SQSTM1, has been identified. ULK1 phosphorylates p62 to promote selective autophagy in response to proteotoxic stress [
14]. Expression of mutant Htt causes an increase in p62 phosphorylation, which can also facilitate autophagic clearance of polyQ protein. Unexpectedly, loss of p62 actually alleviates toxicity in HD mouse models, pointing to a negative impact of p62 in the disease progression [
15]. However, it is not fully understood how ULK1 regulates autophagy, especially in the context of protein aggregate prone neurodegenerative diseases.
Herein, we report a mechanism whereby ATG14-Vps34 activity is regulated by ULK1-mediated phosphorylation of ATG14. This phosphorylation occurs in an mTOR-dependent fashion. In contrast to our previous report of increased ULK1-mediated p62 phosphorylation in animal and cellular HD models [
14], we show that ATG14 phosphorylation and ATG14-Vps34 activity is decreased in Q175 mice. Furthermore, decreased ATG14 phosphorylation is observed during general proteotoxic stress, which we propose is caused by recruitment of ULK1 away from ATG14 by p62.
Methods
Antibodies and reagents
Vps34 (#4263), Myc (#2276), Actin (#3700), and Beclin 1 pS14 (#84966) were purchased from Cell Signaling Technologies (Danvers, MA). Vps15 (#A302-571A), and NRBF2 (#A301-852A) were purchased from Bethyl Laboratories. ATG14 (MBL; #PD026), polyQ (Merck Millipore; #MAB1574), huntingtin aggregates (Merck Millipore, #MAB5374) Beclin 1 (Santa Cruz Biotechnology; #sc-11427), LC3 (Abcam; #ab48394), Flag-M2 (Sigma-Aldrich; #F1804), p62 (American Research Products; #03-GP62-C), p62 pS409 (in house), ULK1 (Sigma-Aldrich, #A7481), GAPDH (Thermofisher; #2D4A7), and GFP (Roche; #11814460001) were purchased from indicated company. The ATG14 antibody used to detect the purified fragment (a.a. 20–73) was made in house [
11]. Anti–phosphorylated ATG14 polyclonal antibody was raised in rabbits using the peptide RDLVD(p
S)VDDAEGC as an antigen by Cocalico Biologicals. MG132 (Calbiochem), isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich), lipofectamine 2000 (Invitrogen), EDTA-free protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche Diagnostics), Dynabeads protein G (Invitrogen), NuPAGE Bis-Tris gel system (Invitrogen), QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies), Hybond-P PVDF membrane (GE Healthcare Life Sciences), BCA Protein Assay Reagent Kit (Pierce), Radioactive [γ-32P]ATP (PerkinElmer), Calf intestinal alkaline phosphatase (New England Biolabs), and TALON metal affinity resin (Clontech) were purchased from the companies listed.
Plasmids
Myc-ULK1 wildtype and kinase inhibited (K46I) mutant were provided by Dr. Sharon Tooze (London Research Institute). Beclin 1-AsRed, ATG14-GFP, FLAG-ATG14, and myc-Vps34-his-Vps15 plasmids were generated as previously described [
11]. ATG14 a.a. 20–73 was provided as a generous gift by Dr. Yanxiang Zhao (Hong Kong Polytechnic University).
Cell culture
MEF, HEK293T, HCT116, and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 50 μg/mL penicillin and streptomycin (Invitrogen). Wild type and p62 KO MEF cells were provided by Dr. Masaaki Komatsu (Niigata University) [
16]. ATG14 KO HCT cells were generously provided by Dr. Richard Youle (National Institute of Neurological Disorders and Stroke) [
17] and were maintained in McCoy’s 5A plus media (Invitrogen) supplemented with 10% fetal bovine serum, 1X non-essential amino acids (Invitrogen), and glutamine (4 mM final concentration). PolyQ-mCFP HeLa cells were maintained as previously described [
13], which were generous gifts from Dr. Ai Yamamoto (Columbia University). Wildtype and ULK1/2 double KO MEFs were a provided by Dr. Mondira Kundu (St. Jude Children’s Research Hospital). Transient DNA transfection of HEK293T and HeLa cells was performed using Lipofectamine 2000 kit according to the manufacturer’s manual (Invitrogen).
Immunoblot analysis
All IP’s were performed using 10 mM Tris–HCl pH 7.5, 2 mM EDTA, 100 mM NaCl, 1% NP-40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). All other lysates were prepared using 50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, an protease/phosphatase inhibitors (Roche). Supernatants were collected after centrifugation at 13,000 g for 15 min at 4 °C. Supernatants were subjected to BCA assay and then resolved by SDS-PAGE.
In vitro lipid kinase assays
Immunoprecipitation was performed with 500 μg protein from post-nuclear supernatant, using antibodies against ATG14 (MBL) overnight at 4 °C. Samples were incubated with 30 μl Dynabeads (Life Technologies) for 1– 2 h at 4 °C and then washed three times with lysis buffer. Two thirds of the beads were reserved for western blotting, while the remaining third was used for the kinase assay. Beads for the kinase assay were washed once with wash buffer (20 mM HEPES pH 7.4, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, and 0.05 mM DTT) and then resuspended to a final volume of 50 μl with reaction buffer (20 mM HEPES pH 7.4, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, and 0.05 mM DTT, 50 mM cold ATP, 5 mM MnCl2, 0.1 mg/ml sonicated phosphatidylinositol). 10 nM wortmannin was included in the indicated controls. The reaction was started with the addition of γ-32P-ATP (5 mCi) and the samples were shaken at 37 °C for 30 min. Reactions were stopped with 120 μl stop buffer (CHCl3/CH3OH/HCl at a 10:20:0.2 volume ratio) and then shaken for another 10 min at room temperature. Samples were centrifuged for 5 min at 1000 g. 15 μl of the lower, organic phase was resolved on silica coated TLC plate (Millipore) using CHCl3/CH3OH/NH4OH/H2O (86:76:10:14 volume ratio) and visualized with the Typhoon 9400 Variable Imager (GE Healthcare Biosciences).
In vitro ULK1 kinase assay
Myc-ULK1 WT, myc-ULK1 kinase inhibited, or myc protein was immunopurified from HEK cells using anti-myc antibodies. IPs were washed once with kinase buffer (20 mM HEPES pH 7.4, 12.5 mM beta-glycerophosphate, 25 mM MgCl2, 5 mM EGTA, 0.25 mM DTT, 100 μM cold ATP, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche)) and then resuspended to a final volume of 50 μl with reaction buffer including 5 μg of substrate (ATG14 a.a. 20–73 or MBP) and 5 μCi 32P-ATP. Reaction was incubated at 37 °C for 30 min. Samples were subject to gel electrophoresis in a 4–12% bis-tris gel. The gel was visualized with the Typhoon 9400 Variable Imager (GE Healthcare Biosciences).
Protein expression and purification
Expression of His-ATG14 a.a. 20–73 was induced in E.coli BL21-CodonPlus (Agilent) cells by growing at room temperature for 16 h with 0.05 mM of IPTG. Bacterial cell lysis and protein purification was done using the TALON metal affinity resin kit in strict accordance with the recommended manufacturers protocol (Clontech).
Triton X-100 soluble and insoluble assays
Samples were lysed in PBS containing 1% Triton X-100 and phosphatase/protease inhibitors on ice for 30 min. After centrifugation at 15,000 g for 30 min at 4
°C, Triton X-100 insoluble fractions were collected. Pellets were washed 4x in lysis buffer. Pellets were further solubilized in PBS containing 1% SDS, 1% Triton X-100, and phosphatase/protease inhibitors at 60
°C for 1 h. Triton X-100 insoluble fractions were collected after centrifugation at 15,000 g for 30 min at 4
°C. A BCA assay was done on the lysates to ensure equal protein concentrations for western blot. This procedure was described elsewhere [
14].
Fluorescence microscopy
Mice were transcardially perfused with ice cold PBS to remove excess blood, then perfused with cold 4% paraformaldehyde. Brain sections were blocked in PBS containing 3% BSA and 0.1% Triton X-100 for 1 h at room temperature. Sections were then incubated with primary antibodies in blocking buffer overnight at 4 °C. After washing 3 times in PBS sections were incubated with Alexa-conjugated secondary antibody for 1 h at room temperature. Secondary antibody was goat anti-mouse Alexa Fluor 555. After 3 more washes with PBS, sections were mounted with mounting medium (ProLong Gold antifade mountant with DAPI, Invitrogen). Cells were examined under Carl Zeiss upright or invert confocal microscopes (LSM780 system). Images were taken with 63X oil immersion objective lens at room temperature and image acquisition was performed by Zen2012 software.
Animals
All animal studies were performed in compliance with IACUC (Institutional Animal Care and Use Committee) at Icahn School of Medicine at Mount Sinai. Heterozygous z_Q175 and littermate controls were obtained from the CHDI colony at the Jackson Laboratories. For imaging analysis, GFP-LC3 expressing mice [
18] were crossed with z_Q175 mice. All mice used were aged 12–15 months.
Statistical analysis
Data are presented as mean ± SEM from at least three independent experiments. Statistical analysis was performed with GraphPad Prism v5.0 (Graphpad Software). For data normalized with control group (a value of 1), one sample t-tests against a hypothetical value of 1 were used to compare the difference between the other group and control group. For other non-normalized values, unpaired Student’s t-tests were used. For multifactorial analysis, a two-way ANOVA was used. Bonferroni’s correction was used for multiple comparisons. P values <0.05 were considered as statistically significant.
Discussion
Autophagy modulation is being pursued as a therapeutic strategy for neurodegenerative diseases associated with protein aggregates. Thus, a better understanding of the autophagy status in the disease process is critical to this endeavor. Our study provides an insight into the mechanism of autophagy regulation whereby ULK1/2 signals to autophagy-specific Vps34 complex to activate autophagy through the mTOR nutrient pathway. We find that ULK1 mediates ATG14 phosphorylation at a novel serine site, S29, in response to amino acid (but not glucose) starvation, which in turn up-regulates Vps34 kinase activity. Importantly, we find that the specific ATG14 phosphorylation and ATG14-associated VSP34 activity, which is autophagy-specific, are impaired in HD cell and animal models. Interestingly, our current assays suggest no obvious impairment of autophagic activity in the brain of Q175 mice. Thus the 35% decrease of ATG14-linked Vps34 activity does not corroborate a significance reduction of basal autophagy in the HD animal models. Note that our autophagy assays are based on GFP-LC3 reporter and protein levels of several autophagy markers including p62 [
14], LC3II, ULK1, and Beclin 1-VPS34-ATG14 protein levels; they are limited in that they do not reflect accurately the autophagy flux, which has been extremely challenging to study in vivo. It is possible that transient, subtle and regional changes of autophagy in Q175 mice are difficult to detect using available approaches with low sensitivity. We thus postulate that monitoring ATG14 phosphorylation and ATG14-Vps34 activity provides a new means for in-vivo autophagy pathway analysis which serves as a useful biomarker for altered autophagy signaling in disease.
Interestingly, a previous study in HdhQ200 knockin model, which manifests an accelerated and robust phenotypes than Q175 mice, has perinulear mHtt foci accompanied by colocalized LC3 and p62 aggregates. The HdhQ200 mice have increased levels of LC3II and p62 levels in the striatal neurons [
3]. Although interpreted as the induction of autophagy, the available evidence indeed informs little regarding the exact direction change of autophagy or autophagy flux, nonetheless shows the alteration of autophagy in HdhQ200 mice. Our study found no perinuclear mHtt in Q175 HD brain, which may explain the lack of GPF-LC3 puncta formation. The lack of GFP-LC3 puncta in control
GFP-LC3 mice is consistent with the idea that basal autophagosome formation is extremely rare in wildtype neurons as described by Mizushima’s group [
3]. Then it is not surprising that no GFP-LC3 puncta formation was found in Q175 neurons, as the reduced ATG14 phosphorylation and Vps34 activity as shown in our study predicts even lower rate for autophagosome formation. It is likely that accelerated accumulation of mHtt aggregates in HdhQ200 mice causes apparent change in LC3 and p62 levels. It would be interesting to investigate the ULK1-mediated ATG14 and Beclin 1 phosphorylation in HdhQ200 mice. Furthermore, future study should examine whether a long term impact (e.g. >15 months) of the decrease of ATG14-linked Vps34 activity can result in detectable reduction of autophagy measured through autophagic markers such as LC3 and p62.
ULK1-mediated phosphorylation of ATG14 may represent an important signaling event for autophagy induction that coordinates the action of the two key components of autophagy machinery, the ULK1 kinase complex and ATG14-Vps34 complex (autophagy specific). During the preparation of this manuscript, Park, et al. published their independent discovery of ATG14 phosphorylation by ULK1 [
32]. Their results are in an agreement with our conclusion that Vps34 activity is regulated by ATG14 phosphorylation and demonstrate its positive effects on autophagosome formation. A previous study showed that ULK1-mediated Beclin 1 phosphorylation contributes to the regulation of Vps34 activity [
23]. However, it showed that ULK1-mediated Beclin 1 phosphorylation is also promoted by both UVRAG and ATG14. However, two separate reports reveal that starvation-induced mTOR inhibition either has no effect on UVRAG-Vps34 activity or decreases it by 60% [
33,
34], raising a question about the precise role for phosphorylation of Beclin 1 in upregulating Vps34 activity. Our current data shows that, although phosphorylation of ATG14 and Beclin 1 occur with the similar kinetics in response to mTOR inhibition, ATG14 phosphorylation status is able to regulate Vps34 activity independent of Beclin 1 phosphorylation. Although it goes beyond the scope of the present study, further clarification of the role of Beclin 1 phosphorylation for UVRAG-Vps34 activity is warranted. Finally, given ATG14’s role at the ER-mitochondria contact site (MAM) and the requirement of ULK1 in ATG14 puncta formation during autophagy induction, it is possible that ULK1 activity is involved in the recruitment of one or more Vps34 complex members to the MAM [
12,
35].
Our detailed study of ULK1 kinase and ATG14-Vps34 lipid kinase show reduced activity of both kinases, suggesting altered regulation of the autophagy pathway in HD models. Both phosphorylated Beclin 1 and ATG14 levels are decreased in HD brains, indicating the lower ULK1 kinase activity, which is regulated by mTOR nutrient pathway. The reduced ULK1-mediated ATG14 or Beclin 1 phosphorylation was initially surprising, given that ULK1-mediated p62 phosphorylation (mTOR-independent) increases in the same models of HD [
14]. Interestingly, this differential ULK1 activity (mTOR vs. non-mTOR regulated) was also seen in cultured cells following proteasomal stress. It is possible that the regulatory mechanism for the aberrant ULK1 activity is shared between proteasomal stress and HD. Our data suggests that autophagy receptor p62 plays a role in regulating ULK1-mediated ATG14 phosphorylation upon proteotoxic stress. From our earlier study, in both HD (expressing polyQ-protein) and proteasomal stress conditions, there is an increased binding between p62 and ULK1, concurrent with competition of ULK1 by p62 oligomerization [
14]. As a potential result, the access of ATG14-Vps34 complex to ULK1 kinase may be reduced. Our result also indicates proteasomal stress condition causes sequestration of the whole ULK1-ATG13-FIP200 complex. A similar competition model for ULK1 binding was also proposed by another group, in which Htt was shown to pull ULK1 away from the inhibitory effects of mTOR during selective autophagy [
36]. Alternatively, optineurin, another autophagy receptor, is involved in aggrephagy and has also been shown to interact with Htt and co-localize with ULK1 [
37‐
40]. Therefore, we are unable to rule out the possibility that other autophagy receptors may also sequester ULK1 away from ATG14 during proteotoxic stress.
Finally, our current work extends our previous report that ULK1-mediated p62 phosphorylation in selective autophagy helps clear polyQ protein [
14] by showing that ATG14 phosphorylation and ULK1 activity per se has beneficial effects in the clearance of mutant Htt. By targeting ULK1 activity to regulate autophagy, the unintended consequences of general mTOR inhibition can be avoided. Future work should be aimed at exploring the modulation of ULK1 activity for the treatment of neurodegenerative diseases associated with protein aggregates.
Conclusions
As the major pathway for protein aggregate clearance, autophagy has been heavily implicated in the HD pathogenesis and considered a therapeutic target. However, the exact molecular mechanisms at work remain elusive. In this work, we identify a critical process during autophagosome biogenesis that is disregulated following mutant Htt-induced proteotoxic stress. We find that in the canonical autophagy pathway, ULK1 phosphorylates ATG14, which promotes Vps34 lipid kinase activity. ATG14 phosphorylation and Vps34 activity, however, are impaired in HD models. Our study highlights ULK1 and Vps34 as potential targets for the treatment of HD.
Acknowledgements
We thank members of the Yue Lab for technical assistance. We would like to thank Dr.’s Yanxiang Zhao and Richard Youle for kindly providing reagents used for this study.