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Keywords:

  • autophagy;
  • dopamine neuron;
  • mTOR ;
  • oxidative stress;
  • Parkinson's disease;
  • TSC2

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
Thumbnail image of graphical abstract

Abnormal autophagy may contribute to neurodegeneration in Parkinson's disease (PD). However, it is largely unknown how autophagy is dysregulated by oxidative stress (OS), one of major pathogenic causes of PD. We recently discovered the potential autophagy regulator gene family including Tnfaip8/Oxi-α, which is a mammalian target of rapamycin (mTOR) activator down-regulated by OS in dopaminergic neurons (J. Neurochem., 112, 2010, 366). Here, we demonstrate that the OS-induced Tnfaip8 l1/Oxi-β could increase autophagy by a unique mechanism that increases the stability of tuberous sclerosis complex 2 (TSC2), a critical negative regulator of mTOR. Tnfaip8 l1/Oxi-β and Tnfaip8/Oxi-α are the novel regulators of mTOR acting in opposition in dopaminergic (DA) neurons. Specifically, 6-hydroxydopamine (6-OHDA) treatment up-regulated Tnfaip8 l1/Oxi-β in DA neurons, thus inducing autophagy, while knockdown of Tnfaip8 l1/Oxi-β prevented significantly activation of autophagic markers by 6-OHDA. FBXW5 was identified as a novel binding protein for Tnfaip8 l1/Oxi-β. FBXW5 is a TSC2 binding receptor within CUL4 E3 ligase complex, and it promotes proteasomal degradation of TSC2. Thus, Tnfaip8 l1/Oxi-β competes with TSC2 to bind FBXW5, increasing TSC2 stability by preventing its ubiquitination. Our data show that the OS-induced Tnfaip8 l1/Oxi-β stabilizes TSC2 protein, decreases mTOR phosphorylation, and enhances autophagy. Therefore, altered regulation of Tnfaip8 l1/Oxi-β may contribute significantly to dysregulated autophagy observed in dopaminergic neurons under pathogenic OS condition.

Dysfunctional autophagy is frequently observed in post-mortem brains of patients and animal models of Parkinson's disease. In dopaminergic neurons of the 6-hydroxydopamine (6-OHDA) model, oxidative stress induces Tnfaip8 l1/Oxi-β, which results in increased autophagy by its exclusive binding with FBXW5 to stabilize TSC2. Thus, altered regulation of Tnfaip8 l1/Oxi-β may contribute to dysregulated autophagy in dopaminergic neurons under pathogenic oxidative stress, implicating both Oxi-β and FBXW5 as potential intervention targets for dysfunctional autophagy in dopaminergic neurons under oxidative stress.

Abbreviations used
6-OHDA

6-hydroxydopamine

DA

dopaminergic

LC-3

microtubule-associated protein 1A/1B-light chain 3

mTOR

mammalian target of rapamycin

OS

oxidative stress

PD

Parkinson's disease

SN

substantia nigra

TSC2

tuberous sclerosis complex 2

Autophagy is a lysosomal-driven cellular degradation process involved in the turnover of protein aggregates and organelles (He and Klionsky 2009). Dysfunctional autophagy is frequently observed in selective neuronal populations afflicted in common neurodegenerative diseases. In Parkinson's disease (PD), altered autophagy was first discovered in the degenerating dopaminergic (DA) neurons of the substantia nigra (SN) by ultrastructural examinations in post-mortem brains (Anglade et al. 1997). The familial PD-associated molecules, α-synuclein, DJ-1, parkin, PTEN-induced putative kinase 1, and Leucine-rich repeat kinase 2 are in part involved in the autophagy pathway (Cuervo et al. 2004; Plowey et al. 2008; Xilouri et al. 2009; Irrcher et al. 2010; Michiorri et al. 2010), suggesting that autophagy may be dysregulated in PD. In fact, dysregulation of autophagy has been observed during DA cell death in neurotoxin-induced animal models of PD (Thiruchelvam et al. 2000; Larsen et al. 2002; Gomez-Santos et al. 2003; Peng et al. 2004; Gonzalez-Polo et al. 2007; Zhu et al. 2007). However, it is unknown how autophagy signaling is altered by oxidative stress (OS), the major pathogenic condition in PD.

Oxidative stress is known to play a significant pathogenic role in the selective loss of DA neurons in human patients (Dexter et al. 1989; Yoritaka et al. 1996; Zhang et al. 1999) as well as experimental models of PD (Kumar et al. 1995; Sriram et al. 1997; Sherer et al. 2002), especially those treated with PD-related neurotoxins, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone or paraquat (Kumar et al. 1995; Sriram et al. 1997; Thiruchelvam et al. 2000; Sherer et al. 2002; Peng et al. 2004). We previously identified and characterized the phylogenetically conserved Oxi gene family [homologs of TNFAIP8 family (Patel et al. 1997; Lou and Liu 2011)], which includes distinct autophagy regulators (Choi et al. 2010). Importantly, OS increases autophagy and cell death in DA neurons by down-regulating expression of Tnfaip8/Oxi-α, as well as up-regulating expression of Tnfaip8 l1/Oxi-β via unknown mechanism. Tnfaip8/Oxi-α encodes an mammalian target of rapamycin (mTOR) activator (Choi et al. 2010), which is expressed in specific brain regions including SN DA neurons, whereas Tnfaip8/Oxi-α extensively expressed in immune and cancer tissues as well as other organs (Patel et al. 1997; Kumar et al. 2000). In our paradigm, OS-induced down-regulation of Tnfaip8/Oxi-α suppresses the activation of mTOR kinase and enhances neuronal susceptibility to OS. Conversely, up-regulation of Tnfaip8/Oxi-α exerts a protective effect against OS by activating mTOR kinase, which may repress autophagic cell death. Of interest, the close temporal relationship between OS-induced Tnfaip8 l1/Oxi-β and autophagy in DA neurons suggests a potential regulatory role of Tnfaip8 l1/Oxi-β on autophagy. However, little is known about the regulatory mechanisms of Tnfaip8 l1/Oxi-β underlying OS-induced autophagy in degenerating DA neurons of various PD models.

The complex formed by tuberous sclerosis complex 1 (TSC1) and tuberous sclerosis complex 2 (TSC2) is a critical negative regulator of mTOR complex 1 (mTORC1), which controls autophagy (Huang and Manning 2008). TSC2 includes GTPase-activating protein activity, which activates Rheb, and it is through this mechanism that the TSC1-TSC2 complex regulates signaling through mTORC1 to control translation, cell growth, proliferation, neural connectivity, and other cellular conditions (Choi et al. 2008; Huang and Manning 2008; Tomasoni and Mondino 2011). Thus, in higher eukaryotes, the TSC1-TSC2 complex has emerged as a central hub of signal transduction to sense and integrate numerous cellular conditions. For instance, TSC2 activity is regulated by phosphorylation by many different kinases (e.g., PI3K/Akt, Erk, AMPK, GSK3 and RSK1), in response to mitogenic signals, energy levels, and amino acid levels (Manning et al. 2002; Roux et al. 2004; Ma et al. 2005; Inoki et al. 2006), and TSC2 activation modulates mTORC1 activity. Moreover, TSC2 stability and complex turnover have been shown to be regulated by CUL4-RING E3 ubiquitin ligase assembly (Hu et al. 2008), which includes the linker protein DDB1 and WD40-containing substrate-specific receptor, FBXW5 (Jin et al. 2004; Jackson and Xiong 2009). At least two dozen proteins are reported to be degraded by a CUL4-RING E3 ligase assembly and TSC2 is a well-known substrate of the CUL4 E3 ligase (Hu et al. 2008; Jackson and Xiong 2009). However, it is largely unknown what physiological conditions signal the CUL4-RING E3 ligase to modulate TSC2 protein turnover.

The current studies investigated the potential molecular mechanism underlying dysregulated autophagy in PD by characterizing the effects of increased Tnfaip8 l1/Oxi-β in DA neurons in culture and in the 6-OHDA model. We first identified the F-box protein FBXW5, the TSC2 binding receptor of CUL4 E3 ligase complex, as a novel Tnfaip8 l1/Oxi-β binding protein. Next, Tnfaip8 l1/Oxi-β was shown to prevent FBXW5-dependent degradation of TSC2. Finally, we demonstrated that 6-OHDA-induced OS up-regulated Tnfaip8 l1/Oxi-β, modulated FBXW5, increased TSC2 stability, and finally increased autophagy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials

Hydrogen peroxide, 6-OHDA, bafilomycin A1 was purchased from Sigma-Aldrich (St.Louis, MO, USA). Antibody for Tnfaip8 l1/Oxi-β (Figure S1) was produced using the protein-specific synthetic peptides (MDTFSTKSLALQAQKK) from AbFrontier (Seoul, South Korea) and other antibodies were purchased from: Clonentech (c-Myc, Mountain View, CA, USA), Cell Signaling (phospho-mTOR/mTOR, phospho-p70S6 kinase/S6 kinase, LC3; Danvers, MA, USA), and Sigma-Aldrich (FLAG). The Tnfaip8 l1/Oxi-β gene-specific siRNAs (two suggested ones) and scrambled siRNA duplexes were synthesized by Dharmacon (ThermoFisher Scientific, Waltham, MA, USA) as described previously (Choi et al. 2010).

Plasmids, Cell culture, Transient transfection, and Gene knockdown

The DA neuronal cell line SN4741 (> passage #7) was cultured at 33°C with 5% CO2 in RF medium that contained Dulbecco's modified Eagle medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, Utah, USA), 1% glucose, Penicillin (100 units/mL)-Streptomycin (100 g/mL), and L-glutamine (2 mM) as described previously (Son et al. 1999; Chun et al. 2001). COS 7 and HEK293T cells were cultured as recommended by the American Type Culture Collection (Manassas, MA, USA). For transient expression analysis, cells were plated in RF medium 1 day before transfection, then were transfected with FuGeneHD (Promega, Madison, WI, USA). For immunoblot or immunoprecipitation, cells were harvested after incubation for 18–36 h. For gene knockdown, the two suggested siRNA duplexes were delivered using the TransIT-TKO Transfection reagent (Mirus, Madison, WI, USA) 4 h before application of stress as described previously (Choi et al. 2010). The efficiency of siRNA delivery was determined by Cy3-labeled siRNA using the siRNA labeling kit (Ambion, Foster city, CA, USA). pMyc-FBXW5 and pFLAG-TSC2 were purchased from Addgene (Cambridge, MA, USA). To construct pFLAG-Oxi-β the pOxi-β was digested with Hind III and inserted into pFLAG-CMV plasmid. To construct Oxi-β promoter–luciferase plasmid, 1766 bp Oxi-β promoter was inserted into pGL3 basic vector (Invitrogen) digested with XhoI.

Stereotaxic injection of 6-OHDA

All experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and procedures were approved by the Animal Care and Use Committee of University of Central Florida and taken to minimize pain and discomfort. Female Sprague Dawley rats (Charles River; 8 weeks old at the time of 6-OHDA treatment, 1 per cage) were maintained in a temperature/humidity-controlled environment under a 12 h light/dark cycle with free access to food and water. Rats were treated as previously described with some modifications (Choi et al. 2012). Briefly, rats were deeply anesthetized (ketamine and xylazine mixture 30 mg/kg, i.p.) and placed in a rat stereotaxic apparatus. Animals received two injections of freshly prepared 6-OHDA 2 μL at 7.5 μg/μL in 0.02% ascorbic acid in the right striatum (coordinate: anteroposterior, + 0.8 mm; mediolateral, −1.7 and −2.7 mm; dorsoventral, −5.0 mm) according to the atlas of Paxinos and Watson (1998) and two injections of vehicle into the left striatum (coordinate: anteroposterior, + 0.8 mm; mediolateral, +1.7 and +2.7 mm; dorsoventral, −5.0 mm). All injections were made using a Hamilton syringe equipped with a 30-gauge beveled needle, attached to a syringe pump (KD Scientific, Holliston, MA, USA). The injection rate was 0.5 μL/min, and the syringe was kept in place for an additional 5 min before being retracted slowly. Rats were killed after 3, 7, or 14 days (n = 5 rats per time point).

Tissue preparation and immunohistochemistry

Rats were deeply anesthetized with sodium pentobarbital (120 mg/kg) and transcardially perfused with saline containing 0.5% sodium nitrite and 10 U/mL heparin sulfate, followed by 4% cold paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.2). Brains were post-fixed in the same solution overnight, then 30% sucrose overnight until they sank. Brains were then freeze sectioned using a sliding microtome into 40-μm coronal sections. All sections were collected in six separate series and processed for immunohistochemistry as previously described with some modifications (Choi et al. 2012). Free-floating serial sections were rinsed twice for 15 min in phosphate-buffered saline (PBS) and then pre-treated for 5 min at 20°C in PBS containing 3% H2O2. Sections were then rinsed twice for 15 min in PBS and blocked for 30 min at 20°C in PBS containing 5% normal serum (Vector Laboratories, Burlingame, CA, USA), 0.2% triton X-100 (Fisher BioReagents, Houston, TX, USA) and 1% bovine serum albumin (BSA) (Fisher BioReagents). After rinsing with PBS containing 0.5% BSA, sections were subsequently incubated overnight at 20°C with Oxi-β (1 : 500) antibody. Sections were then rinsed in PBS containing 0.5% BSA twice for 15 min and incubated for 1 h at 20°C in biotin-conjugated anti-rabbit secondary antibody (1 : 400; Vector Laboratories, Burlingame, CA, USA). Sections were rinsed again in PBS containing 0.5% BSA and incubated for 1 h at 20°C in avidin-biotin complex kit (Vector Laboratories). After rinsing twice in 0.1 M PB, the signal was visualized by incubating sections in 3,3′-diaminobenzidine kit (Vector Laboratories) following the manufacture's protocol. After rinsing with PBS sections were dehydrated in serially diluted ethanol, and cleaned in xylene followed by sequential mounting on glass slides using permanent mounting medium. Mounted slices were evaluated via light microscope (Leica, Tonawanda, NY, USA). For double-immunofluorescence staining, the sections were mounted on glass slides, washed in PBS, incubated for 30 min in PBS containing 5% normal serum, 0.2% triton X-100 and 1% BSA, and subsequently incubated overnight at 20°C with antibodies to tyrosine hydroxylase (TH) (1 : 2000; Millipore, Billerica, MA, USA) and Oxi-β (1 : 500) in PBS containing 0.5% BSA. After rinsing with PBS containing 0.5% BSA, sections were treated simultaneously with a mixture of FITC-conjugated donkey anti-sheep IgG (1 : 1000; Jackson Immuno Research, West Grove, PA, USA) and AlexaFluor 647-conjugated donkey anti-rabbit IgG (1 : 1000; Invitrogen, Grand Island, NY, USA) for 1 h at 20°C, washed in 0.1 M PB, and incubated for 5 min at 20°C with 2 μM Hoechst33342 (Anaspec, Fremont, CA, USA) for nuclear staining. Finally, slices were mounted sequentially using Vectashield medium (Vector Laboratories) and viewed using a Nikon Eclipse E600 microscope (Morrell Instruments Co. Inc., Melville, NY, USA). To determine the localization of the two antibodies in double-stained samples, images were obtained from the same area and were merged using interactive software.

Differential gene chip analysis

Total RNA samples were prepared using the TRIzol reagent (Invitrogen Life Technologies) from control SN4741 DA neuronal cells and SN4741 cell over-expressing Tnfaip8 l1/Oxi-β for 12 h. RNA samples were competitively hybridized to an Illumina Expression Chip/MouseRef-8 (24K) (Illumina, San Diego, CA, USA) with approximately 24 000 genes and further analyzed using an Arrayassist Expression Software (Macrogen, Seoul, South Korea). The raw and normalized data are available from Gene Expression Omnibus with accession number GSE47634.

Measurement of Autophagic flux

Autophagic flux is assessed by comparing LC3-II/Actin ratios, derived from immunoblots, in the absence and presence of bafilomycin A1 (50 nM for 24 h) (Wang et al. 2009) and leupeptin (100 mM for 24 h) (Haspel et al. 2011). For immunoblot, cells were harvested 36 h after transient transfection of Oxi-β. The flux ratios were compared between control and Tnfaip8 l1/Oxi-β over-expressed cells.

Cell death assay

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Trypan blue dye exclusion were used to assess cell death. For MTT assay, Thiazolyl blue tetrazolium bromide (Sigma) was dissolved in PBS and filter sterilized to prepare a 5 mg/mL stock solution. After application of OS, cells were incubated in MTT stock solution with RF medium at a final concentration 0.5 mg/mL for 2 h. The formazan crystals were dissolved in dimethylsulfoxide (Sigma) and the background absorbance was measured at 540 nm and 670 nm. Results were expressed as the percentage of reduced absorbance, assuming absorbance of control cells at 100%. These data were directly correlated with the number determined by Trypan blue dye exclusion.

Immunoprecipitation

COS 7 and HEK293T cells were cotransfected and lysed in M-PER mammalian extraction buffer (Pierce, Rockford, IL, USA) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) for 30 min on ice. Cells were scraped on ice, then centrifuged at 14,000× g for 30 min at 4°C. Lysates were incubated with c-myc antibody-coated Dynabeads Protein A (Invitrogen) overnight at 4°C in 0.1 M sodium phosphate buffer pH 8 with 0.01% Tween 20. After incubation, the beads were washed and boiled in 5X sample buffer for 5 min.

Western blot analysis

Cells grown under experimental conditions were washed with ice-cold PBS and lysed for 30 min on ice in a radioimmunoprecipitation assay buffer containing protease inhibitor cocktails (Roche) and phosphatase inhibitors. Cells were scraped on ice, then centrifuged at 14 000× g for 30 min at 4°C. Protein concentration was determined by the Bradford method using BSA as the standard. After denaturation with 5X sample buffer and boiling at 90°C for 5 min, protein samples (10–30 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto pre-wetted polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA,). The membranes were incubated with 5% non-fat dry milk or 5% BSA in Tris-buffered saline with 0.1% Tween 20, for 1 h to block non-specific binding. Primary antibodies were diluted in 5% non-fat dry milk or 1% BSA in Tris-buffered saline with 0.1% Tween 20 and incubated at 4°C overnight with gentle shaking. Blots were probed with proper secondary antibodies and visualized by enhanced chemiluminescence (Amersham Biosciences, GE Healthcare, Buckinghamshire, UK) as described previously (Chun et al. 2001). Membranes were exposed to X-ray films (AGFA, Greenville, SC, USA), and densitometric quantification of the immunoblotted membranes was performed by an Image Analyzer system (Fujifilm, Tokyo, Japan) using Multi-Gauge version 2.3 software. To obtain protein samples from SN region, rat brain was placed ventral side up and two coronal cuts were made: one just after anterior to the SN through the caudal end of the mammillary bodies and the other, 2 mm posterior to this through the nucleus interpeduncularis. In the resulting brain slice, SN region was dissected out by oblique cuts along the leminiscus medalis and further used for preparation of protein samples and subsequent immunoblotting.

Fluorescence confocal microscopy

SN4741 cells, cultured on poly-L-lysine coated two-well slides, and transfected with the FLAG-tagged Oxi-β and LC3-Green Fluorescent Protein (GFP) plasmids, were fixed with 4% paraformaldehyde for 20 min and washed with PBS. Immunostaining was followed after blocking with 1% BSA in PBS with 0.1% triton X-100, then immunostained. After primary FLAG antibody (Sigma) incubation, the cells were labeled with Alexa Fluor 568 goat anti-mouse. The culture slides were mounted with Vectashield aqueous mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed on a Zeiss LSM510 META laser scanning confocal microscope (Carl Zeiss, Germany). Image processing and analysis were performed with Zeiss LSM510 software version 2.3.

ROS measurement

SN4741 cells were plated in 100 mm dish 24 h before experiment. After appropriate treatment, cells were harvested and followed by 10 μM CM-H2DCFDA (Invitrogen, molecular probes #C6827, Grand Island, NY, USA) staining. After resuspending in HBSS buffer, FACS analysis (BD FACSCalibur) was performed and the fluorescent DCF was assessed by using BD CellQuest Pro software (BD Biosciences, San Jose, CA, USA).

Statistical analysis

For statistical analyses, two-sample comparisons were performed using Student's t-test, and multiple comparisons were performed using one-way anova followed by Bonferroni's multiple comparison test.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Oxidative stress induces Tnfaip8 l1/Oxi-β

In our previous study, Tnfaip8 l1/Oxi-β was significantly up-regulated, whereas Tnfaip8/Oxi-α, an activator of mTOR, was down-regulated during OS-induced autophagy and cell death in SN4741 DA neuronal cells (Choi et al. 2010). As OS is known to be a representative pathological cause in PD patients and animal models, we first investigated whether OS increases Tnfaip8 l1/Oxi-β expression at both transcriptional and translational levels by promoter-reporter assay and immunoblot analysis, respectively. As expected, Tnfaip8 l1/Oxi-β promoter activity was increased about two-fold within 3 h after OS (100 μM H2O2) treatment (Fig. 1a), which coincided with the maximum increase in ROS production within 2–6 h after OS treatment (Figure S2). Tnfaip8 l1/Oxi-β protein levels were significantly increased about 3.5-fold during the period of the greatest cell death and autophagy (i.e., between 12 and 24 h during the OS-induced DA cell death) (Fig. 1b and c). However, 3-MA treatment increased neither Tnfaip8 l1/Oxi-β levels nor cell survival significantly (Figure S3).

image

Figure 1. Oxidative stress (OS) up-regulated Tnfaip8 l1/Oxi-β at both transcriptional and translational levels. (a) Transcriptional induction of Tnfaip8 l1/Oxi-β by OS was measured by promoter–luciferase reporter assay using Tnfaip8 l1/Oxi-β 1.7 kb-luciferase plasmid. Tnfaip8 l1/Oxi-β promoter activity was increased about two-fold within 3 h after OS treatment (100 μM H2O2) in SN4741 dopaminergic neuronal cells. (b) To measure OS-induced Tnfaip8 l1/Oxi-β expression at translational levels, Tnfaip8 l1/Oxi-β protein levels were measured by immunoblot analysis. Tnfaip8 l1/Oxi-β protein levels were significantly increased (~ 3.5 fold) within 12–30 h after OS treatment (100 μM H2O2), when the most significant cell death occurs as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue exclusion assays (Figure S8). (c) Induction of autophagy was assessed by measuring both phosphorylation of S6K [a sensitive substrate of mammalian target of rapamycin (mTOR)] and LC3-II (a representative autophagy marker) by immunoblot analysis during OS-induced cell death. All values are the means ± SEM from at least three independent experiments. * and ** denote difference between control cells and OS-treated cells at < 0.05 and < 0.01, respectively.

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Treatment of 6-OHDA induces the DA neuron-specific up-regulation of Tnfaip8 l1/Oxi-β

We next examined whether 6-OHDA (100 μM), a well-known inducer of OS (Bernstein et al. 2011; Figure S4), exerted a similar effect on the Tnfaip8 l1/Oxi-β expression and autophagy during 6-OHDA-induced cell death in SN4741 DA neuronal cells. Temporal induction of Tnfaip8 l1/Oxi-β protein peaked at the time of maximal 6-OHDA-induceed cell death, and was accompanied by induction of autophagy markers (i.e., S6K, LC3-II and autophagosomes) and apoptotic markers (i.e., cleavage of Poly(ADP-ribose) polymerase (PARP) and activation of caspase 3) in vitro (Fig. 2a,b and c and Figure S5). Then, we determined whether the Tnfaip8 l1/Oxi-β gene knockdown affected cell death by 6-OHDA. As demonstrated in Fig. 2d and Figure S1, gene knockdown increased cell viability by about 15% compared with control cells exposed to 6-OHDA (at 24 h) while the scrambled siRNA did not affect the cell viability, suggesting a causal relationship among Tnfaip8 l1/Oxi-β induction, autophagy, and cell death.

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Figure 2. 6-hydroxydopamine (6-OHDA) increased expression of Tnfaip8 l1/Oxi-β and autophagy in vitro. (a) Maximal induction of Tnfaip8 l1/Oxi-β protein occurred at the peak of cell death between 24 and 30 h after 100 μM 6-OHDA, a well-known inducer of oxidative stress (OS) in Parkinson's disease (PD) model, treatment in SN4741 dopaminergic neuronal cells. (b–c) Temporal induction of Tnfaip8 l1/Oxi-β protein after 6-OHDA (100 μM) treatment was accompanied by induction of autophagy markers (i.e., decreased phosphor-S6K, an mammalian target of rapamycin (mTOR) substrate, and increased LC3-II) and apoptotic markers (i.e., cleavage of PARP and activation of caspase 3). (d) Tnfaip8 l1/Oxi-β gene knockdown increased cell viability by about 15% compared to cells exposed to 6-OHDA for 18 h, whereas the scrambled siRNA (con) did not affect the cell viability. All values are the means ± SEM from at least five independent experiments. ** denotes difference between control (scrambled siRNA treatment) and Tnfaip8 l1/Oxi-β-specific siRNA treatment at < 0.01.

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The observation that Tnfaip8 l1/Oxi-β was induced by 6-OHDA in vitro led us to test whether a similar induction of Tnfaip8 l1/Oxi-β expression would occur in rat SN DA neurons treated with 6-OHDA. To assess this, we injected 6-OHDA or 0.02% ascorbic acid vehicle into the striatum (Fig. 3a) and performed western blot analysis using our Tnfaip8 l1/Oxi-β antibody. Similar to in vitro findings, Tnfaip8 l1/Oxi-β expression was increased by 51.6 ± 12.7% after 7 days in 6-OHDA-treated rat, relative to vehicle-treated controls (Fig. 3b; < 0.05). Consistent with this finding, immunostaining showed Tnfaip8 l1/Oxi-β to be increased at the same time point (Fig. 3c). To examine whether Tnfaip8 l1/Oxi-β is expressed in DA neurons, we performed double fluorescence immunostaining with the Tnfaip8 l1/Oxi-β and TH antibodies in SN DA neurons 7 days after intrastriatal 6-OHDA injection. Double fluorescent immunolabeling showed that Tnfaip8 l1/Oxi-β (red) was coexpressed with TH (green) in SN DA neurons (Fig. 3d). Similar levels of cell type-specific expression of Tnfaip8 l1/Oxi-β in the SN DA neurons were also observed in mice (Figure S10). These data indicate that 6-OHDA enhances Tnfaip8 l1/Oxi-β expression in DA neuron similar to in vitro results.

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Figure 3. 6-hydroxydopamine (6-OHDA) up-regulated Tnfaip8 l1/Oxi-β in rat substantia nigra (SN) dopaminergic (DA) neurons. (a) To assess 6-OHDA-induced Tnfaip8 l1/Oxi-β expression in rat SN DA neurons, animals received two injections of freshly prepared 6-OHDA (2 μL at 7.5 μg/μL in 0.02% ascorbic acid) in the right striatum and two injections of vehicle into the left striatum as detailed in Materials and Methods. (b) Similar to the in vitro findings, intrastriatal injection of 6-OHDA induced Tnfaip8 l1/Oxi-β expression in rat SN DA neurons, as assessed by western blot. Tnfaip8 l1/Oxi-β expression in the SN was increased by about 52% at 7 days after 6-OHDA treatment, which were quantified using Quantity One software and normalized against β-actin. Data are shown as the mean ± SEM. Statistical analysis was performed using one-way anova, followed by Bonferroni's multiple comparison test. *< 0.05. (c) A representative immunohistochemical analysis demonstrated that Tnfaip8 l1/Oxi-β was increased in SN DA neurons at 7 days, of which quantified data assessed by immunoblot analysis are shown in (B). (d) The DA neuron-specific expression of Tnfaip8 l1/Oxi-β was further confirmed by double fluorescence immunostaining with Tnfaip8 l1/Oxi-β and TH antibodies at 7 days. Tnfaip8 l1/Oxi-β (red) was colocalized with TH (green), a dopaminergic neuronal marker. Hoechst 33342 was used as a nuclear marker (blue).

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Tnfaip8 l1/Oxi-β functions as an inhibitor of mTOR

Based on previous observations that Tnfaip8/Oxi-α is an activator of mTOR (Choi et al. 2010) and that 6-OHDA induces autophagy (Fig. 2), we investigated whether OS-induced Tnfaip8 l1/Oxi-β plays a regulatory role in increased autophagy by assessing known biochemical markers of autophagy, such as mTOR, S6K, and LC3-II, as well as accumulation of autophagosomes using GFP-LC3, a fluorescent tagged protein that specifically labels autophagosomes and autophagic isolation membranes (Kabeya et al. 2000; Mizushima et al. 2004). Increased Tnfaip8 l1/Oxi-β levels significantly inhibited mTOR activity and subsequently, decreased phosphorylation of S6K and increased accumulation of LC3-II (Fig. 4a and Figure S6) and autophagosomes (Fig. 4b). Increased accumulation of LC3-II by Tnfaip8 l1/Oxi-β was further confirmed by a leupeptin-based assay (Fig. 4a, Bottom panel). This notion was further tested by comparing the pharmacological effect of bafilomycin A1, which inhibits fusion between autophagosome and lysosome, in the presence and absence of Tnfaip8 l1/Oxi-β over-expression. The autophagic flux was increased significantly (~30%) by Tnfaip8 l1/Oxi-β over-expression compared with the steady-state control (Fig. 4c), confirming the inductive function of Tnfaip8 l1/Oxi-β in autophagy. We next determined whether Tnfaip8 l1/Oxi-β gene knockdown could suppress 6-OHDA-induced activation of autophagy. As expected, Tnfaip8 l1/Oxi-β knockdown decreased LC3-II level by about 50% compared to that in control cells (Fig. 4d), confirming an inhibitory function of Tnfaip8 l1/Oxi-β in the regulation of mTOR activity, contrary to the mTOR activator role of Tnfaip8/Oxi-α (Choi et al. 2010).

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Figure 4. Tnfaip8 l1/Oxi-β is a novel mammalian target of rapamycin (mTOR) inhibitor and has an inducible function in autophagy. (a) We investigated whether Tnfaip8 l1/Oxi-β regulates autophagy by assessing the representative biochemical markers of autophagy, such as phospho-mTOR, phospho-S6K, and LC3-II, by immunoblot analysis in SN4741 dopaminergic (DA) cells. Increased Tnfaip8 l1/Oxi-β significantly inhibited mTOR activity (i.e., decreased p-mTOR) and subsequently, decreased S6K phosphorylation and increased LC3-II levels (Top panel). LC3-II increases were further confirmed by a leupeptin-based assay (Bottom panel), indicating that increased Tnfaip8 l1/Oxi-β level exerts a direct regulatory role in enhanced autophagy. Leup: leupeptin. (b) The regulatory role of Tnfaip8 l1/Oxi-β in autophagy was further confirmed by confocal microscopy using GFP-LC3, a fluorescent tagged protein that specifically labels autophagosomes. Increased Tnfaip8 l1/Oxi-β also caused accumulation of autophagosomes, demonstrated by significant increase in GFP-LC3 punctae, which was quantified by counting at least 100 cells with more than five GFP-LC3 punctae. (c) The autophagic flux was measured in the presence of Tnfaip8 l1/Oxi-β over-expression by treatment of bafilomycin A1 (50 nM for 24 h). The autophagic flux was increased significantly (about 30%) by Tnfaip8 l1/Oxi-β over-expression compared with the steady-state control, confirming the inductive function of Tnfaip8 l1/Oxi-β in autophagy. * and ** denote difference between control cells and Tnfaip8 l1/Oxi-β over-expressed cells at < 0.05 and < 0.01, respectively. (d) Then, we tested whether Oxi-β gene knockdown could suppress 6-hydroxydopamine (6-OHDA)-induced activation of autophagy by immunoblot analysis of a representative autophagy marker, LC3-II. As expected, Tnfaip8 l1/Oxi-β knockdown decreased LC3-II by about 50% compared to that in control cells treated with scrambled siRNA, confirming the inducible function of Tnfaip8 l1/Oxi-β in autophagy. All values are the means ± SEM from three independent experiments. *, ** and *** denote difference between control cells treated with scrambled siRNA and cells treated with Tnfaip8 l1/Oxi-β siRNA at < 0.05, < 0.01 and < 0.005, respectively.

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FBXW5 is identified as a Tnfaip8 l1/Oxi-β binding protein

Despite being a primary candidate binding protein for Tnfaip8 l1/Oxi-β, mTOR did not interact directly with Tnfaip8 l1/Oxi-β (data not shown). Significant cell death caused by the over-expressed Tnfaip8 l1/Oxi-β might interfere with co-IP of proteins in low abundance. Instead, differential gene chip analysis demonstrated that total 30 genes exhibited more than 1.5-fold change of their transcription by Tnfaip8 l1/Oxi-β over-expression in SN4741 DA neuronal cells (Gene Expression Omnibus accession # GSE47634; Figure S7). Among those, FBXW5, Ubadc1, and Psma5 were initially chosen as potential target(s) and/or interacting protein(s) of Tnfaip8 l1/Oxi-β, mainly because of their known functional roles in protein degradation and/or metabolism and potential relevance to autophagy and very significant up-regulation. To our surprise, the most up-regulated FBXW5 (about 25-fold) was identified and confirmed as a binding partner of Tnfaip8 l1/Oxi-β by co-IP assay (Fig. 5a). Reverse IP assay using Tnfaip8 l1/Oxi-β and FBXW5 further confirmed their interaction (Fig. 5b).

image

Figure 5. FBXW5 is a novel binding protein for Tnfaip8 l1/Oxi-β. (a) Differential gene chip analysis followed by Tnfaip8 l1/Oxi-β over-expression help identify FBXW5 as a protein that may interact with Tnfaip8 l1/Oxi-β. Co-IP assay using myc-tagged FBXW5 and Flag-tagged Tnfaip8 l1/Oxi-β confirmed FBXW5 as a binding partner of Tnfaip8 l1/Oxi-β (Top panel). Tnfaip8/Oxi-α was added as a negative control, which did not interact with FBXW5 at all (Bottom panel). (b) Reverse IP assay using Flag-tagged Tnfaip8 l1/Oxi-β and myc-tagged FBXW5 further confirmed their interaction.

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Tnfaip8 l1/Oxi-β competes with TSC2 for FBXW5 binding in CUL4-RING E3 ligase

As both Tnfaip8 l1/Oxi-β and TSC2 bind to the same WD domain of FBXW5 in the CUL4-RING E3 ligase complex, we investigated whether Tnfaip8 l1/Oxi-β can prevent TSC2 from binding with FBXW5 and subsequently cause TSC2 accumulation. As expected, Tnfaip8 l1/Oxi-β competed with TSC2 to bind to FBXW5 (Fig. 6a; please compare lane 3 and lane 4 on the right panel), which further prevented TSC2 from being ubiquitinated by CUL4-RING E3 ligase (left panel in Fig. 6b). Moreover, over-expression of Tnfaip8 l1/Oxi-β resulted in the intracellular accumulation of endogenous TSC2 (Fig. 6c), suggesting that OS-induced Tnfaip8 l1/Oxi-β outcompetes TSC2 for binding with FBXW5 in CUL4 E3 ligase and increases TSC2 stability by protecting TSC2 from degradation.

image

Figure 6. Tnfaip8 l1/Oxi-β competed with tuberous sclerosis complex 2 (TSC2) to bind FBXW5. (a) As both Tnfaip8 l1/Oxi-β and TSC2 bind to the same WD domain of FBXW5, it was investigated whether Tnfaip8 l1/Oxi-β can prevent TSC2 from binding with FBXW5 by Co-IP analysis. Indeed, Tnfaip8 l1/Oxi-β was able to compete with TSC2 to bind FBXW5, resulting in the preferential binding of FBXW5 with Tnfaip8 l1/Oxi-β (lane 4), not with TSC2 (i.e., compare lanes 3 and 4) in HEK293T cells. (b) Then, it was determined whether the binding of Tnfaip8 l1/Oxi-β with FBXW5 causes the reduced ubiquitination of TSC2 by CUL4-RING E3 ligase. The Co-IP analysis demonstrated that the over-expressed Tnfaip8 l1/Oxi-β prevented significantly the accumulation of ubiquitinated TSC2 ranging from 180 to 220 kDa (Right panel) and subsequently resulted in the increased level of TSC2 (left panel). (c) Over-expression of Tnfaip8 l1/Oxi-β caused the significant accumulation of TSC2 compared with the control, suggesting that oxidative stress (OS-induced) Tnfaip8l1/Oxi-β could outcompete TSC2 to bind FBXW5 and prevent ubiquitination and degradation of TSC2. Note: the average M.W. of TSC2 is about 180 kDa and those of ubiquitinated TSC2 range from 180 to 220 kDa, which are quite difficult to separate clearly and tend to aggregate. At least three independent experiments were performed.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Dysregulated autophagy affects intercellular communication and neural connectivity within the brain and subsequently, contributes to neurodegeneration. However, it is unknown how autophagy is dysregulated by OS conditions, such as in PD. We have discovered that the first pair of homologous autophagy regulator proteins, Tnfaip8/Oxi-α and Tnfaip8 l1/Oxi-β, each has opposing effects on mTOR in DA neurons. Whereas Tnfaip8/Oxi-α is an mTOR activator, Tnfaip8 l1/Oxi-β is a novel mTOR inhibitor, which is significantly up-regulated in SN DA neurons by 6-OHDA treatment. Up-regulated Tnfaip8 l1/Oxi-β induces autophagy by competitive binding to FBXW5, which protects TSC2 from CUL4-RING E3 ligase-mediated ubiquitination and subsequent proteasomal degradation. Thus, the OS condition in PD may cause Tnfaip8 l1/Oxi-β up-regulation, followed by the increased accumulation of TSC2 (a negative regulator of mTOR) and increased autophagy in SN DA neurons.

Tight control of autophagy is essential for neuronal integrity and survival, as it prevents intraneuronal accumulation of protein aggregates and dysfunctional organelles (Son et al. 2012). Although a pathogenic role of dysregulated autophagy is supported by findings from genetic and toxin-induced models, as well as from post-mortem PD brains (Anglade et al. 1997; Thiruchelvam et al. 2000; Larsen et al. 2002; Gomez-Santos et al. 2003; Cuervo et al. 2004; Peng et al. 2004; Gonzalez-Polo et al. 2007; Zhu et al. 2007; Plowey et al. 2008; Xilouri et al. 2009; Irrcher et al. 2010; Michiorri et al. 2010), it is largely unknown how OS alters mTOR activity. Previously, we discovered the phylogenetically conserved Oxi gene family [homologs of TNFAIP8 family (Patel et al. 1997; Lou and Liu 2011)], including the distinct pair of homologous proteins, Tnfaip8/Oxi-α and Tnfaip8 l1/Oxi-β, which are temporally regulated by OS in DA neurons (Choi et al. 2010). Furthermore, OS dramatically increases autophagy in DA neurons with simultaneous down-regulation of Tnfaip8/Oxi-α and up-regulation of Tnfaip8 l1/Oxi-β. To investigate their regulatory role in autophagy, we first analyzed how they regulate mTOR activity by using over-expression and 6-OHDA treatment. As expected, Tnfaip8/Oxi-α functioned as an mTOR activator via an unknown mechanism (Choi et al. 2010), whereas Tnfaip8 l1/Oxi-β acted as an mTOR inhibitor via accumulation of TSC2 as shown in Fig. 6c. Moreover, down-regulated Tnfaip8/Oxi-α and up-regulated Tnfaip8 l1/Oxi-β exerted similar effects on autophagy, such as increased autophagy markers (i.e., mTOR, S6K, and LC3-II) and autophagic vacuoles (i.e., LC3-GFP labeled autophagosomes) with increased autophagic flux (Choi et al. 2010; Fig. 4c). Furthermore, 6-OHDA treatment was associated with up-regulation of Tnfaip8 l1/Oxi-β in SN DA neurons (Fig. 3d). Therefore, pathogenic OS conditions in PD models appear to induce enhanced autophagy by concomitantly up-regulating Tnfaip8 l1/Oxi-β and down-regulating Tnfaip8/Oxi-α (Choi et al. 2010), which would inhibit mTOR activity and result in an additive increase in autophagy. Our data suggest that this pathway may be a potential target for intervention to treat the dysregulated autophagy in DA neurons in PD.

To elucidate the mechanism underlying the inhibition of mTOR activity by Tnfaip8 l1/Oxi-β, we performed differential gene chip analysis followed by co-IP analysis. Of interest, FBXW5 was identified as the primary binding partner of Tnfaip8 l1/Oxi-β, showing 25-fold increase in response to over-expressed Tnfaip8 l1/Oxi-β (Fig. 5a). The FBXW5 is an F-box protein and a WD40-containing substrate receptor of the CUL4-RING E3 ligase assembly (Jackson and Xiong 2009), which regulates TSC2 stability and TSC2 complex turnover by ubiquitin–proteasome pathway (Hu et al. 2008). In particular, the TSC1 and TSC2 complex is a critical negative regulator of mTORC1, as TSC2 containing GTPase-activating protein activity, and activates to regulate signaling mTORC1 signaling, thereby mediating autophagy and other cellular conditions (Choi et al. 2008; Huang and Manning 2008; Tomasoni and Mondino 2011). Previously, TSC2 activity was thought to be regulated mainly by many different kinases, such as PI3K/Akt, Erk, AMPK, GSK3, and RSK1 (Manning et al. 2002; Roux et al. 2004; Ma et al. 2005; Inoki et al. 2006), by which specific phosphorylation of TSC2 modulates mTORC1 activity. However, it is largely unknown what physiological condition signals the CUL4-RING E3 ligase to modulate TSC2 protein turnover. Here, we demonstrate for the first time that TSC2 protein turnover is regulated by OS-induced Tnfaip8 l1/Oxi-β via its competition with TSC2 to bind FBXW5. Thus, Tnfaip8 l1/Oxi-β inhibits the ubiquitination of TSC2 by CUL4-RING E3 ligase, resulting in intracellular accumulation of TSC2, inhibition of mTOR activity and increased autophagy as summarized in Fig. 7. In fact, the inhibition of mTORC1 signaling was observed by over-expression of TSC2 (Choi et al. 2008), while the autosomal dominant mutations in TSC2 lead to the constitutively active mTORC1 signaling, leading to the development of benign tumors in the inherited tuberous sclerosis (Sampson 2009) and some human tumors (Mieulet and Lamb 2010), which suggest the Tnfaip8/Oxi-α and Tnfaip8 l1/Oxi-β pathway as potential intervention target in specific disease conditions, such as neurodegeneration, cancer, and tuberous sclerosis, caused by defective mTORC1 signaling.

image

Figure 7. A schematic diagram for Tnfaip8 l1/Oxi-β mechanism of action under oxidative stress (OS). Up-regulated Tnfaip8 l1/Oxi-β induces autophagy by competitive binding to FBXW5, which prevents CUL4-RING E3 ligase-dependent ubiquitination of tuberous sclerosis complex 2 (TSC2) and subsequent proteasomal degradation. Thus, in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease (PD), pathogenic OS condition up-regulates Tnfaip8l1/Oxi-β, increases the negative mammalian target of rapamycin (mTOR) regulator TSC2, and increases autophagy in substantia nigra (SN) dopaminergic (DA) neurons.

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In summary, Tnfaip8 l1/Oxi-β is a novel mTOR inhibitor, which is significantly up-regulated in SN DA neurons by OS and 6-OHDA. Up-regulated Tnfaip8 l1/Oxi-β induces autophagy by competitive binding to FBXW5, which stabilizes TSC2 by preventing ubiquitination of TSC2 by CUL4-RING E3 ligase and subsequent proteasomal degradation (Fig. 7). As pathogenic OS conditions in PD up-regulate Tnfaip8 l1/Oxi-β, the negative regulator of mTOR, TSC2 accumulates and the increased autophagy occurs in SN DA neurons. Furthermore, the fact that OS-induced Tnfaip8 l1/Oxi-β can outcompete TSC2 to bind FBXW5 within CUL4-RING E3 ligase, implicates Tnfaip8 l1/Oxi-β and FBXW5 as potential candidates of intervention targets for dysregulated autophagy in DA neurons under pathogenic OS conditions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

All experiments were approved by NRF and Ewha Global Top 5 Project, the Republic of Korea and were conducted in compliance with the ARRIVE guidelines. Funding was provided by NRF (2013-008773 and 2010-0009233) and Ewha Global Top 5 (2012-1781-001). The authors have no conflict of interest to declare.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12643-sup-0001-FigS1-S10.pdfapplication/PDF3170K

Figure S1. Specificity of the purified Oxi-β antibody in mouse brain and other tissues.

Figure S2. ROS measurement by FACS analysis after OS treatment (100 μM H2O2) in SN4741 cells.

Figure S3. Effect of 3-MA (1 mM) on both Oxi-β expression and cell death.

Figure S4. ROS measurement after 6-OHDA treatment (100 μM): the maximum ROS activity occurred within 2 h.

Figure S5. 6-OHDA (after 8 h) induces the significant increase in endogenous Oxi-β (red), which promotes autophagosome formation (green) in LC3-GFP-transfected cells.

Figure S6. Comparison of autophagy induction capability of Oxi-β with β -galactosidase.

Figure S7. Differential gene expression analysis demonstrated that Tnfaip8l1/Oxi-β has the greatest effect on FBXW5 mRNA expression.

Figure S8. Measurement of cell death by Trypan blue exclusion assay, which is comparable to the cell death rate measured by MTT assay in Fig. 1B.

Figure S9. The similar rate of protection was obtained even after longer period of gene knockdown (for 48 h) by Oxi-β siRNA.

Figure S10. Cell type-specific expression of Oxi-β in mouse SN dopaminergic neurons: colocalization of Oxi-β in TH-+ neurons in SN (Substantia nigra) region.

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