Autophagy induced by the class III histone deacetylase Sirt1 prevents prion peptide neurotoxicity

Neurobiology of Aging 34 (2013) 146 –156 www.elsevier.com/locate/neuaging

Autophagy induced by the class III histone deacetylase Sirt1 prevents prion peptide neurotoxicity Jae-Kyo Jeong, Myung-Hee Moon, You-Jin Lee, Jae-Won Seol, Sang-Youel Park* Center for Healthcare Technology Development, Korea Zoonoses Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk, South Korea Received 12 December 2011; received in revised form 12 March 2012; accepted 4 April 2012

Abstract Sirtuin 1 (Sirt1) is a class III histone deacetylase that mediates the protective effects of neurons in neurodegenerative disorders, including Alzheimer’s and prion disease. However, the mechanism directly involved in neuroprotection is still poorly understood. Recent evidence has demonstrated that activating Sirt1 induces autophagy, and that activating autophagy protects neurons against neurodegenerative disorders by regulating mitochondrial homeostasis. Thus, we focused on the mechanism of the Sirt1-mediated neuroprotective effect that was associated with regulating mitochondrial homeostasis via autophagy. Adenoviral-mediated Sirt1 overexpression prevented prion protein (PrP)(106 –126)-induced neurotoxicity via autophagy processing. Moreover, Sirt1-induced autophagy protected against the PrP(106 –126)mediated decrease in the mitochondrial membrane potential value. Additionally, Sirt1 overexpression decreased PrP(106 –126)-induced Bax translocation to the mitochondria and cytochrome c release into the cytosol. Sirt1 knockdown using small interfering (si) RNAs induced downregulation of Sirt1 protein expression and sensitized neuron cells to PrP(106 –126)-induced cell death and mitochondrial dysfunction. Knockdown of autophagy-related 5 (ATG5) using small interfering RNA decreased autophagy-related 5 and autophagy marker microtubuleassociated protein 1 light chain 3-II protein levels and blocked the effect of a Sirt1 activator against PrP(106 –126)-induced mitochondrial dysfunction and neurotoxicity. Taken together, this study is the first report demonstrating that autophagy induced by Sirt1 activation plays a pivotal role protecting against prion-induced neuron cell death and also suggests that regulating autophagy including which by Sirt1 activation may be a therapeutic target for neurodegenerative disorders including the prion disease. © 2013 Elsevier Inc. All rights reserved. Keywords: Sirt1; Autophagy; PrP(106 –126); Neurotoxicity; Mitochondrial dysfunction

1. Introduction Transmissible spongiform encephalopathies or prion diseases are a family of progressive disorders caused by aggregating the scrapie isoform of the prion protein (PrPsc), which accumulates to form central nervous system plaque (Wickner, 2011). Mitochondrial failure caused by aggregation of misfolded proteins is a key mechanism of neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and prion disease (Borger et al., 2011;

* Corresponding author at: College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756, South Korea. Tel.: ⫹82 63 270 3886; fax: ⫹82 63 270 3780. E-mail address: [email protected] (S.-Y. Park). 0197-4580/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2012.04.002

Coskun et al., 2012; Sisková et al., 2010). Recent studies have shown that activating the sirtuin 1 (Sirt1) pathway prevents Alzheimer’s and Parkinson’s disease (Vingtdeux et al., 2010; Wu et al., 2011). Additionally, cortical neuron cells treated with the prion protein (PrP) fragment (106 – 126) become neurotoxic, develop dysfunctional mitochondria, and increase prion-mediated neurotoxicity associated with the induction of Bax translocation to the mitochondria (O’Donovan et al., 2001). The synthetin PrP(106 –126) contains the amino acid residues 106 –126 of the cellular prion protein and possesses many PrPsc characters including the ability to cause neurotoxicity, which converts cellular prion protein (PrPc) into scrapie isoform aggregates, resulting in fibril accumulation and mitochondrial damage in vivo and in vitro (Jeong et al.,

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2011b; Singh et al., 2002). These properties of PrP(106 – 126) are useful for the in vitro study of PrPsc pathogenesis. The mammalian silent information regulator 2 homolog, Sirt1, is a NAD⫹-dependent histone deacetylase that plays a major role in the regulation of gene expression, metabolism, and aging (Asher et al., 2008; Herranz et al., 2010). Sirt1 is a regulator of proteins and genes involved with tumor suppressor p53 and nuclear factor-kappa B (NF-␬B) and is a member of the forkhead transcription factor (FOXO) family (Elangovan et al., 2011; Wang et al., 2011). It has also been shown to regulate mitochondrial functions and has an essential role in neuronal plasticity, learning, and memory (Aquilano et al., 2010; Gao et al., 2010; Michán et al., 2010). Furthermore, recent studies have shown that the Sirt1 pathway might be indispensable for neuroprotection against neurodegenerative disorders including Alzheimer’s and prion disease (Kim et al., 2007; Seo et al., 2012). Sirt1 modulates key target proteins to maintain mitochondrial function, including peroxisome proliferator-activated receptor gamma coactivator (PGC)-1␣, adenosine monophosphate-activated protein kinase, and FOXO3a (Cantó et al., 2009; Gerhart-Hines et al., 2007; Kobayashi et al., 2005; Nemoto et al., 2005). Mudò et al. (2012), studying 1-methyl 4-phenyltetrahydropyridine (MPTP)-mouse model of Parkinson’s disease, showed that MPTP-induced cell degeneration and mitochondrial-oxidative damage is prevented by activating the Sirt1/PGC-1␣ pathways (Mudò et al., 2012). This observation supports the idea that modulating Sirt1 activity may be beneficial to protect against neurodegenerative disease by regulating mitochondrial homeostasis. Sirt1 induces the autophagy-lysosome pathway (Lee et al., 2008; Salminen and Kaarniranta, 2009). Autophagy is a mechanism for degradation of organelles and protein aggregates through the lysosomal pathway (Pan et al., 2008; Singh et al., 2009). As such, autophagy plays a pivotal role in anticancer, antiaging, antimicrobial defense, and neuroprotection (Alirezaei et al., 2011; Chen and Karantza-Wadsworth, 2009; Deretic and Levine, 2009; Rubinsztein et al., 2011). In particular, autophagy protects against neurodegeneration by degrading misfolded proteins and impaired mitochondria (Khandelwal et al., 2011; Moreira et al., 2010; Vingtdeux et al., 2011). The Sirt1 activator resveratrol protects against rotenone-induced neurotoxicity in an in vitro model of Parkinson’s disease through the autophagy-lysosome pathway (Wu et al., 2011). However, the influence of the Sirt1/autophagy pathway on mitochondrial dysfunction as a cause for neurodegenerative diseases including prion disease is unclear. Our previous study showed that resveratrol protects against prion-mediated neurotoxicity by activating Sirt1 and that activating Sirt1 enzymes decrease PrP(106 –126)-induced acetylation of p53 and activation of the p65(RelA/ p65) subunit of nuclear factor-kappa B (NF-␬B) (Seo et al., 2012). However, the neuroprotective effect of the Sirt1/

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autophagy pathway on mitochondrial dysfunction-mediated neurodegenerative diseases including prion diseases has not been reported. Thus, the present study focused on the influence of the Sirt1/autophagy pathway on PrP(106 –126)induced cytotoxicity and mitochondrial damage in neuronal cells. The results showed that regulating the Sirt1/autophagy pathway appeared to prevent neurotoxicity caused by the prion peptide. These results suggest that regulating the Sirt1/autophagy pathway may be a viable therapeutic strategy for neurodegenerative disorders such as prion disease caused by mitochondrial dysfunction. 2. Methods 2.1. Cell culture The human neuroblastoma cell line SH-SY5Y was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in Minimum Essential Medium (MEM; Hyclone Laboratories, Logan, UT, USA) containing 10% fetal bovine serum (Invitrogen-GIBCO, Grand Island, NY, USA) and gentamycin (0.1 mg/mL) in a humidified incubator maintained at 37 °C and 5% CO2. 2.2. PrP(106 –126) treatment Synthetic PrP(106 –126) (sequence, Lys-Thr-Asn-MetLys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-ValGly-Gly-Leu-Gly) was synthesized by Peptron, Inc. (Seoul, Korea). The peptide was dissolved in sterile dimethyl sulfoxide at 10 mM and stored at ⫺80 °C. 2.3. Annexin V assay Apoptosis was assessed by the annexin V assay in detached cells using an annexin V Assay kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer’s protocol. Annexin V levels were determined by measuring fluorescence at 488-nm excitation and 525/30 emission using a Guava EasyCyte HT System (Millipore, Bedford, MA, USA). 2.4. Lactate dehydrogenase assay Cytotoxicity was assessed in supernatants using a lactate dehydrogenase (LDH) cytotoxicity Detection kit (Takara Bio, Inc., Tokyo, Japan) according to the manufacturer’s protocol. LDH activity was determined by measuring the absorbance at 490 nm using a microplate reader (Spectra Max M2, Molecular Devices, Sunnyvale, CA, USA). 2.5. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis was performed to measure the degree of cellular apoptosis using an in situ Apo-BrdU DNA Fragmentation Assay kit (BioVision, San Francisco, CA, USA) as previously described (Jeong et al., 2011a). Briefly, cells were washed with phosphate buffered saline

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(PBS), fixed with paraformaldehyde for 15 minutes, preincubated with 50 ␮L of DNA-labeling solution (10 ␮L TdT reaction buffer, 0.75 ␮L TdT enzyme, 8 ␮L Br-dUTP) for 1 hour at 37 °C, and incubated with 5 ␮L fluorescein isothiocyanate (FITC) conjugated anti-BrdU monoclonal antibody for 0.5 hours at room temperature. Finally, cells were mounted with Dako Fluorescent mounting medium (Dako, Glostrup, Denmark) and visualized using fluorescence microscopy. Cells were counterstained with propidium iodide to show all cell nuclei. 2.6. Western blot analysis After SH-SY5Y cells were lysed in buffer (25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 100 mM NaCl, 1 mM EDTA (Ethylene Diamine Tetra Acetic acid), 5 mM MgCl2, 0.1 mM DTT (Dithiothreitol), and a protease inhibitor mixture), proteins were electrophoretically resolved on a 10%–15% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoreactivity was detected through sequential incubations with horseradish peroxidaseconjugated secondary antibodies and enhanced chemiluminescence reagents. The antibodies used for immunoblotting were LC3 (Novus Biologicals, Littleton, CO, USA), Bax (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (BD BioScience, San Jose, CA, USA), autophagy related 5 (ATG5) (Abcam, Cambridge, MA, USA), Sirt1 (Santa Cruz, CA, USA) and ␤-actin (Sigma Aldrich, St. Louis, MO, USA). Images were examined using a Fusion FX7 imaging system (Vilber Lourmat, Torcy Z.I. Sud, France). 2.7. Cellular fractionation SH-SY5Y cells were resuspended in mitochondrial buffer (210 mM sucrose, 70 mM mannitol, 1 mM EDTA, and 10 mM HEPES), broken with a 26-gauge needle, and subjected to centrifugation at 700g for 10 minutes. The postnuclear supernatant was centrifuged at 10,000g for 30 minutes. The pellet was used as the mitochondrial fraction, and the supernatant was used as the cytosolic fraction. Total protein was obtained and subjected to Western blot analysis. 2.8. Mitochondrial transmembrane potential assay The change in mitochondrial transmembrane potential (MTP) was evaluated by the JC-1 cationic fluorescent indicator (Molecular Probes, Eugene, OR, USA) which aggregates in intact mitochondria (red fluorescence), indicating high, normal, or low MTP when it remains in monomeric form in the cytoplasm (green fluorescence). SH-SY5Y cells were incubated in MEM containing 10 ␮M JC-1 at 37 °C for 15 minutes, washed with PBS, and then transferred to a clear 96-well plate. JC-1 aggregate fluorescent emissions were measured at 583 nm with an excitation wavelength of 526 nm, and the JC-1 monomer fluorescence intensity was measured with excitation and emission at 525 and 530 nm,

respectively using a Guava EasyCyte HT System (Millipore) or a microplate reader (Spectra Max M2, Molecular Devices). SH-SY5Y cells were cultured on cover slips in a 24-well plate, incubated in MEM containing 10 ␮M JC-1 at 37 °C for 15 minutes, and then washed with PBS. Finally, cells were mounted with Dako Fluorescent mounting medium and visualized via Kikon Eclipse 80i fluorescence miscropy (Nikon, Tokyo, Japan). 2.9. Construction of recombinant adenoviruses The Sirt1 over-expressing adenovirus (Ad-Sirt1) was kindly provided by Professor Byung-Hyun Park (Chonbuk National University, Jeonju, Jeonbuk, South Korea). The lacZ-bearing adenovirus (Ad-lacZ) was used as a control. Recombinant adenoviruses were amplified in human embryonic kidney (HEK)-293 cells and purified using the Vivapure AdenoPACK kit (Sartorius AG, Göttingen, Germany) according to the manufacturer’s instructions. 2.10. RNA interference SH-SY5Y cells were transfected with ATG5 small interfering RNA (siRNA; oligoID HSS114104; Invitrogen, Carlsbad, CA, USA) or Sirt1 siRNA (oligoID VHS50608; Invitrogen) using Lipofectamine 2000 according to the manufacturer’s instructions. After a 48-hour culture, knockdown efficiency was typically measured at the protein level by immunoblot analysis. Nonspecific siRNA (oligoID 12935–300; Invitrogen) was used as a negative control. 2.11. Sirt1 deacetylase activity assay Nuclear proteins were extracted from SH-SY5Y cells using a Nuclear/Cytosol Fractionation kit (BioVision, California, USA) to measure cellular Sirt1 deacetylase activity. Sirt1 deacetylase activity was quantified following the protocols of the Sirt1 Fluorometric Assay kit (Sigma-Aldrich, St. Louis, MO, USA). Fluorescence intensities were measured with a microplate fluorometer (excitation wavelength ⫽ 360 nm, emission wavelength ⫽ 450 nm). The fluorescence intensities of Sirt1 deacetylase activity were normalized to protein levels measured in the cell samples. 2.12. Statistical evaluation All data are expressed as means ⫾ standard deviations and compared using the Student t test, analysis of variance, and Duncan’s test using SAS statistical software version 9.1 (SAS Institute, Cary, NC, USA). Results were considered significant at * p ⬍ 0.05, ** p ⬍ 0.001 and # p ⬍ 0.01. 3. Results 3.1. An increase in Sirt1 activity induced autophagy As Sirt1 prevents neurodegeneration in neuron cells via mitochondrial homeostasis (Gomes et al., 2012), and a recent study showed that activating autophagy was the main factor preventing mitochondrial damage (Rambold and Lip-

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Fig. 1. Overexpression of sirtuin 1 (Sirt1) increased expression of autophagy marker LC3-II and activity of Sirt1. (A) Sirt1 over-expressing adenovirus (Ad-Sirt1) or lacZ-bearing adenovirus (Ad-lacZ) transfected SH-SY5Y cells were incubated with or without autophagy inhibitor (3-MA and wortmannin) for 24 hours. The treated cells were assessed for LC3-1/2 and Sirt1 production by Western blot analysis. Results were normalized with ␤-actin. (B) Sirt1 activity conversion in the SH-SY5Y cells treated as described in (A). * p ⬍ 0.05, ** p ⬍ 0.001; significant differences between control and each treatment group.

pincott-Schwartz, 2011), we examined whether activating the Sirt1 enzyme could induce autophagy. First, we investigated whether Sirt1 overexpression after adenoviral transfection resulted in Sirt1 activity. Transfecting Ad-Sirt1 at a multiplicity of infection (MOI) of 50 and 200 increased Sirt1 activity, but cells transfected with Ad-lacZ at 200 MOI did not affect Sirt1 activity (Fig. 1A). We next assessed whether the increase in Sirt1 activity using an adenoviral transfection system affected the induction of autophagy. At 48 hours after transfection with different Ad-Sirt1 MOI (0, 50, 100, and 200 MOI), the transfected cells showed a virus dose-dependent increase in LC3-II, a late light chain 3 autophagosome marker, levels compared to that of the AdlacZ transfected control group (Fig. 1B). Additionally, the autophagy inhibitors 3-MA, bafilomycin A1, and wortmannin blocked Sirt1-induced LC3-II levels, whereas they were not blocked in Ad-Sirt1 transfected cells (Fig.1). These data indicate that Sirt1 overexpression induced the autophagy pathway by increasing Sirt1 activity. 3.2. Neuroprotective effect of Sirt1 against PrP(106 – 126)-induced mitochondrial apoptotic pathway was related to autophagy regulation Mitochondrial impairment is related with prion-mediated neuronal cell death (O’Donovan et al., 2001); therefore, we examined whether an increase in Sirt1 activity affected

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prion-mediated mitochondrial dysfunction. Ad-Sirt1 transfected cells were treated with 50 ␮M PrP(106 –126) (Fig. 2A and B). PrP(106 –126)-exposed cells showed increased JC-1 monomer positive cells (41.56%), whereas transfection with Ad-Sirt1 decreased PrP(106 –126)-induced JC-1 monomer positive cells, but, these preventative effects of Sirt1 were blocked by treatment with autophagy inhibitors (Fig. 2A). Consistent with these results, fluoroscopy (Fig. 2B) revealed cells with green fluorescence after PrP(106 – 126) treatment indicating lower MTP, whereas the negative control cells and Sirt1 transfected cells fluoresced red, indicating high MTP values. We also examined the effect of Sirt1 on PrP(106 –126)-mediated Bax translocation and cytochrome c release. PrP(106 –126)-treated cells were induced by Bax translocation to the mitochondria, which increased cytochrome c release to the cytosol. However, PrP(106 –126)-induced Bax translocation and cytochrome c release decreased following Sirt1 transfection of SH-SY5Y cells. Furthermore, these protective effects of Sirt1 were blocked by treatment with the autophagy inhibitor 3-MA (Fig. 2C). To determine whether Sirt1 operated by activating autophagy to block PrP(106 –126)-induced neuronal cell death, cells were transfected with Ad-Sirt1 then exposed to PrP(106 –126) with or without the autophagy inhibitor 3-MA. Ad-Sirt1 transfected SH-SY5Y cells showed decreased PrP(106 –126)-induced apoptosis, whereas treatment with the autophagy inhibitor blocked the protective effect of Sirt1 on PrP(106 –126)-induced apoptosis (Fig. 3A). Consistent with these results, the LDH assay (Fig. 3B) showed that activating Sirt1 inhibited PrP(106 –126)-induced apoptosis by activating autophagy. We also examined the influence of Sirt1 overexpression on Sirt1-mediated autophagy induction in PrP(106 –126)-treated cells. As shown in Fig. 3C and D, Ad-Sirt1 transfection increased Sirt1 protein expression and Sirt1 activity, whereas transfection with Ad-lacZ did not. Additionally, treatment with PrP(106 –126) decreased the Sirt1 activity caused by Sirt1 overexpression. Protein expression of the autophagy marker LC3-II was determined by Western blot assay. The LC3-II protein increased following Ad-Sirt1 transfection, whereas the Ad-lacZ transfection and PrP(106 –126) treatment did not affect LC3-II protein expression (Fig. 3C). Collectively, these results are consistent with the idea that activating Sirt1 inhibits PrP(106 –126)-induced mitochondria-mediated apoptosis by activating autophagy. The Sirt1 RNAinterference (RNAi) oligomer was used to knockdown Sirt1 gene expression to verify that Sirt1 plays a protective role in prion-mediated mitochondrial cell death by activating autophagy. Sirt1 activity and Sirt1 expression increased in SH-SY5Y cells exposed to the Sirt1 activator resveratrol, but Sirt1 gene knockdown using the Sirt1 RNAi oligomer blocked Sirt1 expression (Fig. 4A and B). LC3-II protein expression increased in resveratrol-treated cells but not in Sirt1 knockdown cells (Fig. 4A).

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Fig. 2. Protective effect of overexpression of sirtuin 1 (Sirt1) on prion protein (PrP)(106 –126)-mediated mitochondrial dysfunction. (A) Sirt1 over-expressing adenovirus (Ad-Sirt1) or lacZ-bearing adenovirus (Ad-lacZ) transfected SH-SY5Y cells exposed to 50 ␮M of PrP(106 –126) with or without autophagy inhibitor (2 mM of 3-MA, 100 nM bafilomycin A1, or 10 ␮M of wortmannin) for 24 hours. The treated cells were measuring JC-1 mono form (green) by flow cytometry. M1 represents the population of JC-1 monomeric cells. (B) Representative images of J-aggregate formation in cells treated as described in (A). The treated cells were measuring JC-1 aggregates form (red) and mono form (green) by confocal microscopy analysis. Scale bar ⫽ 50 ␮m. (C) Cells were homogenized in a mitochondrial buffer, as described in Methods. The separation of cytosol and mitochondrial extracts were analyzed by Western blot using antibodies against cytochrome c and Bax protein.

Next, we examined the influence of Sirt1 knockdown on the neuroprotective effects of the Sirt1 activator against PrP(106 – 126)-mediated neurotoxicity in SH-SY5Y cells by the LDH assay. SH-SY5Y cells were exposed to resveratrol with or without PrP(106 –126). Cells were responsive to resveratrol treatment and resveratrol blocked PrP(106 –126)-induced neurotoxicity in SH-SY5Y cells (Fig. 4C). In contrast, treatment with the Sirt1 RNAi oligomer (Sirt1 siRNA) blocked the protective effect of the Sirt1 activator resveratrol on PrP(106 – 126)-induced neurotoxicity (Fig. 4C).

We next assessed whether the protective effect of activating Sirt1 activator resveratrol-mediated autophagy on PrP(106 –126)-mediated neurotoxicity was related to preventing mitochondrial dysfunction. Fluorescence microscopy images showed red fluorescing resveratrol-treated cells with J-aggregates form of JC-1 after PrP(106 –126) treatment, indicating high MTP values, whereas the Sirt1 siRNA transfection displayed green fluorescence (JC-1 monomer form) after PrP(106 –126) treatment. Consistent with these results, resveratrol-treated cells were blocked by

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Fig. 3. Overexpression of Sirt1 blocked prion protein (PrP)(106 –126)-induced apoptotic pathway via regulation of autophagy. (A) Sirt1 over-expressing adenovirus (Ad-Sirt1) or lacZ-bearing adenovirus (Ad-lacZ) transfected SH-SY5Y cells were exposed to autophagy inhibitor 3-MA (2 mM). Cell viability was measured by annexin V assay. (B) Western blot analysis of LC3-1/2 and Sirt1 conversion in the SH-SY5Y cells treated as described in (A). Results were normalized with ␤-actin. (C) Bar graph indicated the averages of Sirt1 activity. * p ⬍ 0.05, ** p ⬍ 0.001; significant differences between control and each treatment group. (D) Ad-Sirt1 or Ad-lacZ-transfected SH-SY5Y cells were exposed to 50 ␮M of PrP(106 –126) with or without 2 mM of 3-MA (autophagy inhibitor) for 24 hours, and then release of lactate dehydrogenase into the cell culture supernatant from damaged cells was measured. * p ⬍ 0.05, ** p ⬍ 0.001; significant differences between control and each treatment group, and # p ⬍ 0.01; significantly different when compared with PrP(106 –126)-treated group.

the PrP(106 –126)-induced increase in the JC-1 monomer/ oligomer ratio, indicating low MTP values, whereas melatonin treatment reduced the PrP(106 –126)-induced JC-1 monomer/oligomer ratio (Fig. 4E). Bax translocation into mitochondria is a main factor in the mitochondrial apoptotic

pathway. Thus, we tested the influence of Sirt1 siRNA on Sirt1 activation against PrP(106 –126)-induced Bax translocation and cytochrome c release. PrP(106 –126)-induced Bax translocation and cytochrome c release were inhibited by the Sirt1 activator resveratrol, whereas trans-

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Fig. 4. Sirtuin 1 (Sirt1) knockdown break the Sirt1-mediated neuroprotective effect against prion protein (PrP)(106 –126). (A) Sirt1 small interfering RNA (siRNA) or NC siRNA (negative control siRNA) transfected SH-SY5Y cells were incubated with 50 ␮M PrP(106 –126) for 24 hours after exposure to Sirt1 activator (resveratrol) for 12 hours. The treated cells were assessed for LC3-1/2 and Sirt1 production by Western blot analysis. Results were normalized with ␤-actin. (B) Cells were treated with to 2 ␮M of resveratrol (12 hours) and then exposed to 50 ␮M of PrP(106 –126) for 24 hours. The graph indicated the averages of Sirt1 activity. * p ⬍ 0.05, significant differences between control and each treatment group. (C) Bar graph indicated that the release of lactate dehydrogenase into the cell culture supernatant from damaged cells was measured. * p ⬍ 0.05, ** p ⬍ 0.001; significant differences between control and each treatment group. (D) Representative images of J-aggregate formation in cells treated as described in (A). The treated cells were measuring JC-1 aggregates form (red) and mono form (green) by confocal microscopy analysis. Scale bar ⫽ 50 ␮m. (E) Bar graph indicated the J-aggregate formation. * p ⬍ 0.05, ** p ⬍ 0.001; significant differences between control and each treatment group. (F) Cells were homogenized in a mitochondrial buffer, as described in Methods. The separation of cytosol and mitochondrial extracts were analyzed by Western blot using antibodies against cytochrome c and Bax protein.

fection of Sirt1 siRNA blocked the protective effect of resveratrol (Fig. 4F). Collectively, these results are consistent with the notion that the regulation of Sirt1 modulates PrP(106 –126)-induced mitochondrial apoptosis by activating autophagy. 3.3. ATG5 knock-down blocked the neuroprotective effect of a Sirt1 activator against PrP-mediated neurotoxicity An ATG5 RNAi oligomer was utilized to knockdown ATG5 gene expression to examine the protective role of Sirt1-mediated autophagy in PrP-mediated neuronal cell death. Treatment with the ATG5 RNAi oligomer (ATG5 siRNA) inhibited LC3-II and ATG5 protein expression (Fig. 5A). In contrast, ATG5 siRNA had no effect on

Sirt1 expression or Sirt1 activity. Additionally, the protective effect of Sirt1 activation on the PrP(106 –126)induced reduction in MTP values was inhibited by transfection with ATG5 siRNA (Fig. 5C). Consistent with these results, fluorescence microscopy images showed that PrP(106 –126)-treated cells fluoresced red (JC-1 aggregates form) after Sirt1 activator treatment, whereas the ATG5 siRNA transfected cells displayed green fluorescence (JC-1 monomer form) (Fig. 5D). Also, ATG5 siRNA transfected cells blocked the protective effect of Sirt1 activator on PrP(106 –126)-induced apoptosis (Fig. 6). These results suggest that activating Sirt1 prevents PrP(106 –126)-induced mitochondrial dysfunction and apoptosis by regulating autophagy.

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4. Discussion

Fig. 5. Autophagy-related (ATG) knockdown blocked the sirtuin 1 (Sirt1)mediated mitochondrial protective effect against prion protein (PrP)(106 – 126). (A) ATG5 small interfering RNA (ATG5 siRNA) or NC siRNA (negative control siRNA) transfected SH-SY5Y cells were incubated with 50 ␮M PrP(106 –126) for 24 hours after exposure to Sirt1 activator (resveratrol) for 12 hours. The treated cells were assessed for ATG5 and LC3-1/2 production by Western blot analysis. Results were normalized with ␤-actin. (B) Cells were treated with 2 ␮M of resveratrol (12 hours) and then exposed to 50 ␮M of PrP(106 –126) for 24 hours. The graph indicated the averages of Sirt1 activity. * p ⬍ 0.05; significant differences between control and each treatment group. (C) Bar graph indicated that the release of lactate dehydrogenase into the cell culture supernatant from damaged cells was measured. * p ⬍ 0.05, ** p ⬍ 0.001; significant differences between control and each treatment group. (D) Representative images of J-aggregate formation in cells treated as described in (A). The treated cells were measuring JC-1 aggregates form (red) and mono form (green) by confocal microscopy analysis. Scale bar ⫽ 50 ␮m.

The present study demonstrated that activating Sirt1 prevented prion-mediated mitochondrial damage by activating autophagy, and that inducing autophagy by activating Sirt1 played a pivotal role in the protective effect of Sirt1 against prion-mediated neuronal cell death. Notably, Sirt1-mediated induction of autophagy lead to decreased mitochondrial impairment in neuron cells, which, in turn, conferred neuroprotection. Several lines of evidence support a protective role for Sirt1 in the cellular response to energy metabolism, including caloric restriction, which is a common method to increase life span (Herranz et al., 2010; Nemoto et al., 2005). In particular, caloric restriction increases cell adaptation to hypoxia through Sirt1-dependent mitophagy (mitochondrial autophagy) in mouse kidneys (Kume et al., 2010). Additionally, activating neuronal Sirt1 prevents Alzheimer’s disease amyloid neuropathology through caloric restriction (Qin et al., 2006; Wang et al., 2010). Sirt1 expression and activation in the brain tissue of caloric restriction prevented against Alzheimer’s disease amyloid neuropathology (Wang et al., 2010). Also, activation of Sirt1 suppresses amyloidbeta (A␤) production is through regulation of the nonamyloidogenic processing of amyloid precursor protein (APP) by means of inhibition of serine/threonine Rho kinase ROCK1 expression (Qin et al., 2006). In addition, Sirt1 activator resveratrol prevents MPTP-mediated cell degeneration and mitochondrial-oxidative damage by activating the Sirt1/PGC-1␣ pathways (Mudò et al., 2012). For this reason, regulation of Sirt1 activation may be considered a key factor for prevention of neurodegenerative disorders. Similarly, our previous study showed that activation of Sirt1 protects against prion-mediated neurotoxicity (Seo et al., 2012), but, we cannot find the relation for prion-mediated neurotoxicity and Sirt1-regulated mechanisms. Some reports showed that Sirt1 induces the autophagylysosome pathway (Lee et al., 2008; Salminen and Kaarniranta, 2009). Autophagy is a major catabolic process which is for the recycling and degradation of cell organelles and protein aggregates (Garcia et al., 2011; Glick et al., 2010). A basal autophagy plays a physiological role in caloric restriction, immunity, and neuroprotection (Hartman, 2011; Kang et al., 2011; Nicoletti et al., 2011). In particular, food restriction provides a neuroprotective effect by regulating the Sirt1 activation and autophagy pathway, respectively (Alirezaei et al., 2011; Qin et al., 2006). Additionally, resveratrol prevents prevents rotenone-mediated neurotoxicity in Parkinson’s disease through the autophagy-lysosome pathway (Wu et al., 2011). However, the relation for Sirt1mediated protective effect and autophagy in prion-mediated neurotoxicity is not clear. Therefore, we investigated the relationship between the Sirt1 and autophagy activation in prion-induced neuronal cell death. To understand the mechanisms by which induced Sirt1mediated autophagy prevents prion-mediated neurotoxicity,

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Fig. 6. Autophagy-related (ATG) knockdown blocked the sirtuin 1 (Sirt1)-mediated neuroprotective effect against prion protein (PrP)(106 –126). ATG5 small interfering RNA (ATG5 siRNA) or NC siRNA (negative control siRNA) transfected SH-SY5Y cells were incubated with 50 ␮M PrP(106 –126) for 24 hours after exposure to Sirt1 activator (resveratrol) for 12 hours. Cell viability was measured by annexin V assay.

our first experiment examined the relationship between Sirt1 activity and the induction of autophagy on PrP(106 – 126)-induced apoptosis. Sirt1 overexpression increased LC3-II autophagy marker levels, Sirt1 expression (Fig. 1A), and Sirt1 activity (Fig. 1B). Moreover, the autophagy inhibitor blocked LC3-II expression without any effect on Sirt1 activity (Fig. 1). Additionally, PrP(106 –126)-induced neuronal cell death (Fig. 3) was protected by Sirt1 overexpression; however, treatment with autophagy inhibitors attenuated the protective effect of Sirt1 (Fig. 3). These observations suggest that activating Sirt1 may protect against PrP(106 –126)-induced neurotoxicity by activating autophagy. Regulation of mitochondrial function affects the progression of neurodegenerative diseases including Alzheimer’s and prion diseases (O’Donovan et al., 2001; Pagani and Eckert, 2011). Recent studies have shown that the resveratrol prevents amyloid beta toxicity in Alzheimer’s disease models (Vingtdeux et al., 2010). However, the effect of activating Sirt1 on mitochondrial dysfunction in prion disease has not been reported. In the present study, Sirt1 overexpression prevented PrP(106 –126)-induced low MTP values (Fig. 2A and B), translocated the Bax protein to the mitochondria, and released cytochrome c from mitochondria (Fig. 2C); however, treatment with autophagy inhibitors blocked the protective effects of Sirt1 (Fig. 2). These observations support the idea that activating Sirt1 prevents PrP-mediated mitochondrial dysfunction by inducing autophagy. Sirt1 RNAi oligomer-treated cells showed reduced Sirt1 protein expres-

sion (Fig. 4A) and Sirt1 activity (Fig. 4B) and inhibited the neuroprotective effect of the Sirt1 activator on PrP(106 –126)-induced mitochondrial dysfunction (Fig. 4D and E) and apoptosis (Fig. 4A and C). In contrast, autophagy inhibitors inhibited the protective effect of melatonin on prion-mediated neurotoxicity (Fig. 5) and mitochondrial dysfunction (Fig. 4). These observations also support the hypothesis that regulating of Sirt1 activity modulates prion-mediated neuronal cell death via the mitochondrial apoptotic pathway. An examination of neuron cells, in which ATG5 expression was deviated, demonstrated that resveratrol treatment increased Sirt1 protein (Fig. 4A) and activity (Fig. 5B) but did not inhibit PrP(106 –126)-induced neuronal cell death (Fig. 6) or mitochondrial damage (Fig. 5C and D). These data indicate that activating Sirt1 had a protective effect against prion-mediated mitochondrial damage and neuronal cell death by regulating the induction of autophagy. Some reports showed that interaction of Sirt1 with the FOXOs, p53, and PGC-1␣ signaling can regulate both the autophagy pathway and lifespan extension (Leibiger and Berggren, 2006; Salminen and Kaarniranta, 2009). Thus, the protective effect of Sirt1-mediated autophagy against prion-induced apoptotic pathway is thought to be associated with a downstream signaling network. Further detailed studies of the Sirt1-mediated downstream signaling network including FOXOs, p53, and PGC-1 alpha will be important in understanding neuroprotective mechanism of Sirt1-mediated autophagy against prion-mediated neurotoxicity and mitochondrial damage.

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This is the first report demonstrating that inducing Sirt1mediated autophagy may be the main mechanism for neuroprotection against the prion peptide-induced mitochondrial apoptotic pathway. These results suggest that Sirt1 may be involved in the pathogenesis and, as such, may be a valid therapy target for prion-related neurodegenerative diseases.

Disclosure statement The authors disclose no conflicts of interest.

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