O-GlcNAc regulation of autophagy and α-synuclein homeostasis; implications for Parkinson’s disease
© The Author(s). 2017
Received: 11 April 2017
Accepted: 29 June 2017
Published: 19 July 2017
Post-translational modification on protein Ser/Thr residues by O-linked attachment of ß-N-acetyl-glucosamine (O-GlcNAcylation) is a key mechanism integrating redox signaling, metabolism and stress responses. One of the most common neurodegenerative diseases that exhibit aberrant redox signaling, metabolism and stress response is Parkinson’s disease, suggesting a potential role for O-GlcNAcylation in its pathology. To determine whether abnormal O-GlcNAcylation occurs in Parkinson’s disease, we analyzed lysates from the postmortem temporal cortex of Parkinson’s disease patients and compared them to age matched controls and found increased protein O-GlcNAcylation levels. To determine whether increased O-GlcNAcylation affects neuronal function and survival, we exposed rat primary cortical neurons to thiamet G, a highly selective inhibitor of the enzyme which removes the O-GlcNAc modification from target proteins, O-GlcNAcase (OGA). We found that inhibition of OGA by thiamet G at nanomolar concentrations significantly increased protein O-GlcNAcylation, activated MTOR, decreased autophagic flux, and increased α-synuclein accumulation, while sparing proteasomal activities. Inhibition of MTOR by rapamycin decreased basal levels of protein O-GlcNAcylation, decreased AKT activation and partially reversed the effect of thiamet G on α-synuclein monomer accumulation. Taken together we have provided evidence that excessive O-GlcNAcylation is detrimental to neurons by inhibition of autophagy and by increasing α-synuclein accumulation.
Autophagy is important for recycling or removing damaged proteins and organelles in response to nutrient deprivation and stress [1, 2]. Deficient autophagy can lead to accumulation of dysfunctional intracellular proteins [3, 4] and have a significant impact on the pathogenesis and progression of neurodegenerative diseases . The coupling of autophagy with metabolism is critical in neurons and may be related to their susceptibility to the proteotoxicity associated with neurodegenerative diseases such as Parkinson’s disease. A key nutrient sensing pathway is protein O-GlcNAcylation which is an evolutionarily conserved process in which proteins are reversibly modified at Ser/Thr residues in response to nutrient availability and stress [6–10]. The attachment of O-GlcNAc to proteins is catalyzed by O-GlcNAc transferase (OGT), and its removal by O-GlcNAcase (OGA) [11–13]. This protein post-translational modification process modifies Ser/Thr residues and can occur as rapidly as phosphorylation, thereby modulating protein function and signaling [10, 14–16]. Approximately 2–5% of glucose taken up by cells is consumed by the hexosamine biosynthetic pathway to generate uridine diphosphate N-acetyl-glucosamine (UDP-GlcNAc), the substrate for OGT [10, 11, 14–17].
The impact of O-GlcNAcylation has been studied in the context of neurodegenerative diseases, focusing mainly on O-GlcNAc modification of aggregation-prone proteins . In the context of Alzheimer’s disease, O-GlcNAcylation may be beneficial because pharmacological inhibition of OGA increases tau O-GlcNAcylation, decreases tau phosphorylation, and decreases neurodegenerative phenotypes [19–24]. However, in a C. elegans model of neurodegeneration, an OGA inactive mutant that results in increased O-GlcNAcylation was shown to increase proteotoxicity . In cell and fly models, increased O-GlcNAcylation has been shown to be associated with increased mutant huntingtin toxicity . These observations suggest that changes in protein O-GlcNAcylation are an important contributor to the pathogenesis of neurodegenerative diseases but its effects are highly context-dependent . Pertinent to Parkinson’s disease, it has been shown that α-synuclein, a protein involved in the pathophysiology of the disease, can be O-GlcNAcylated [28, 29]. It has been shown in vitro that O-GlcNAcylation at T72 decreases both the propensity of α-synuclein to aggregate and its toxicity when added to cultured cells . Despite the fact that both autophagy and the O-GlcNAc pathway share nutrient and stress sensing properties, whether the O-GlcNAc pathway also contributes to autophagy regulation is only now being investigated [30, 31]. For example, it has shown in C. elegans and HeLa cells that O-GlcNAc modification of the protein SNAP-29 regulates autophagosome maturation . We and others have previously shown that the O-GlcNAc pathway is active in the brain and that O-GlcNAcylated proteins are abundant in nerve terminals [12, 33–35]. O-GlcNAcylation levels in the brain have been shown to increase by 30% from 5 to 24 months, suggesting an involvement in age-dependent neuronal function [22, 33]. Furthermore, we have demonstrated that increased O-GlcNAc levels lead to impaired autophagic signaling and that key regulators of autophagy, Beclin-1 and Bcl-2, are O-GlcNAcylated in response to nutrient deprivation in cardiomyocytes . In the present study we provide evidence that O-GlcNAcylation levels are significantly increased in Parkinson’s disease postmortem brains, and that pharmacological inhibition of OGA and thereby increasing O-GlcNAc levels in neuronal cultures decreases autophagic flux and induces α-synuclein accumulation.
Pharmacological inhibition of OGA by thiamet G increases O-GlcNAcylated proteins in primary neurons.
Prior studies in rat cardiac myocytes have demonstrated effects of high glucose on O-GlcNAcylation of mitochondrial proteins and impairment of complex I, III and IV activity in isolated mitochondria, which can be reversed by overexpression of OGA [37–40], how O-GlcNAcylation affects mitochondrial function in neurons is unknown. We observed that complex IV activity in the presence of ascorbate and TMPD was modestly decreased while complex I and II substrate linked oxidation in the presence of either ADP or FCCP was unchanged 24 h after thiamet G at 0.25 μM (Additional file 1: Figure S1A). Only after 7 d of thiamet G exposure, complex II substrate linked oxidation in the presence of FCCP was also decreased (Additional file 1: Figure S1B). A mitochondrial stress test on intact cells after either 24 h or 7 d of thiamet G exposure demonstrated that in intact cells, no significant changes occurred in basal, maximal, ATP-linked, proton leak and non-mitochondrial oxygen consumption rates (data not shown). Furthermore, thiamet G did not affect mitochondrial DNA copy number (Additional file 1: Figure S1C); or overall mtDNA damage (Additional file 1: Figure S1D). There was also no change in PGC1α after either 24 h or 7 d thiamet G exposure (Additional file 1: Figure S1E–F), suggesting mitochondrial biogenesis is not affected. Therefore, the levels of protein O-GlcNAcylation in response to the sub-lethal thiamet G concentration over the time period in our experiments are not sufficient to decrease mitochondrial quality.
OGA inhibition decreases autophagic flux in primary neurons
Increased protein O-GlcNAcylation in Parkinson’s disease postmortem brains
Inhibition of OGA increases α-synuclein levels in primary neurons
Inhibition of OGA increases MTOR activation
To investigate the possible mechanisms of thiamet G-dependent inhibition of autophagy, we first performed real-time RT-PCR analyses of selective autophagy and lysosomal genes. We found that there was a modest increase in p62 mRNA at 24 h but not at 7 d, although p62 protein levels remain unchanged (Additional file 4: Figure S4, Additional file 5: Figure S5). There was no change of Becn mRNA after 24 h or 7 d thiamet G exposure (Additional file 4: Figure S4A-B), while a modest increase of BECN protein was seen after 7 d (Additional file 4: Figure S4E-F, 4H). mRNA and protein levels of Hsc70 and Lamp2a, which are involved in chaperone mediated autophagy, were unchanged (Additional file 4: Figure S4A-B, 4I–L), and there was no change in Lamp1 mRNA (Additional file 4: Figure S4A-B).
Inhibition of OGA decreases autophagic flux even in the presence of rapamycin
MTOR activation and AKT signaling
In this study we investigated the role of O-GlcNAcylation on neuronal autophagy and α-synuclein homeostasis. We show that inhibition of O-GlcNAc removal in primary neurons attenuates autophagic flux. Over time, hyper-O-GlcNAcylation also causes α-synuclein monomer accumulation prior to cell death, while mitochondrial function is largely intact. Of importance, we report for the first time an increase in global protein O-GlcNAcylation in postmortem temporal cortical samples obtained from Parkinson’s disease patients compared to controls. Since α-synuclein inclusions do not appear in the temporal cortex until stage IV, our results suggest that increased overall O-GlcNAcylation precedes α-synucleinopathy.
In this study we inhibited O-GlcNAc removal acutely using a highly specific inhibitor of O-GlcNAcase, thiamet G . Although this is a highly specific inhibitor, off-target effects cannot be ruled out and future studies using a conditional knockout of OGA, or overexpressing OGT in neurons in vivo are planned. We found no changes of overall ubiquitinated proteins nor proteasome activities after 24 h or 7 d of thiamet G, suggesting that neurons rely more heavily on macroautophagy rather than the proteasome for clearance of damaged proteins. Our observation that both increased MTOR activation and increased LC3-II accumulation are associated with thiamet G exposure suggests that thiamet G inhibits both initiation and completion of autophagy. This observation is further substantiated by the decreased autophagic flux in rapamycin exposed neurons in the presence of thiamet G. Regarding mechanisms of autophagy regulation, although our study focused on effects of inhibition of OGA on MTOR activation, it is conceivable that O-GlcNAc modification of SNAP-29 may also occurs in neurons to regulate autophagosome maturation . Future studies with sensitive and quantitative methods such as mass spectrometry are needed to determine whether SNAP-29 O-GlcNAcylation also occurs in neurons and contributes to α-synuclein accumulation in response to thiamet G.
At present the exact mechanisms of how thiamet G increases both MTOR and AKT phosphorylation and how rapamycin decreases both MTOR and AKT phosphorylation are not well defined. One possibility is that increasing O-GlcNAc levels activate MTOR through Sp1 activation of transcription of glycerol-3-phosphate acyltransferase-1 (GPAT1) which activates MTOR . Whether in neurons thiamet G induced MTOR activation is mediated by Sp1 activation of GPAT1 will need to be further investigated. In addition to being activated by MTOR inhibition , AKT-S473 phosphorylation has been shown to inhibit autophagosome formation via phosphorylation of BECN .
O-GlcNAcylation has been shown to play a protective role against tau phosphorylation in HEK cells, rats and transgenic mice carrying pathogenic tau [21, 24]. While these prior findings support the potential benefit of developing pharmacological agents that increase O-GlcNAcylation and thereby decreasing tau phosphorylation for the treatment of Alzheimer’s disease, our observations that α-synuclein accumulates in response to thiamet G suggest that such an approach may be detrimental to α-synuclein homeostasis. This is important as α-synuclein accumulation occurs in ~60% of Alzheimer’s disease cases and in the majority of Parkinson’s disease cases. We found that in the presence of thiamet G, overall protein O-GlcNAcylation is not decreased by rapamycin, whereas α-synuclein and p62 are decreased, suggesting that rapamycin-dependent increase in autophagic flux is sufficient to overcome the inhibitory effects of increased O-GlcNAcylation on autophagy of specific substrates such as α-synuclein and p62. This finding has important implications for the clinical applications of rapamycin since rapamycin can be beneficial in decreasing α-synuclein even in the presence of pathologic suppression of autophagy with increased O-GlcNAcylation.
Neurobasal medium (21103–049), B-27 supplement (17504044–044), L-glutamine (25030–081) and penicillin-streptomycin (15140–122) were purchased from Life Technologies. Chloroquine (C6628), oligomycin (75351), FCCP (C2920), rotenone (R8875), antimycin-A (A8674), pyruvate (P5280), malate (M6413), ADP (A2754), succinate (S2378), L-ascorbic acid (A5960), N,N,N′,N′-Tetramethyl-p-phenylenediamine (TMPD; T7394), poly-L-lysine, rapamycin (R8781) were all purchased from Sigma. PMP (102504) was from Seahorse Bioscience. Thiamet G was purchased from SD Chem Molecules LLC. (Owings Mills, MD). Papain was from Worthington.
Postmortem brain analyses
Temporal cortex specimens from No-disease (ND) controls and Parkinson’s disease (PDII-IV stage) were obtained from the Arizona Parkinson’s disease Consortium (Dr. Thomas G. Beach, Director) and the Michael J Fox Foundation, classified by a unified staging system [56, 57]. Robust cortical synucleinopathy is evident at stage IV. Tissues were lysed in lysis buffer containing 50 mM Tris pH 7.4, 175 mM NaCl, 5 mM EDTA pH 8.0. The lysates were centrifuged at 10,000 x g for 15 min at 4 °C. Supernatants were subjected to BCA protein assay, and 15 μg protein was loaded per lane for western blotting analyses.
Primary neuron cultures
Primary rat cortical neurons were obtained from embryonic day 18 embryos [58, 59]. Timed-pregnant Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were sacrificed by CO2 inhalation and embryos were collected in a petri dish and placed on ice. Dissections were performed in ice-cold Hanks’ balanced sodium salts (without Ca2+ and Mg2+). Cerebral cortices were isolated and collected in 15-ml Falcon tubes. The tissues were incubated for 15 min at 37 °C with papain (Worthington), followed by mechanical dissociation by pipetting. Cells were briefly exposed to DNase I and finally concentrated by centrifugation at ~200 x g for 5 min and resuspended in Neurobasal medium containing 2% B27 supplement (Invitrogen), 1% Pen–Strep (10,000 U/ml, 10,000 mg/ml), and 0.5 mM L-glutamine. Cell preparations were plated in XF96 plates and 48-well plates coated with 0.1 mg/ml poly-L-lysine (Sigma) for mitochondrial bioenergetics studies and western blotting respectively. The cultures were kept in a humid incubator (5% CO2, 37 °C). 7 d in vitro (DIV7) cultures were used for all experiments. All animal studies have been approved by IACUC University of Alabama at Birmingham.
Western blot analysis
250,000 cells were seeded on poly-L-lysine coated 48-well plates and treated for either 24 h or 7 d with different concentrations of thiamet G (0.25–25 μM). Then cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.1% SDS, 50 mM Tris, pH 8.0), and protein extracts were separated by SDS-PAGE and probed with the antibodies listed below. Since we had a lot of samples from human postmortem brains, we had to run more than one gels. Thus, all the samples for the control and disease conditions were run at the same time on two separate gels using a common loading control to compare between the 2 gels for quantification. Relative levels of protein were quantified using Image J software from the NIH (Bethesda, MA, USA) and normalized to loading control.
O-GlcNAcylated proteins (UAB hybridoma core, CTD110.6,1:1000), p62 (Abnova, H00008878-M02,1:2000), Microtubule-associated protein 1 light chain 3 alpha/LC3 (Sigma, L8918, 1:2000), β-actin (Sigma, A5441, 1:5000), OGT (Sigma, O6264, 1:2000), α-synuclein (Santa Cruz, sc7011R, 1:1000), PGC1α (Santa Cruz, sc13067,1:1000), HSC70 (abcam,19,136–100, 1:5000), LAMP2a (abcam, 37,024, 1:1000), Ubiquitin (DAKO, Z0458, 1:1000), GAPDH (Millipore, MAB374,1:5000), MTOR (Cell Signaling, 2983S, 1:1000), p-MTOR-S2448 (Cell Signaling 2971S, 1:1000), p-AKT-S473 (cell signaling, 587F11, 1:1000), AKT (cell signaling, 4691S,1:1000).
Goat anti-rabbit IgG-HRP (sc2004, 1:5000), Goat anti-mouse IgG-HRP (sc2005, 1:5000), Goat anti-mouse IgM-HRP (Calbiochem, 1:10,000) and Goat anti-rat IgG-HRP (sc-2006, 1:5000).
Mitochondrial respiration in intact and permeabilized neurons
Respiration in intact neurons was measured using a Seahorse XF96 Analyzer (Seahorse Bioscience) [60–62]. Cells were switched to XF media 30 min before measurement of oxygen consumption rate (OCR), followed by sequential injection of oligomycin (1 μg/ml), FCCP (1 μM), and antimycin A (10 μM) and measurements of OCR between injections. For the mitochondrial activity assay cells were switched to MAS buffer (70 mM Sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, adjusted pH to 7.2) , OCR was measured after injection with 20 μg/ml PMP (1 nM) to permeabilize the cells. Initially, complex I substrates were included with the PMP plus FCCP for complex I activity assays, then injected with 2 μM rotenone to inhibit complex I activity, followed by injection of complex II substrates then injected with Antimycin A. In a separate assay, complex IV activity was measured in PMP permeabilized neurons by injection of ascorbate and TMPD, followed by sequential injections of rotenone and azide. Complex I activity was calculated as the difference in OCR before and after rotenone injection. Complex II activity was calculated as the difference between before and after antimycin injection. Complex IV activity was calculated as the difference after ascorbate injection and after azide injection, n = 3–6 independent incubations. The experiments were repeated with >3 independent cultures and data are presented as representative experiments.
Assessment of cell viability
Primary cortical neurons were plated at 80,000 cells per well in 96-well plates, and following treatment, trypsinized and resuspended in Neurobasal media. Trypan blue was added, and the cells non-permeable to trypan blue were counted as viable [64, 65].
Autoclaved glass coverslips were placed in 24-well culture plates and seeded with primary cortical rat neurons at a density of 240,000 cells per well. Cells were exposed to thiamet G and/or chloroquine for 24 h, followed by fixation with 4% paraformaldehyde and 4% sucrose. After fixation, cells were blocked with 5% BSA and 10% horse serum and then probed for LC3 (Sigma L8918). AlexaFluor 488 and Hoechst were added before slides were mounted and visualized with a Leica TCS SP5 V confocal laser scanning microscope. Cells with LC3 puncta were then counted.
Mitochondrial copy number
The mitochondrial and nuclear 18S DNA were amplified using RT Real-Time SYBR Green PCR master mix (Invitrogen) in an ABI 7500 PCR machine . The primer sequences used for mtDNA were mtDNA-F (5′-CCAAGGAATTCCCCTACACA-3′) and mtDNA-R (5′- GAAATTGCGAGAATGGTGGT-3′). The primer sequences for the nuclear DNA were 18S–F (5′-CGAAAGCATTTGCCAAGAAT-3′) and 18S–R (5′-AGTCGGCATCGTTTATGGTC-3′) and targeted nuclear 18S DNA. Cycling conditions were as follows: 94 °C for 15 s, followed by 40 cycles at 94 °C for 15 s, 60 °C for 1 min, then and 72 °C for 10 min. The mtDNA copy number was normalized to the amplification of the 18S nuclear amplicon.
Mitochondrial DNA damage
Quantitative real-time RT-PCR
Proteasome activity assays
We analyzed the proteasome activities in 25–50 μg protein extracts. The assay buffer consists of 50 mM Tris (pH 7.5), 2.5 mM EGTA, 20% glycerol, 1 mM DTT, 0.05% NP-40, and 25 μM substrate. Substrates Ac-nLPnLD-AMC, Bz-VGR-AMC and Suc-LLVY-AMC were from Enzo life science [43, 68]. MG132 was used at a final concentration of 10 μM to block proteasome activities as negative controls. Fluorescence was read at 5 min intervals for 2 h, at an excitation wavelength of 380 nm and an emission wavelength of 460 nM, n ≥ 3 per group. Data were activities normalized to total protein and control in the assay.
Data are reported as mean ± SEM. Comparisons between groups were performed with unadjusted, unpaired Student’s t-tests or Wilcoxon for non-parametric comparison or ANOVA and Bonferroni’s Multiple Comparison post-hoc test, as noted. A p value of less than 0.05 was considered statistically significant.
We are grateful to the Banner Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona for the provision of human biological materials (or specific description, e.g. brain tissue, cerebrospinal fluid). The Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson’s Disease and Related Disorders), the National Institute on Aging (P30 AG19610 Arizona Alzheimer’s Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer’s Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the Arizona Parkinson’s Disease Consortium) and the Michael J. Fox Foundation for Parkinson’s Research.
This work was supported by Michael J Fox Foundation (Access Data Biospecimens Program) (JJS), UAB AMC21 reload multi-investigator grant (VDU, JCC, JZ), HL101192, HL-110366 (JCC), and NIHR01-NS064090 (JZ). The funding body did not participate in the design of the study and collection, analysis, interpretation of data or writing the manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this published article (and its Additional files).
WYW performed the majority of the experiments and wrote the manuscript. XO performed RT-PCR for mtDNA and RNA. GAB performed all seahorse analyses of mitochondrial bioenergetics. MR assisted in neuronal cultures. SC assisted in the statistical analyses. JJS provided key reagents. JCC, VDU and JZ provided expertise, conceptualization, data analyses and interpretation of this study. All authors read and approved the manuscript.
Ethics approval and consent to participate
All subjects sign Institutional Review Board-approved informed consents allowing both clinical assessments during life and several options for brain and/or bodily organ donation after death in Banner Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona. No individual person’s data in any form was available to researchers involved in preparation of this manuscript.
Consent for publication
The authors declare that they have no competing interests.
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