Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway

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Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway

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Benxu Cheng a,d,⇑, Alex Anthony Martinez c, Jacob Morado e, Virginia Scofield a, James L. Roberts c,f, Shivani Kaushal Maffi a,b,⇑ a

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Regional Academic Health Center-Edinburg (E-RAHC), Medical Research Division, 1214 W. Schunior St., Edinburg, TX 78541, United States Department of Molecular Medicine, University of Texas Health Science Center, 15355 Lambda Dr. San Antonio, TX 78245, United States Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229, United States d Department of Cellular & Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229, United States e The University of Pan American, Cooperative Pharmacy Program, Edinburg, TX 78541, USA f Department of Biology, Division of Neuroscience, Trinity University, One Trinity Place, San Antonio, TX 78212, United States b c

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Article history: Received 6 April 2012 Received in revised form 22 October 2012 Accepted 30 October 2012 Available online xxxx

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Keywords: Programmed cell death Oxidative stress Epoxomicin Retinoic acid SH-SY5Y cells Ubiquitin Proteasome inhibition AKT

a b s t r a c t Inhibition of proteasome activity and the resulting protein accumulation are now known to be important events in the development of many neurological disorders, including Alzheimer’s and Parkinson’s diseases. Abnormal or over expressed proteins cause endoplasmic reticulum and oxidative stress leading to cell death, thus, normal proteasome function is critical for their removal. We have shown previously, with cultured SH-SY5Y neuroblastoma cells, that proteasome inhibition by the drug epoxomicin results in accumulation of ubiquitinated proteins. This causes obligatory loading of the mitochondria with calcium (Ca2+), resulting in mitochondrial damage and cytochrome c release, followed by programmed cell death (PCD). In the present study, we demonstrate that all-trans-retinoic acid (RA) pretreatment of SHSY5Y cells protects them from PCD death after subsequent epoxomicin treatment which causes proteasome inhibition. Even though ubiquitinated protein aggregates are present, there is no evidence to suggest that autophagy is involved. We conclude that protection by RA is likely by mechanisms that interfere with cell stress-PCD pathway that otherwise would result from protein accumulation after proteasome inhibition. In addition, although RA activates both the AKT and ERK phosphorylation signaling pathways, only pretreatment with LY294002, an inhibitor of PI3-kinase in the AKT pathway, removed the protective effect of RA from the cells. This finding implies that RA activation of the AKT signaling cascade takes precedence over its activation of ERK1/2 phosphorylation, and that this selective effect of RA is key to its protection of epoxomicin-treated cells. Taken together, these findings suggest that RA treatment of cultured neuroblastoma cells sets up conditions under which proteasome inhibition, and the resultant accumulation of ubiquitinated proteins, loses its ability to kill the cells and may likely play a therapeutic role in neurodegenerative diseases. Ó 2012 Published by Elsevier Ltd.

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1. Introduction

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During the development of Parkinson’s disease (PD), there is progressive loss of dopaminergic neurons in the Substantia Nigra pars compacta. These cells contain protein aggregates, containing a-synuclein and ubiquitin, which are called Lewy bodies (Gerlach and Riederer, 1996; McNaught et al., 2004). The specific mecha-

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⇑ Corresponding authors. Addresses: UTHSC Regional Academic Health Center-

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Edinburg (E-RAHC), Medical Research Division, 1214 W. Schunior St., Edinburg, TX 78541, United States. Tel.: +1 (956) 393 6424; fax: +1 (956) 393 6402 (B. Cheng), Department of Molecular Medicine, University of Texas Health Science Center, 15355 Lambda Dr. San Antonio, TX 78245, United States. Tel.: +1 (210) 854 7406; fax: +1 (956) 393 6402 (S.K. Maffi). E-mail addresses: [email protected] (B. Cheng), maffi@uthscsa.edu (S.K. Maffi).

nisms that cause death in these cells are not well understood. However, events that cause the appearance of protein aggregates in these cells are associated with dysfunction of the ubiquitin–proteasome system, mitochondrial dysfunction, intracellular calcium imbalance, endoplasmic reticulum stress and oxidative stress leading to apoptosis (Belal et al., 2012; Koch et al., 2009; McNaught and Jenner, 2001; Moore et al., 2005). Apoptosis or PCD type-I is one of the three forms of programmed cell death (PCD) (Hengartner, 2000; Nagley et al., 2010). The other two forms of PCD are autophagy (or PCDII) and programmed necrosis (PCDIII). The role of mitochondria is central to each of these cell death pathways, however, the distinction lies in the activation of specific set of proteins to achieve cell death (Chinta et al., 2008; Nagley et al., 2010; Yang et al., 2009b). In the absence of a reliable animal model for PD (Kordower et al., 2006; McNaught and Olanow, 2003; McNaught et al., 2004),

0197-0186/$ - see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.neuint.2012.10.014

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some recent investigations have focused on primary mesanchephalic neuron cultures (Huang and Chuang, 2010; Kikuchi et al., 2003; Yamamoto et al., 2007) or cell lines derived from the dopaminergic neurons involved in PD (Koch et al., 2009; Lu et al., 2006). In these studies, such cells have been exposed to either PD-initiating drugs, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Lee et al., 2011; Mann and Tyndale, 2010), or to its active metabolite methylpyridinium (MPP) (Gomez-Santos et al., 2002; Halvorsen et al., 2002; Huang and Chuang, 2010), or to proteasome inhibitors such as epoxomicin (Canu et al., 2000; Cheng et al., 2011; Koch et al., 2009). In eukaryotic cells, the ubiquitin–proteasome system removes unwanted, damaged, senescent or misfolded proteins. This occurs via a two-step process, in which specific proteins first get ubiquitinated which targets them to the proteasomes, and then are broken down in the proteasomes for recycling of their amino acids. In cultured cells that are dividing, proteasome inhibition activates apoptosis, while proteasome inhibition in nondividing cells has antiapoptotic effects (Drexler, 1997; Drexler et al., 2000; Grimm et al., 1996; Sadoul et al., 1996). A number of studies, including our own, have used dividing dopaminergic neuroblastoma cell lines, in which proteasome inhibition was induced either by (a) exposure of the cells to pro-oxidants, or (b) treatment of the cells with the proteasome inhibitor, epoxomicin (Cheng et al., 2011; Keller and Markesbery, 2000; Lopes et al., 1997; Okada et al., 1999; Reinheckel et al., 1998). After either of these two regimens, the cells accumulate aggregated proteins in the cytoplasm, and this is followed by mitochondrial dysfunction, antioxidant depletion and apoptosis (Boukhtouche et al., 2006; Canu et al., 2000; Ding and Keller, 2001; Keller and Markesbery, 2000; Kikuchi et al., 2003; Qiu et al., 2000). Taken together, work from many laboratories has ubiquitin–proteasome system dysregulation to be centrally involved and highly relevant to the pathogenesis of the several neurodegenerative disorders (Kikuchi et al., 2003; Lopez Salon et al., 2000; McNaught et al., 2002; McNaught and Jenner, 2001). All-trans-retinoic acid (RA) is a naturally occurring and biologically active metabolite of vitamin A. RA is a potent regulator of morphogenesis, cell proliferation, and cell differentiation (Canon et al., 2004; De Luca, 1991; Konta et al., 2001; Love and Gudas, 1994). RA plays a critical role during normal neuronal development (Durston et al., 1989; Hunter et al., 1991; Jackson et al., 1991; Maden, 2001), where it induces neurite outgrowth and neuronal differentiation (Bain et al., 1995; Corcoran and Maden, 1999; Kobori et al., 2004; Lopez-Carballo et al., 2002; Pennypacker et al., 1989; Rebhan et al., 1994). In cultured neuroblastoma cell lines, including SH-SY5Y, high concentrations of RA treatment induces neuronal differentiation over 3–10 days in culture (Encinas et al., 1999; Miloso et al., 2004; Pan et al., 2005; Rebhan et al., 1994; Schneider et al., 2011). For our studies, it is important to note that in some cell types and in some tissues, RA has anti-apoptotic and antioxidant properties. For example, it inhibits induction of lipid peroxidation in rat brain slices (Das, 1989), decreases staurosporine-induced apoptosis in neuronal cells, reduces mitochondrial ROS production in stressed neurons, and inhibits glutathione depletion in the same cells (Ahlemeyer et al., 2001; Ahlemeyer and Krieglstein, 1998). In cultured hippocampal cells from neonatal Fischer rats, RA treatment maintains SOD levels during oxidative stress, under conditions where SOD levels normally fall (Ahlemeyer et al., 2001). RA also reduces H2O2-induced apoptosis in PC12 cells (Jackson et al., 1991), mesangial cells, and fibroblasts (Moreno-Manzano et al., 1999). RA even has anti-inflammatory effects in some cells; for instance, it attenuates TNF-a and iNOS mRNA production in rat microglia exposed to b-amyloid peptide (Ab) and lipopolysacharide (LPS) (Dheen et al., 2005), and it slows the development of

performance deficits when administered to aged mice (Etchamendy et al., 2001). More recent work has confirmed that RA protects cultured hippocampal and retinal neurons, (Sakamoto et al., 2010; Shinozaki et al., 2007), and there is emerging evidence that RA treatment may be effective in slowing or reversing Alzheimer’s disease (AD) (Lee et al., 2009). SH-SY5Y is a neuroblastoma cell line that represents substantia nigra cells as a model for Parkinson’s disease (Cheng et al., 2011). In these cells, proteasome inhibition by the drug epoxomicin causes programmed cell death in association with protein aggregation and mitochondrial oxidative stress. However, when these cells are treated with RA prior to epoxomicin treatment, PCD is prevented and oxidative stress is reduced, even though proteasome inhibition is sustained and protein aggregates are present. Observations in this study conclusively uncouple proteasome impairment and abnormal protein accumulation from PCD and oxidative stress in neuronal cells, and they identify the PI3/AKT pathway as the signaling cascade by which this takes place. Our results identify RA as an effective candidate drug for the treatment of neurological disorders in which proteasome impairment activates PCD pathway leading to neuronal death.

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2. Materials and methods

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2.1. Reagents

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Minimum essential medium (MEM) and F-12 nutrient mixture (HAM) were purchased from Gibco BRL (Gaithersburg, MD), and fetal bovine serum was from ATCC (Manassas, VA). Retinoic acid, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], epoxomicin were obtained from Sigma (St. Louis, MO), Hoechst 33342 and 20 ,70 -dichlorodihydrofluorescein diacetate (DCF-DA) and Propidum Iodide from Life Technologies (Eugene, OR). The caspase-3 inhibitor, Z-DEVD-FMK, was purchased from Biovision (Mountain View, CA). LY294002 and PD098059, were obtained from Calbiochem-EMD4Biosciences, CA.

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2.2. Cell culture

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Human SH-SY5Y cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained at 5% CO2 at 37 °C in MEM:Ham’s F12 (1:1) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 lg/ml). Cells were sub-cultured weekly. All experiments were performed between passages 25 and 35 and at 80% cell confluence. The cells were seeded onto 60 mm tissue culture dishes or into multi-well plates. The cell culture medium was replaced every 2–3 days.

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2.3. Inhibitor treatments

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Proteasome inhibition was induced in SH-SY5Y cells as we have described previously (Cheng et al., 2011), using epoxomicin, a natural product isolated from an Actinomycetes species. This compound is an irreversible inhibitor of the chymotrypsin-like (CT-L), trypsin-like (T-L), and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities of 26S/20S proteasome (Meng et al., 1999). It functions by modifying the proteasomal catalytic subunits LMP-7, MECL1, and Z. Epoxomicin does not affect activities of non-proteasomal cellular proteases such as trypsin, cathepsin B, or chymotrypsin (Meng et al., 1999; Princiotta et al., 2001). Epoxomicin was dissolved in dimethyl sulfoxide (DMSO). SH-SY5Y cells were usually treated with epoxomicin at 100 nM concentration for 24 h. In certain experiments, cells were treated for 30 min either with 20 lM LY294002, a PI3/AKT inhibitor, or a MAP Kinase inhibitor, 10 lM PD098059, followed by addition of retinoic acid to the same

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media. SH-SY5Y cells were also treated with 50 lM of a caspase-3 specific inhibitor (Z-DEVD-FMK) for two hours prior to 100 nM Q2 epoxomicin exposure. In order to avoid any reversible action, the inhibitors were present throughout the duration of the experiment. Respective control cultures received a DMSO concentration which was equivalent to DMSO in the inhibitor treated cultures.

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2.4. Cell viability

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Morphological changes in control and treated cells were monitored using phase-contrast microscopy. Changes in cell viability were tracked, and final cell viability data was collected after each treatment regimen, by measuring the conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to formazan, as detailed in (Cheng et al., 2011). Briefly, cells were treated with 0.25 mg/ml MTT in fresh media for 2 h at 37 °C. The medium was removed and the formazan precipitate solubilized with DMSO, 100 ll aliquots transferred to 96-well plates and optical densities were measured using a microplate reader at 540 nm absorption.

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2.5. Western blotting

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Changes in amounts of cell signaling intermediates were measured by Western blotting. Protein homogenates were prepared as follows: cells were lysed in ice-cold lysis buffer (50 mM Tris– HCl (pH 7.5), 100 mM NaCL, 0.1% Triton X-100, 0.1% SDS and 1 mM EDTA) containing protease and phosphatase inhibitor cocktail (1:100, Sigma, (P8340)). Clear lysates were obtained by centrifugation at 4 °C for 20 min at 13,000 rpm in a refrigerated microcentrifuge. Protein concentrations were determined using the Micro-BCA assay (Pierce, Rockford, IL) according to the manufacturer’s instruction. Equal amounts (20 g) of the protein samples were separated on a 10% polyacrylamide gel and transferred to nitrocellulose membranes, blocked either for 1hour at room temperature or overnight at 4 °C with Tris buffer saline containing 0.1% Tween 20 (TBST, pH 7.4) and 5% (w/v) nonfat dried milk. In some instances, protein samples were run on precast gels (10%) purchased from BioRad. The antibodies used in these experiments were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) – Erk (sc-16982), Akt (sc-1619) and p-Erk (sc-7383), NFkB(65) (sc372) and Ubiquitin (sc-9133), Cell Signaling (Beverly, MA) – pAKT (9271), p-NFkB (3031), LC3B (2775) and Caspase-3 (9661), Biovision- Cytochrome c (3026) and PARP (93001), and Sigma (St. Louis, MO) – b-Actin (A5441). The blotted membranes were incubated with specific primary antibodies for 1 h at room temperature or overnight at 4 °C, after which the membranes were washed again and incubated for another 1 h with the appropriate horseradish peroxidase-conjugated secondary antibodies. Bands were detected using a chemiluminescent method (ECL), (GE Healthcare), according to the manufacture’s instructions. Band densities were analyzed by densitometry using the NIH image software. To measure the amounts of cytochrome c release from the mitochondria, cytosolic protein lysates of controls and treated cells were collected and run on 12% polyacrylamide gels (Bianchi et al., 2003; Cheng et al., 2011; Lu et al., 2003). Briefly, the cells were washed twice with PBS, scraped, cell pellets were collected and resuspended in extraction buffer (220 nM mannitol, 58 mM sucrose, 50 mM PIPES-KOH (pH 7.4), 50 mM KCL, 5 mM EDTA, 2 mM DTT, plus protease inhibitor cocktail) and incubated on ice for 30–45 min. Then, cells were homogenized by 20 strokes with a glass dounce homogenizer. The homogenates obtained were centrifuged at 10,000g for 15 min, supernatants collected and subjected to protein quantitation, followed by Western blot analysis using an anti-cytochrome c antibody, as noted above.

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2.6. Confocal microscopy

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2.6.1. Oxidative stress, mitochondrial membrane potential and immunofluorescence A cell-permeant fluorophore, 20 ,70 -dichlorodihydrofluorescein diacetate (DCFDA), was used to detect the generation of reactive oxygen species (ROS), as previously described (Cheng et al., 2011; Maffi et al., 2008). Cell death was measured by simultaneous labeling of the cells with propidium iodide. In most experiments, cells were grown to 70–80% confluence in 4-well Nunc cover glass chambers, and then treated with DMSO alone (control) or 100 nM epoxomicin for 24 h. In other groups cultures were treated with 500 nM RA for 18 h, or untreated cells were first treated with RA and then treated with epoxomicin. 1hM DCF-DA and 1hg/ml propidium iodide were added to all cultures 35 and 5 min prior to terminating the experiment, respectively. The cells were then washed Q3 once with PBS and immediately imaged on a FV1000 confocal microscope equipped with a 20 objective NA0.75 with an electronic zoom of 4.4, using 488 nm Argon and 543 nm HeNe laser settings. Laser exposure for image acquisition was attenuated to minimize photobleaching and phototoxicity to the cells. For reproducibility and comparison purposes, all experimental conditions as well as microscope settings were kept identical across experiments. To measure mitochondrial membrane potentials, cells were plated on glass chambers, treated as described above, and then labeled with a 20 nM final concentration of tetramethylrhodamine methylester (TMRM) for 15 min. Multiple random Z-stack images of each culture were captured using an Olympus FV1000 confocal microscope equipped with a 543 nm laser and a 40  NA 1.3 oil objective, with a 2.2 electronic zoom. For immunofluorescence experiments, SH-SY5Y cells were plated onto Nunc Lab-TekII 8-chamber slides (Fisher Scientific) and subjected to treatment regimens as mentioned above. At the end of treatment duration, cells were fixed in 4% paraformaldehyde for 10 min at room temperature, washed several times with PBS, permeabilized with 0.2% Triton-X-100/BSA buffer for 1 h and followed by several PBS washes. Cells were then probed overnight with the primary ubiquitin antibody (1:250), washed three times and incubated with a secondary antibody conjugated to AlexaFluor 488, Life Technologies (1:1000) for one hour. Cells were mounted using VectaShield mounting medium, and images were collected using an Olympus FV1000 confocal microscope with a 40 objective, NA 1.30 along with a 3.4 electronic zoom, using standard inbuilt 488 nm Argon laser settings (Ex. 488 nm/Em. 520 nm) along with Differential Interference Contrast (DIC) images. Laser exposure for image acquisition was attenuated to minimize photobleaching and also set using appropriate iso-controls. Moreover, for comparison purposes, microscope settings were kept identical across each treatment group.

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2.7. Data analysis Data were analyzed using one-way analysis of variance and Tukey’s comparison test, using either the Instat biostatistic program (GraphPad software) or Sigma Plot. Data are presented as the mean ± SEM of at least three independent experiments. In the figures, asterisks indicate the degree of significance for different treated cell cultures when compared with to treatment controls or as mentioned (⁄P < 0.05 and ⁄⁄P < 0.01, respectively).

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3.1. Retinoic acid pretreatment protects against proteasome inhibition-induced cell death We have shown previously that exposure of SH-SY5Y cells for 24 h to varying concentration of epoxomicin (10–200 nM) results

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in a dose-dependent and significant loss in cell viability (Cheng et al., 2011). Measurable ubiquitination of proteins accompanied by diminished proteasome activity was observed only at epoxomicin treatment concentrations of 100 nM or higher; thus, in this study, we used a concentration of 100 nM epoximicin. Fig. 1A shows that under these conditions of epoxomicin treatment, cell viability was reduced to 55%, while treatment with 500 nM RA for 18 h prior to epoxomicin exposure reduces this viability loss to 86%, with RA treatment alone having no significant effect on viability. Fig. 4D shows photomicrographs of SH-SY5Y cells treated as described above. In addition to the pyknosis and membrane blebbing evident in the treated cells (Fig. 4D), nuclear condensation was observed when the cells were stained with Hoechst 33342, a fluorophore that specifically binds to nuclear DNA (data not shown). To confirm and quantitate PCD in these cultures, immunoblots were prepared from the control and treated cells, and these were probed to assess expression levels of two cell death markers, poly-(ADP-ribose) polymerase (PARP) and cleaved caspase-3. Fig. 1B shows that treatment of the cells with 100 nM of epoxomicin for 24 h causes cleavage and activation of caspase-3, which subsequently leads to cleavage of the 89 kDa (PARP) fragment from its native 116 kDa complex, rendering it inactive. This result is consistent with similar findings reported by us and others (Cheng et al., 2011; Feuillette et al., 2005). However, when the cells were pretreated with 500 nM RA for 18 h prior to their 24 h epoxomicin exposure, the protein expression of PARP cleavage and activation of caspase-3 was significantly decreased in relation to treatment with epoxomicin alone. The activation of cleaved caspase-3 although reduced is significant when compared to control thought not significant when compared to epoxomicin alone. When the cells were loaded with DCFDA (a ROS marker), images captured

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using confocal microscopy of cells exposed to epoxomicin alone show a marked increase in DCFDA fluorescence intensity as compared to untreated cells, while DCFDA-loaded cells treated with RA prior to epoxomicin, as described above, contain significantly lower ROS levels, as indicated by the diminished intracellular fluorophore accumulation (Fig. 2A). Similarly, epoxomicin treated cells died after treatment (as observed by the number of propidium iodide positively labeled cells), while cells pretreated with 500 nM RA prior to epoxomicin did not. To determine whether RA protects epoxomicin-treated cells by preventing mitochondrial dysfunction, SH-SY5Y cells were cultured in the presence or absence of RA for 18 h prior to epoxomicin exposure for 24 h, as described above, followed by preparation of lysates, electrophoresis and immunoblotting for cytochrome c levels. The immunoblot and densitometry data in Fig. 2C show that RA significantly reduces the release of cytochrome c from the mitochondria. Further, mitochondrial membrane integrity was also measured by labeling control or treated cells with the fluorophore TMRM (Fig. 2B). Confocal images from the vehicle untreated group showed the bright TMRM labeling characteristic of healthy reticular and punctuate mitochondira, whereas, mitochondrial fluorescence was appreciably diffuse and reduced reflecting loss of mitochondrial membrane potential, in cells treated with 100 nM epoxomicin. By contrast, more mitochondria appear intact and normal in cells pretreated with RA prior to epoxomicin (Fig. 2B). We next asked whether RA directly affects intracellular protein ubiquitination. For this, SH-SY5Y cells were treated with vehicle (control), 500 nM RA for 18 h, 100 nM epoxomicin alone for 24 h, or pretreatment with 500 nM RA followed by treatment with 100 nM epoxomicin for 24 h. Total protein lysates from cells of the different treatment groups were subjected to electrophoresis

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Fig. 1. RA protects SH-SY5Y cells against epoxomicin-induced apoptosis. (A) MTT assays were performed to compare the viability of SH-SY5Y cells subjected to four different treatments: DMSO control (Ctrl), 500 nM RA only (RA), 100 nM epoxomicin (EPX), or pretreatment with 500 nM RA for 18 h followed by 100 nM epoxomicin (RA + EPX). Results are expressed as means ± SEM of controls from four independent experiments. (B) Cell lysates from each of the four treatment groups were collected for Western blot analysis, and probed with antibodies for PCD markers (cleavage of the PARP protein from its 116 kDa native length to its 89 kDa fragment and cleaved caspase-3 17–19 kDa fragment). b-Actin served as the loading control. The right panel is a densitometry graph of cleaved PARP (89 kDa) and cleaved caspase-3 (17–19 kDa), as normalized to bactin expression. The level of significance is denoted as ⁄P < 0.05 and ⁄⁄P < 0.01.

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Fig. 2. RA reduces oxidative stress and cell death, and allows maintenance of mitochondrial integrity, in SH-SY5Y cells exposed to Epoxomicin. (A) Cells were cultured in the absence (DMSO control) or presence of epoxomicin, alone, or with RA by itself or RA followed by epoxomicin. Following incubation, the cells were loaded with the fluorescent redox dye DCF-DA and propidum iodide (PI), to identify cells containing elevated levels of reactive oxygen species (ROS). Images were captured using a confocal microscope. Cells having elevated levels of ROS are green (left panel), which denotes DCF-DA fluorescence in response to epoxomicin treatment, and cells that have died are red, identified by PI fluorescence (middle panel). Scale bar shown represents 10 lm. (B) To assess mitochondrial function, cells were treated as mentioned above in (A) and loaded with 20 nM TMRM. Confocal microscopy images were captured to record mitochondrial membrane potential (DWm) fluorescence intensity generated from each of the four treatment groups. Images shown are representative of three independent experiments. (C) Western blot analysis for cytochrome c expression (reflective of mitochondrial damage) was performed using cytosol lysates collected as mentioned in materials and methods. b-Actin served as the loading control. Right panel depicts densitometry graph of relative values of cytochrome c expression normalized to b-actin. The level of significance is denoted as ⁄P < 0.05.

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and immunoblots probed for ubiquitin protein expression. As noted by others (Cheng et al., 2011; Feuillette et al., 2005), and shown here, an increase in protein ubiquitination occurs if epoxomicin treatment is used alone. However, in this case prior pretreatment with RA does not alter the accumulation of ubiquitinated proteins (Fig. 3A). Ubiquitin accumulation in cells after epoxomicn exposure, both in the presence and absence of RA was also determined with immunofluorescence experiments performed under identical conditioned as mentioned above. Here too, prior treatment with RA did not diminish the intracellular accumulation of ubiquitin (Fig. 3B). Thus, suggesting that RA affords its protective effect on

cells via an indirect mechanism that bypasses the proteasomeubiquitination system. Immuno blotting experiments shown in Fig. 3C (top panel) using a known autophagy marker, LC3 show that despite ubiquitin accumulation, there is no increase in autophagy across different treatment groups and when compared to controls. In addition, we tested the expression of nuclear factor kappa B (NFkB) which can influence expression of both apoptotic and anti-apoptotic gene and subsequent induction of its phosphorylated form (Ghosh et al., 2007). Bottom panel of Fig. 3C shows a modest activation of NFkB caused by epoxomicin treatment for 24 h, although its

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phosphorylation (P-NFkB) is dramatically increased within the same duration. RA pretreatment followed by epoxomicin exposure does not alter activation of NFkB and increased NFkB phosphorlyation when compared to epoxomicin treatment alone. NFkB phosphorlylation generally occurs within a few minutes of injury, however short term exposure cells to epoxomicin and RA treatments had no effect (data not shown).

3.2. Retinoic acid increases cell survival via PI3/AKT pathway Both the PI3 kinase and MAPK (ERK) pathways participate in neuronal cell growth and survival (Xia et al., 1995), however, their effects vary in different cell types. To determine whether one or both pathways are involved in RA protection of epoxomicin-treated SH-SY5Y cells, we therefore assessed the effect of RA on signaling intermediates for these two cascades. To start, we set to determine whether AKT/protein kinase B phosphorylation as well as mitogen-activated protein kinases, ERK1/ERK2 phosphorylation is changed as a function of time following treatment of SH-SY5Y cells with RA (500 nM). The immunoblots in Fig. 4A show that, RA treatment evoked a potent upregulation in levels of phosphorylated AKT and of phosphorylated ERK1/2. Maximum AKT and ERK phosphorylation is observed around 18 h after RA administration and P-ERK1/2 levels are still significantly elevated even at 24 h of exposure. To further determine whether RA protection involves both signaling pathways, we selected specific inhibitors for each pathway, and treated the SH-SY5Y cells with these inhibitors to disable their respective target systems. The cells were treated for 30 min either with a PI3/AKT inhibitor, 20 lM LY294002, or a MAPK inhibitor, 10 lM PD098059, followed by addition of 500 nM RA for 18 h in the same media, due to the reversible nature of these inhibitors. At the end of RA treatment, cell lysates were prepared for Western blotting. The immunoblot in Fig. 4B shows that the PI3K inhibitor almost completely blocked AKT phosphorylation, but only partially decreased ERK phosphorylation induced by RA. On the other hand, 10 lM PD098059 inhibited only MAPK/ERK phosphorylation, and had little effect on AKT activation

induced by RA, when compared to control and RA treated cells, respectively (Fig. 4B). With this information, we next asked whether RA activation of either of these two signaling pathways by RA was involved in neuroprotection against epoxomicin-induced toxicity. As described in for 4B, SH-SY5Y cells were first pretreated for 30 min with a PI3/ AKT inhibitor, 20 lM LY294002, or a MAPK inhibitor, 10 lM PD098059, followed by RA exposure for 18 h, subsequently by epoxomicin (100 nM) for 24 h, and compared to their respective controls. The MTT assay results in Fig. 4C indicate that LY294002, but not PD098059 (an upstream activator of ERK), reduces RA-induced neuroprotection against epoxomicin-induced toxicity. Neither LY294002 nor PD098059 exposure alone for 24–42 h had any effect on cell viability when compared to vehicle treated controls (Fig. 4C right panel), but only LY294002 reduced the ability of RA to protect.

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3.3. Cell survival is partially protected with caspase-3 inhibiton Previous studies have demonstrated caspase-3 activation is associated with the pathogenesis of PD (Hartmann et al., 2000; Tatton et al., 2000). Since proteasome inhibition causes activation of caspase-3, PARP cleavage, and eventual cell death, we tested the extent to which caspase-3 activation is functionally involved in epoxomicin-mediated PCD. For these experiments, cell viability was assessed by MTT assay in SH-SY5Y cells pretreated with caspase-3 inhibitor (Z-DEVD-FMK) for 2 h followed by 100 nM epoxomicin exposure for 24 h, and compared it to vehicle controls. Results in Fig. 5A show that while epoximicin treatment alone caused (55%) loss in cell viability, these cells could be partially rescued (65%) by pre-treatment with the caspase-3 specific inhibitor. To confirm this neuroprotective effect, Western blotting experiments further demonstrated that caspase-3 inhibitor attenuate caspase-3 and PARP proteolytic cleavage induced by epoxomicin insult (Fig. 5B). Thus, these results indicate that proteasome inhibition associated programmed cell death is at best only partially Caspase-3-dependent.

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Q1 Please cite this article in press as: Cheng, B., et al. Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway. Neurochem. Int. (2012), http://dx.doi.org/10.1016/j.neuint.2012.10.014

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Fig. 3. RA treatment does not prevent accumulation of ubiquitinated proteins in epoxomicin-treated SH-S5Y cells. (A) Cells were treated as follows: DMSO (control), 500 nM RA, 100 nM epoxomicin, and pretreatment with 500 nM RA prior to 100 nM epoxomicin exposure. Lysates were collected and Western blot analysis performed to detect amounts of accumulated ubiquitinated proteins. b-Actin served as the loading control. Blot shown is a representative of three independent experiments. (B) Cells were treated as above, immuno-fluorescently labeled with Ubiquitin conjugated to Alexa 488 and images collected using a confocal microscope. Epoxomicin as well as RA plus epoxomicin treatment shows an increased accumulation of Ubiquitin expression (green) when compared to controls or RA treatment alone. The bottom panel depicts Ubiquitinated protein labeling merged with DIC images, confirming the presence of aggregates in the cytosolic region. (C) To determine whether ubiquitinated proteins were removed by autophagy, Western blotting experiments were performed using cells treated as mentioned in (A) and probed with LC3. As shown in the upper panel, LC3 (I & II) protein expression remains unchanged across any of the treatment groups when compared to controls. Moreover, bottom panel shows that epoxomicin treatment in the presence or absence of RA augments phosphorylation of NFkB and to a lesser extent expression of NFkB. b-Actin served as the loading control. All blots shown are a representative of three independent experiments.

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Under normal conditions, the ubiquitin–proteasome system is an extremely efficient intracellular machinery whose main purpose is to remove unwanted, senescent, abnormal or damaged proteins. In most cell types, activity of the ubiquitin–proteasome system

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declines with age, particularly in metabolically active cells, including neurons. It is now generally accepted that abnormal protein accumulation is central to the pathophysiology of AD and PD (Cook and Petrucelli, 2009; Keller and Markesbery, 2000; Lopez Salon et al., 2000; McNaught and Olanow, 2003). Recent reports have suggested the involvement of autophagy, either independently or in

Q1 Please cite this article in press as: Cheng, B., et al. Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway. Neurochem. Int. (2012), http://dx.doi.org/10.1016/j.neuint.2012.10.014

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Fig. 4. RA treatment of SH-SY5Y cells results in the appearance of early markers for two stress pathways: the PI3/Akt cascade, and ERK signal transduction (A) Levels of phosphorylated vs. unphosphorylated AKT and ERK1/2 protein expression was assessed by immunoblotting in SH-SY5Y cells treated with 500 nM RA for times indicated. Both p-AKT and p-ERK levels increase steadily, peak at 18 h and remain high even after 24 h of RA treatment. Right panel is an average densitometry plot of three independent experiments. (B) SH-SY5Y cells were treated as follows: vehicle controls, 500 nM RA, 20 lM LY294003 alone for 30 min prior to 500 nM RA, or pretreated with 10 lM PD098059 for 30 min, followed by 500 nM RA. Cell lysates were used for Western blot analysis to determine whether RA regulates phosphorylation of AKT. The right panel is a corresponding densitometry plot of normalized p-AKT/AKT ratio and p-ERK/ERK1–2 values from three independent experiments. The level of significance is denoted as ⁄ P < 0.05 and ⁄⁄P < 0.01, respectively. (C) Cells were treated as follows- vehicle DMSO (Cntrl), 500 nM RA only (RA), 100 nM epoxomicin (EPX), pretreatment with 500 nM RA followed by 100 nM epoxomicin exposure (R + E), prior treatment with 20 lM LY294003 for 30 min followed by 500 nM RA followed by 100 nM epoxomicin (L + R + E) and pretreatment with 10 lM PD098059 for 30 min plus 500 nM RA followed by 100 nM epoxomicin (P + R + E). Cell viability was determined by MTT assay and expressed as percentage means ± SEM of controls (treated with DMSO alone) and are averaged data for four independent experiments. Level of significance is denoted as ⁄⁄P < 0.01. (D) Morphological changes in SH-SY5Y cell cultures were compared after treatment as mentioned in (C): (D.a) control; (D.b) RA; (D.c) epoxomicin (EPX); (D.d) pretreatment with RA followed by epoxomicin exposure (RA + EPX); (D.e) prior treatment with 20 lM LY294003 for 30 min followed by RA followed epoxomicin (LY + RA + EPX); (D.f) pretreatment with 10 lM PD098059 for 30 min, then RA followed by epoxomicin (PD + RA + EPX).

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conjunction with apoptosis in PD (Cheung and Ip, 2009; GonzalezPolo et al., 2005; Yang et al., 2009a). To develop therapeutic strategies for these diseases, it will be important both to identify and characterize mechanisms that regulate the proteasome–ubiquitination system, and to find ways to prevent dysfunction and cell death where abnormal protein aggregation has occurred. To this end, we have shown here that RA pretreatment protects SH-SY5Y neuroblastoma cells from mitochondrial stress, oxidative stress and programmed cell death following their exposure to the proteasome inhibitor epoxomicin. (Fig. 1–3). RA is an active metabolite of Vitamin A. It acts through a family of nuclear transcription factors known as retinoic acid receptors (Marill et al., 2003), which regulate the transcription of specific target genes (Boukhtouche et al., 2006), and control the rate of protein synthesis (Carta et al., 2006). RA is also a neuronal mitogen, and several groups have shown that it protects neurons against both oxidative stress and apoptosis induced by various stimuli (Ahlemeyer and Krieglstein, 1998; Dheen et al., 2005; Hoehner and Prabhakaran, 2003; Jackson et al., 1991; Moreno-Manzano et al., 1999; Ronca et al., 1999). RA induced signaling pathways are shown to play an important role in repair of spinal cord (Boukhtouche

et al., 2006; Mey, 2006) and the survival of hippocampal neurons from oxygen-glucose deprived cell death (Shinozaki et al., 2007). More recently, others have shown that retinoic acid receptor-related orphan receptor a1 overexpression protects neurons against oxidative stress-induced apoptosis (Boukhtouche et al., 2006). As expected in light of these findings, retinoid-deficient adult rats show degeneration of motor neurons, abnormal accumulation of neurofilaments and astrocytosis, resulting in motor neuron disease (Corcoran et al., 2002; Corcoran et al., 2004). Other Vitamin A deficiency studies with rats have identified motor impairments and striatal cholinergic dysfunction (Carta et al., 2006). RA has been shown by others to protect healthy cells from PCD caused by environmental toxicants, and these effects have been shown to involve both PI3/AKT and MAPK/ERK signaling pathway-mediated suppression of cell death signals even in cancer cells (Encinas et al., 1999; Gomez-Santos et al., 2002; Halvorsen et al., 2002; Wei et al., 2002; Xia et al., 1995). For example, AKT activation intersects with apoptotic signaling pathways to promote cell survival (Datta et al., 1999). SH-SY5Y treated cells with RA begin neurite outgrowth in association with ERK-independent/PKC dependent signaling (Miloso et al., 2004). Here, we show that even

Q1 Please cite this article in press as: Cheng, B., et al. Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway. Neurochem. Int. (2012), http://dx.doi.org/10.1016/j.neuint.2012.10.014

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a low dose of RA treatment of these same cells causes phosphorylation of both AKT and ERK. However, LY294002 treatment, but not PD098059 treatment, reduced RA-mediated protection of the cells. Thus, PI3/AKT pathway activation participates in RA-evoked cell survival of proteasome inhibition-induced toxicity, even under conditions where ERK1/2 phosphorylation has occurred and activated the MAPK pathway. Our earlier work (Cheng et al., 2011), identified IGF-1 as having a potent protective effect for SH-SY5Y cells when added prior to their treatment with the proteasome inhibitor epoxomicin. Here, we extend and confirm that RA also protects these cells in a similar manner, i.e., when added prior to epoxomicin. RA is known to be a free radical scavenger, which may account for some of its cell-protective activity, however, it also activates two cell stress pathways: the PI3/AKT pathway, and the MAPK pathway. Using IGF-1 pretreatment, we discovered that the PI3/AKT pathway must be functional for protection to occur. In the present investigation, this pathway was identified as necessary for RA protection of epoxomicin-treated SH-SY5Y cells (summarized in a schematic representation in Fig. 6). Notably, in both studies all epoxomicin-treated cells, whether protected or not, contained large amounts of ubiquitinated proteins, which implies that the presence of ubiquitinated

proteins may not be toxic to proteasome-inhibited cells. It is well established that expression of NFkB may regulate both apoptotic and anti-apoptotic factors in cells and is a sensor of oxidative stress. In the present study, phosphorylation of NFkB does not occur within the first few minutes, but at a later stage and thus likely contributes in cell death process. Our results also highlight two additional points. One, that autophagy is not involved either in the cell death or rescue process. Activation of caspase-3 has been shown to be one of the key steps in the execution process of apoptotic cell death and its inhibition can prevent apoptotic cell death (Qiu et al., 2000). Second, although epoxomicin activates caspase-3 which likely led to PARP cleavage causing cell death. However and interestingly, the extent of protection observed by caspase-3 inhibition against epoxomicin-induced cytotoxicity was only partial, thus suggesting that RA shields proteasome inhibition-induced cell death via a different signaling pathway. In light of the involvement of the PI3/AKT pathway in IGF-1 protection (Cheng et al., 2011) and RA protection in cultured neuroblastoma cells, we propose that the cells are actually protected by responses that reduce or prevent toxicity of ubiquitinated but undigested proteins in epoxomicin-treated cells. One such protective cellular stress response is the upregulation of the protein p62,

Q1 Please cite this article in press as: Cheng, B., et al. Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway. Neurochem. Int. (2012), http://dx.doi.org/10.1016/j.neuint.2012.10.014

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Fig. 5. Cell death induced by proteasome inhibition is only partially regulated by Caspase-3 expression. (A) SH-SY5Y cells were treated either with vehicle (DMSO control), or 100 nM epoxomicin, or pretreated with 50 lM caspase-3 specific inhibitor (Z-VAD) for 2 h followed by 100 nM epoxomicin exposure. Cell viability was measured by MTT assay and results expressed as percentages of mean ± SEM of controls and are from three independent experiments. The level of significance is denoted as ⁄P < 0.05, ⁄⁄P < 0.01, respectively. (B) SH-SY5Y cells were treated as described above in (A), and cell lysates collected for Western blot analysis. The immunoblots probed for ubiquitin, cleaved PARP, and cleaved caspase-3 protein expression. b-Actin served as the internal loading control. The immunoblot shown is representative of three independent experiments.

Fig. 6. Schematic diagram showing the involvement of PI3/AKT and MAPK pathways in proteasome inhibition-induced cell death.

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which binds to aggregated ubiquitinated proteins in the cytoplasm and forms a scaffold, or aggresome that targets them to lysosomes for degradation, thereby protecting the cells from the mitochondrial damage and oxidative stress-induced apoptosis that proteinopathies typically cause (Zatloukal et al., 2002). In most cell types where p62-related aggresome activity has been studied, its protective effect has been shown to require an intact and functioning PI3/ AKT pathway (Kim et al., 2008). With respect to our studies, it will be important next to determine whether these proteins have gathered into aggresomes in IGF-1 and RA-protected cells, and also determine whether these aggresomes, like those from other p62protected cells, contain PI3 kinase and p62 (Kim et al., 2008; Ramesh Babu et al., 2008; Sanchez-Margalet et al., 1995). We conclude that the protein aggregates by themselves may not be lethal

for the cells, and that their accumulation is unlikely to cause cell death provided a functioning PI3/AKT pathway has been activated and positioned to prevent cell death that otherwise would occur via the activated MAPK pathway. In summary, this study shows for the first time that RA pretreatment can prevent apoptosis in SH-SY5Y cells that have been treated with epoxomicin, even though the protein aggregates that appear in epoxomicin cells are still present. Though RA does not prevent the accumulation of ubiquitinated proteins, it affords cell survival by activating the PI3/AKT signaling pathway. Our findings strongly imply that RA administration could have similar protective effects for living neurons in the mammalian brain that are affected by agents or conditions that cause neurodegenerative disease via proteasome inhibition.

Q1 Please cite this article in press as: Cheng, B., et al. Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway. Neurochem. Int. (2012), http://dx.doi.org/10.1016/j.neuint.2012.10.014

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Acknowledgments

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This work was supported in part by funds from the E-RAHC to S.K.M., and research Grants AG08538 and NS 42080 to J.L.R. All fluorescent microscopy experiment images were generated in the Optical Imaging Core Facility at Edinburg – RAHC, Medical Research Division.

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References

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Ahlemeyer, B., Bauerbach, E., Plath, M., Steuber, M., Heers, C., Tegtmeier, F., Krieglstein, J., 2001. Retinoic acid reduces apoptosis and oxidative stress by preservation of SOD protein level. Free Radic. Biol. Med. 30 (10), 1067–1077. Ahlemeyer, B., Krieglstein, J., 1998. Retinoic acid reduces staurosporine-induced apoptotic damage in chick embryonic neurons by suppressing reactive oxygen species production. Neurosci. Lett. 246 (2), 93–96. Bain, G., Kitchens, D., Yao, M., Huettner, J.E., Gottlieb, D.I., 1995. Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168 (2), 342–357. Belal, C., Ameli, N.J., El Kommos, A., Bezalel, S., Al’khafaji, A.M., Mughal, M.R., Mattson, M.P., Kyriazis, G.A., Tyrberg, B., Chan, S.L., 2012. The homocysteineinducible endoplasmic reticulum (ER) stress protein Herp counteracts mutant alpha-synuclein-induced ER stress via the homeostatic regulation of ERresident calcium release channel proteins. Hum. Mol. Genet. 21 (5), 963– 977. Bianchi, P., Seguelas, M.H., Parini, A., Cambon, C., 2003. Activation of pro-apoptotic cascade by dopamine in renal epithelial cells is fully dependent on hydrogen peroxide generation by monoamine oxidases. J. Am. Soc. Nephrol. 14 (4), 855– 862. Boukhtouche, F., Vodjdani, G., Jarvis, C.I., Bakouche, J., Staels, B., Mallet, J., Mariani, J., Lemaigre-Dubreuil, Y., Brugg, B., 2006. Human retinoic acid receptor-related orphan receptor alpha1 overexpression protects neurones against oxidative stress-induced apoptosis. J. Neurochem. 96 (6), 1778–1789. Canon, E., Cosgaya, J.M., Scsucova, S., Aranda, A., 2004. Rapid effects of retinoic acid on CREB and ERK phosphorylation in neuronal cells. Mol. Biol. Cell 15 (12), 5583–5592. Canu, N., Barbato, C., Ciotti, M.T., Serafino, A., Dus, L., Calissano, P., 2000. Proteasome involvement and accumulation of ubiquitinated proteins in cerebellar granule neurons undergoing apoptosis. J. Neurosci. 20 (2), 589–599. Carta, M., Stancampiano, R., Tronci, E., Collu, M., Usiello, A., Morelli, M., Fadda, F., 2006. Vitamin A deficiency induces motor impairments and striatal cholinergic dysfunction in rats. Neuroscience 139 (4), 1163–1172. Cheng, B., Maffi, S.K., Martinez, A.A., Acosta, Y.P., Morales, L.D., Roberts, J.L., 2011. Insulin-like growth factor-I mediates neuroprotection in proteasome inhibition-induced cytotoxicity in SH-SY5Y cells. Mol. Cell Neurosci. 47 (3), 181–190. Cheung, Z.H., Ip, N.Y., 2009. The emerging role of autophagy in Parkinson’s disease. Mol. Brain 2, 29. Chinta, S.J., Rane, A., Poksay, K.S., Bredesen, D.E., Andersen, J.K., Rao, R.V., 2008. Coupling endoplasmic reticulum stress to the cell death program in dopaminergic cells: effect of paraquat. Neuromolecular Med. 10 (4), 333–342. Cook, C., Petrucelli, L., 2009. A critical evaluation of the ubiquitin-proteasome system in Parkinson’s disease. Biochim. Biophys. Acta 1792 (7), 664–675. Corcoran, J., Maden, M., 1999. Nerve growth factor acts via retinoic acid synthesis to stimulate neurite outgrowth. Nat. Neurosci. 2 (4), 307–308. Corcoran, J., So, P.L., Maden, M., 2002. Absence of retinoids can induce motoneuron disease in the adult rat and a retinoid defect is present in motoneuron disease patients. J. Cell Sci. 115 (Pt 24), 4735–4741. Corcoran, J.P., So, P.L., Maden, M., 2004. Disruption of the retinoid signalling pathway causes a deposition of amyloid beta in the adult rat brain. Eur. J. Neurosci. 20 (4), 896–902. Das, N.P., 1989. Effects of vitamin A and its analogs on nonenzymatic lipid peroxidation in rat brain mitochondria. J. Neurochem. 52 (2), 585–588. Datta, S.R., Brunet, A., Greenberg, M.E., 1999. Cellular survival: a play in three Akts. Genes Dev. 13 (22), 2905–2927. De Luca, L.M., 1991. Retinoids and their receptors in differentiation, embryogenesis, and neoplasia. FASEB J. 5 (14), 2924–2933. Dheen, S.T., Jun, Y., Yan, Z., Tay, S.S., Ling, E.A., 2005. Retinoic acid inhibits expression of TNF-alpha and iNOS in activated rat microglia. Glia 50 (1), 21–31. Ding, Q., Keller, J.N., 2001. Proteasome inhibition in oxidative stress neurotoxicity: implications for heat shock proteins. J. Neurochem. 77 (4), 1010–1017. Drexler, H.C., 1997. Activation of the cell death program by inhibition of proteasome function. Proc. Natl. Acad. Sci. USA 94 (3), 855–860. Drexler, H.C., Risau, W., Konerding, M.A., 2000. Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells. Faseb J. 14 (1), 65–77. Durston, A.J., Timmermans, J.P., Hage, W.J., Hendriks, H.F., de Vries, N.J., Heideveld, M., Nieuwkoop, P.D., 1989. Retinoic acid causes an anteroposterior transformation in the developing central nervous system. Nature 340 (6229), 140–144. Encinas, M., Iglesias, M., Llecha, N., Comella, J.X., 1999. Extracellular-regulated kinases and phosphatidylinositol 3-kinase are involved in brain-derived neurotrophic factor-mediated survival and neuritogenesis of the neuroblastoma cell line SH-SY5Y. J. Neurochem. 73 (4), 1409–1421.

604 605 606

11

Etchamendy, N., Enderlin, V., Marighetto, A., Vouimba, R.M., Pallet, V., Jaffard, R., Higueret, P., 2001. Alleviation of a selective age-related relational memory deficit in mice by pharmacologically induced normalization of brain retinoid signaling. J. Neurosci. 21 (16), 6423–6429. Feuillette, S., Blard, O., Lecourtois, M., Frebourg, T., Campion, D., Dumanchin, C., 2005. Tau is not normally degraded by the proteasome. J. Neurosci. Res. 80 (3), 400–405. Gerlach, M., Riederer, P., 1996. Animal models of Parkinson’s disease: an empirical comparison with the phenomenology of the disease in man. J. Neural Transm. 103 (8–9), 987–1041. Ghosh, A., Roy, A., Liu, X., Kordower, J.H., Mufson, E.J., Hartley, D.M., Ghosh, S., Mosley, R.L., Gendelman, H.E., Pahan, K., 2007. Selective inhibition of NF-kappaB activation prevents dopaminergic neuronal loss in a mouse model of Parkinson’s disease. Proc. Natl. Acad. Sci. USA 104 (47), 18754–18759. Gomez-Santos, C., Ferrer, I., Reiriz, J., Vinals, F., Barrachina, M., Ambrosio, S., 2002. MPP+ increases alpha-synuclein expression and ERK/MAP-kinase phosphorylation in human neuroblastoma SH-SY5Y cells. Brain Res. 935 (1– 2), 32–39. Gonzalez-Polo, R.A., Boya, P., Pauleau, A.L., Jalil, A., Larochette, N., Souquere, S., Eskelinen, E.L., Pierron, G., Saftig, P., Kroemer, G., 2005. The apoptosis/ autophagy paradox: autophagic vacuolization before apoptotic death. J. Cell Sci. 118 (Pt 14), 3091–3102. Grimm, L.M., Goldberg, A.L., Poirier, G.G., Schwartz, L.M., Osborne, B.A., 1996. Proteasomes play an essential role in thymocyte apoptosis. Embo J. 15 (15), 3835–3844. Halvorsen, E.M., Dennis, J., Keeney, P., Sturgill, T.W., Tuttle, J.B., Bennett Jr., J.B., 2002. Methylpyridinium (MPP(+))- and nerve growth factor-induced changes in proand anti-apoptotic signaling pathways in SH-SY5Y neuroblastoma cells. Brain Res. 952 (1), 98–110. Hartmann, A., Hunot, S., Michel, P.P., Muriel, M.P., Vyas, S., Faucheux, B.A., MouattPrigent, A., Turmel, H., Srinivasan, A., Ruberg, M., Evan, G.I., Agid, Y., Hirsch, E.C., 2000. Caspase-3: a vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. Proc. Natl. Acad. Sci. USA 97 (6), 2875–2880. Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407 (6805), 770– 776. Hoehner, J.C., Prabhakaran, K., 2003. Induced differentiation affords neuroblastoma cells protection from hypoxic injury. J. Pediatr. Surg. 38 (7), 1069–1074. Huang, J.Y., Chuang, J.I., 2010. Fibroblast growth factor 9 upregulates heme oxygenase-1 and gamma-glutamylcysteine synthetase expression to protect neurons from 1-methyl-4-phenylpyridinium toxicity. Free Radic. Biol. Med. 49 (6), 1099–1108. Hunter, K., Maden, M., Summerbell, D., Eriksson, U., Holder, N., 1991. Retinoic acid stimulates neurite outgrowth in the amphibian spinal cord. Proc. Natl. Acad. Sci. USA 88 (9), 3666–3670. Jackson, G.R., Morgan, B.C., Werrbach-Perez, K., Perez-Polo, J.R., 1991. Antioxidant effect of retinoic acid on PC12 rat pheochromocytoma. Int. J. Dev. Neurosci. 9 (2), 161–170. Keller, J.N., Markesbery, W.R., 2000. Proteasome inhibition results in increased polyADP-ribosylation: implications for neuron death. J. Neurosci. Res. 61 (4), 436– 442. Kikuchi, S., Shinpo, K., Tsuji, S., Takeuchi, M., Yamagishi, S., Makita, Z., Niino, M., Yabe, I., Tashiro, K., 2003. Effect of proteasome inhibitor on cultured mesencephalic dopaminergic neurons. Brain Res. 964 (2), 228–236. Kim, P.K., Hailey, D.W., Mullen, R.T., Lippincott-Schwartz, J., 2008. Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes. Proc. Natl. Acad. Sci. USA 105 (52), 20567–20574. Kobori, N., Waymire, J.C., Haycock, J.W., Clifton, G.L., Dash, P.K., 2004. Enhancement of tyrosine hydroxylase phosphorylation and activity by glial cell line-derived neurotrophic factor. J. Biol. Chem. 279 (3), 2182–2191. Koch, A., Lehmann-Horn, K., Dachsel, J.C., Gasser, T., Kahle, P.J., Lucking, C.B., 2009. Proteasomal inhibition reduces parkin mRNA in PC12 and SH-SY5Y cells. Parkinsonism Relat. Disord. 15 (3), 220–225. Konta, T., Xu, Q., Furusu, A., Nakayama, K., Kitamura, M., 2001. Selective roles of retinoic acid receptor and retinoid x receptor in the suppression of apoptosis by all-trans-retinoic acid. J. Biol. Chem. 276 (16), 12697–12701. Kordower, J.H., Kanaan, N.M., Chu, Y., Suresh Babu, R., Stansell 3rd, J., Terpstra, B.T., Sortwell, C.E., Steece-Collier, K., Collier, T.J., 2006. Failure of proteasome inhibitor administration to provide a model of Parkinson’s disease in rats and monkeys. Ann. Neurol. 60 (2), 264–268. Lee, D.H., Kim, C.S., Lee, Y.J., 2011. Astaxanthin protects against MPTP/MPP+induced mitochondrial dysfunction and ROS production in vivo and in vitro. Food Chem. Toxicol. 49 (1), 271–280. Lee, H.P., Casadesus, G., Zhu, X., Lee, H.G., Perry, G., Smith, M.A., Gustaw-Rothenberg, K., Lerner, A., 2009. All-trans retinoic acid as a novel therapeutic strategy for Alzheimer’s disease. Expert Rev. Neurother. 9 (11), 1615–1621. Lopes, U.G., Erhardt, P., Yao, R., Cooper, G.M., 1997. P53-Dependent induction of apoptosis by proteasome inhibitors. J. Biol. Chem. 272 (20), 12893– 12896. Lopez-Carballo, G., Moreno, L., Masia, S., Perez, P., Barettino, D., 2002. Activation of the phosphatidylinositol 3-kinase/Akt signaling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J. Biol. Chem. 277 (28), 25297–25304. Lopez Salon, M., Morelli, L., Castano, E.M., Soto, E.F., Pasquini, J.M., 2000. Defective ubiquitination of cerebral proteins in Alzheimer’s disease. J. Neurosci. Res. 62 (2), 302–310.

Q1 Please cite this article in press as: Cheng, B., et al. Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway. Neurochem. Int. (2012), http://dx.doi.org/10.1016/j.neuint.2012.10.014

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Love, J.M., Gudas, L.J., 1994. Vitamin A, differentiation and cancer. Curr. Opin. Cell Biol. 6 (6), 825–831. Lu, J., Park, C.S., Lee, S.K., Shin, D.W., Kang, J.H., 2006. Leptin inhibits 1-methyl-4phenylpyridinium-induced cell death in SH-SY5Y cells. Neurosci. Lett. 407 (3), 240–243. Lu, S., Hoestje, S.M., Choo, E., Epner, D.E., 2003. Induction of caspase-dependent and -independent apoptosis in response to methionine restriction. Int. J. Oncol. 22 (2), 415–420. Maden, M., 2001. Role and distribution of retinoic acid during CNS development. Int. Rev. Cytol. 209, 1–77. Maffi, S.K., Rathinam, M.L., Cherian, P.P., Pate, W., Hamby-Mason, R., Schenker, S., Henderson, G.I., 2008. Glutathione content as a potential mediator of the vulnerability of cultured fetal cortical neurons to ethanol-induced apoptosis. J. Neurosci. Res. 86 (5), 1064–1076. Mann, A., Tyndale, R.F., 2010. Cytochrome P450 2D6 enzyme neuroprotects against 1-methyl-4-phenylpyridinium toxicity in SH-SY5Y neuronal cells. Eur. J. Neurosci. 31 (7), 1185–1193. Marill, J., Idres, N., Capron, C.C., Nguyen, E., Chabot, G.G., 2003. Retinoic acid metabolism and mechanism of action: a review. Curr. Drug Metab. 4 (1), 1–10. McNaught, K.S., Bjorklund, L.M., Belizaire, R., Isacson, O., Jenner, P., Olanow, C.W., 2002. Proteasome inhibition causes nigral degeneration with inclusion bodies in rats. Neuroreport 13 (11), 1437–1441. McNaught, K.S., Jenner, P., 2001. Proteasomal function is impaired in substantia nigra in Parkinson’s disease. Neurosci. Lett. 297 (3), 191–194. McNaught, K.S., Olanow, C.W., 2003. Proteolytic stress: a unifying concept for the etiopathogenesis of Parkinson’s disease. Ann Neurol 53 (3), S73–S84, Discussion S84–S76. McNaught, K.S., Perl, D.P., Brownell, A.L., Olanow, C.W., 2004. Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson’s disease. Ann. Neurol. 56 (1), 149–162. Meng, L., Mohan, R., Kwok, B.H., Elofsson, M., Sin, N., Crews, C.M., 1999. Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc. Natl. Acad. Sci. USA 96 (18), 10403–10408. Mey, J., 2006. New therapeutic target for CNS injury? The role of retinoic acid signaling after nerve lesions. J. Neurobiol. 66 (7), 757–779. Miloso, M., Villa, D., Crimi, M., Galbiati, S., Donzelli, E., Nicolini, G., Tredici, G., 2004. Retinoic acid-induced neuritogenesis of human neuroblastoma SH-SY5Y cells is ERK independent and PKC dependent. J. Neurosci. Res. 75 (2), 241–252. Moore, D.J., West, A.B., Dawson, V.L., Dawson, T.M., 2005. Molecular pathophysiology of Parkinson’s disease. Annu. Rev. Neurosci. 28, 57–87. Moreno-Manzano, V., Ishikawa, Y., Lucio-Cazana, J., Kitamura, M., 1999. Suppression of apoptosis by all-trans-retinoic acid. Dual intervention in the c-Jun n-terminal kinase-AP-1 pathway. J. Biol. Chem. 274 (29), 20251–20258. Nagley, P., Higgins, G.C., Atkin, J.D., Beart, P.M., 2010. Multifaceted deaths orchestrated by mitochondria in neurones. Biochim. Biophys. Acta 1802 (1), 167–185. Okada, K., Wangpoengtrakul, C., Osawa, T., Toyokuni, S., Tanaka, K., Uchida, K., 1999. 4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress. Identification of proteasomes as target molecules. J. Biol. Chem. 274 (34), 23787–23793. Pan, J., Kao, Y.L., Joshi, S., Jeetendran, S., Dipette, D., Singh, U.S., 2005. Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogenactivated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells. J. Neurochem. 93 (3), 571–583. Pennypacker, K.R., Kuhn, D.M., Billingsley, M.L., 1989. Changes in expression of tyrosine hydroxylase immunoreactivity in human SMS-KCNR neuroblastoma following retinoic acid or phorbol ester-induced differentiation. Brain Res. Mol. Brain Res. 5 (4), 251–258. Princiotta, M.F., Schubert, U., Chen, W., Bennink, J.R., Myung, J., Crews, C.M., Yewdell, J.W., 2001. Cells adapted to the proteasome inhibitor 4-hydroxy- 5-iodo-3-

nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically active proteasomes for continued survival. Proc. Natl. Acad. Sci. USA 98 (2), 513–518. Qiu, J.H., Asai, A., Chi, S., Saito, N., Hamada, H., Kirino, T., 2000. Proteasome inhibitors induce cytochrome c-caspase-3-like protease-mediated apoptosis in cultured cortical neurons. J. Neurosci. 20 (1), 259–265. Ramesh Babu, J., Lamar Seibenhener, M., Peng, J., Strom, A.L., Kemppainen, R., Cox, N., Zhu, H., Wooten, M.C., Diaz-Meco, M.T., Moscat, J., Wooten, M.W., 2008. Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration. J. Neurochem. 106 (1), 107–120. Rebhan, M., Vacun, G., Bayreuther, K., Rosner, H., 1994. Altered ganglioside expression by SH-SY5Y cells upon retinoic acid-induced neuronal differentiation. Neuroreport 5 (8), 941–944. Reinheckel, T., Sitte, N., Ullrich, O., Kuckelkorn, U., Davies, K.J., Grune, T., 1998. Comparative resistance of the 20S and 26S proteasome to oxidative stress. Biochem. J. 335 (Pt 3), 637–642. Ronca, F., Yee, K.S., Yu, V.C., 1999. Retinoic acid confers resistance to p53-dependent apoptosis in SH-SY5Y neuroblastoma cells by modulating nuclear import of p53. J. Biol. Chem. 274 (25), 18128–18134. Sadoul, R., Fernandez, P.A., Quiquerez, A.L., Martinou, I., Maki, M., Schroter, M., Becherer, J.D., Irmler, M., Tschopp, J., Martinou, J.C., 1996. Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons. Embo J. 15 (15), 3845–3852. Sakamoto, K., Hiraiwa, M., Saito, M., Nakahara, T., Sato, Y., Nagao, T., Ishii, K., 2010. Protective effect of all-trans retinoic acid on NMDA-induced neuronal cell death in rat retina. Eur. J. Pharmacol. 635 (1–3), 56–61. Sanchez-Margalet, V., Zoratti, R., Sung, C.K., 1995. Insulin-like growth factor-1 stimulation of cells induces formation of complexes containing phosphatidylinositol-3-kinase, guanosine triphosphatase-activating protein (GAP), and p62 GAP-associated protein. Endocrinology 136 (1), 316–321. Schneider, L., Giordano, S., Zelickson, B.R., Johnson, M., Benavides, G., Ouyang, X., Fineberg, N., Darley-Usmar, V.M., Zhang, J., 2011. Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress. Free Radic. Biol. Med. 51 (11), 2007–2017. Shinozaki, Y., Sato, Y., Koizumi, S., Ohno, Y., Nagao, T., Inoue, K., 2007. Retinoic acids acting through retinoid receptors protect hippocampal neurons from oxygenglucose deprivation-mediated cell death by inhibition of c-jun-N-terminal kinase and p38 mitogen-activated protein kinase. Neuroscience 147 (1), 153–163. Tatton, W.G., Chalmers-Redman, R.M., Elstner, M., Leesch, W., Jagodzinski, F.B., Stupak, D.P., Sugrue, M.M., Tatton, N.A., 2000. Glyceraldehyde-3-phosphate dehydrogenase in neurodegeneration and apoptosis signaling. J. Neural Transm. 60, 77–100. Wei, W., Wang, X., Kusiak, J.W., 2002. Signaling events in amyloid beta-peptideinduced neuronal death and insulin-like growth factor I protection. J. Biol. Chem. 277 (20), 17649–17656. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J., Greenberg, M.E., 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270 (5240), 1326–1331. Yamamoto, N., Sawada, H., Izumi, Y., Kume, T., Katsuki, H., Shimohama, S., Akaike, A., 2007. Proteasome inhibition induces glutathione synthesis and protects cells from oxidative stress: relevance to Parkinson disease. J. Biol. Chem. 282 (7), 4364–4372. Yang, F., Yang, Y.P., Mao, C.J., Cao, B.Y., Cai, Z.L., Shi, J.J., Huang, J.Z., Zhang, P., Liu, C.F., 2009a. Role of autophagy and proteasome degradation pathways in apoptosis of PC12 cells overexpressing human alpha-synuclein. Neurosci. Lett. 454 (3), 203– 208. Yang, Y.X., Wood, N.W., Latchman, D.S., 2009b. Molecular basis of Parkinson’s disease. Neuroreport 20 (2), 150–156. Zatloukal, K., Stumptner, C., Fuchsbichler, A., Heid, H., Schnoelzer, M., Kenner, L., Kleinert, R., Prinz, M., Aguzzi, A., Denk, H., 2002. P62 Is a common component of cytoplasmic inclusions in protein aggregation diseases. Am. J. Pathol. 160 (1), 255–263.

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Q1 Please cite this article in press as: Cheng, B., et al. Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway. Neurochem. Int. (2012), http://dx.doi.org/10.1016/j.neuint.2012.10.014