Resveratrol Protects DAergic PC12 Cells from High Glucose-Induced Oxidative Stress and Apoptosis: Effect on p53 and GRP75 Localization
Resveratrol (RESV), a polyphenolic natural compound, has long been acknowledged to have cardioprotective and antiinflammatory actions. Evidence suggests that RESV has antioxidant properties that reduce the formation of reactive oxygen species leading to oxidative stress and apoptotic death of dopaminergic (DAergic) neurons in Parkinson’s disease (PD). Recent literature has recognized hyperglycemia as a cause of oxidative stress reported to be harmful for the nervous system. In this context, our study aimed (a) to evaluate the effect of RESV against high glucose (HG)-induced oxidative stress in DAergic neurons, (b) to study the antiapoptotic properties of RESV in HG condition, and c) to analyze RESV’s ability to modulate p53 and GRP75, a p53 inactivator found to be under expressed in postmortem PD brains. Our results suggest that RESV protects DAergic neurons against HG-induced oxidative stress by diminishing cellular levels of superoxide anion. Moreover, RESV significantly reduces HG-induced apoptosis in DAergic cells by modulating DNA fragmentation and the expression of several genes implicated in the apoptotic cascade, such as Bax, Bcl-2, cleaved caspase-3, and cleaved PARP-1. RESV also prevents the pro-apoptotic increase of p53 in the nucleus induced by HG. Such data strengthens the correlation between hyperglycemia and neurodegeneration, while providing new insight on the high occurrence of PD in patients with diabetes. This study enlightens potent neuroprotective roles for RESV that should be considered as a nutritional recommendation for preventive and/or complementary therapies in controlling neurodegenerative complications in diabetes.
KeywordsResveratrol High glucose Apoptosis Oxidative stress Neuroprotection GRP75
Glucose is the essential energy substrate of the central nervous system and large amounts of it are required to fill the high energetic needs of neurons. Unlike muscle cells or adipocytes that depend on insulin, glucose uptake in neurons depends mainly on its extracellular concentration (Tomlinson and Gardiner 2008). Persistent episodes of long-term glucose exposure may induce oxidative stress that results in cellular damage (Giaccari et al. 2009), such as neuropathic complications resulting from hyperglycemia in uncontrolled diabetes (Rajabally 2011). Accumulating evidence has enlightened the relationship between diabetes and neurodegenerative disorders, including Alzheimer’s disease (AD) (Vignini et al. 2013) and Parkinson’s disease (PD) (Jagota et al. 2012). Recent literature has reported an increased risk of developing PD in patients with type 2 diabetes mellitus (Hu et al. 2007; Sun et al. 2012).
PD is a neurodegenerative disorder characterized by the progressive loss of nigrostriatal dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). DAergic neurons in this region are selectively lost due to the high activity of monoamine oxidase and elevated levels of iron which both lead to increased generation of reactive oxygen species (ROS) (Pearce et al. 1997; Cui et al. 2012). At the cellular level, mechanisms of high glucose (HG)-induced toxicity are similarly sustained by oxidative stress in vitro (Bournival et al. 2012; Cao et al. 2012) as well as in vivo (Styskal et al. 2012). By increasing aerobic respiration, raised sugar metabolism promotes excessive formation of ROS which, jointly with insufficient antioxidant defences, may damage cells (Apel and Hirt 2004). Indeed, generation of mitochondrial superoxide is increased and is thought to be at the origin of several HG-induced complications (Brownlee 2001). Currently, it is well known that oxidative stress may lead to apoptosis (Circu and Aw 2010) and increased production of ROS in HG conditions may account for glucose neurotoxicity duly observed.
In addition, several genes are known to be implicated in the pathogenesis of PD, such as PINK1 and DJ-1. Glucose-regulated protein 75 (GRP75, also called mortalin/mtHSP70/mot-2), a member of the cytoprotective Hsp70 family of chaperons, interacts with both PINK1 (Jin et al. 2006, 2007; Li et al. 2005; Rakovic et al. 2011) and DJ-1 (Jin et al. 2005; Li et al. 2005). GRP75 is mainly localized within the mitochondria matrix of neurons where it accomplishes several functions such as mitochondrial import and oxidative stress management (Yaguchi et al. 2007). Overexpression of GRP75 leads to extended life span in nematodes and human cells. On the other hand, it serves as a major target for oxidation and it was shown to be involved in aging of nerve cells and in particular in the degeneration of DAergic neurons (Burbulla et al. 2010). In mitotic cells, GRP75 localized in the cytoplasm sequestrates and inactivates p53 preventing its nuclear translocation and apoptosis (Kaul et al. 2001, 2005; Wadhwa et al. 2002). Indeed, p53 is a tumor suppressor protein known to play an important role in evoking apoptosis when located in the nucleus by encouraging the transcription of several pro-apoptotic genes such as Bax (B cell lymphoma 2 [Bcl-2]-associated X protein, Macip et al. 2003). p53 activity is stabilized in response to oxidative stress through posttranslational modifications disrupting interactions with negative regulators (Neilson et al. 2008). It is also a recurrent target in PD given the involvement of oxidative stress in p53 activation (Nair 2006) and the evidence of DNA fragmentation and chromatin condensation in DAergic neurons of the SNpc in PD patients (Hartmann and Hirsch 2001; Tatton 2000).
Prevention of neuronal loss in PD has not yet been addressed by existing symptomatic treatments. Neuroprotection by dietary polyphenols may be an interesting avenue in current attempts to overcome oxidative stress induced by hyperglycemia. We have recently shown that quercetin and sesamin, antioxidant polyphenols, exert neuroprotective effects in neurons exposed to HG (Bournival et al. 2012). The stilbene resveratrol (RESV) is another polyphenol, primarily found in red wine, known for its potent cardioprotective, antiinflammatory, and anticarcinogenic actions (Rosa et al. 2012; Aluyen et al. 2012). Our group, as well as others, has highlighted its potential in defending neurons against oxidative assaults induced by a spectrum of treatments, including neurotoxins (Gélinas and Martinoli 2002; Blanchet et al. 2008; Bournival et al. 2009; Peritore et al. 2012; Wu et al. 2012) or cerebral ischemic injury (Morris et al. 2011; Simão et al. 2012). Abundant literature suggests that RESV plays a protective role in several neurodegenerative diseases including PD, AD, and Huntington’s disease (Albani et al. 2010; Hung et al. 2010) as well as against neuroinflammation (Foti Cuzzola et al. 2011).
Although the beneficial properties of RESV in neurodegenerative diseases are extensively depicted in the literature, its role in defending neurons against HG-induced damage has yet to be elucidated. The present study was designed to examine the neuroprotective effects of the polyphenol RESV in differentiated DAergic PC12 cells maintained in HG condition. NGF-differentiated PC12 cells are a reliable model for the investigation of oxidative stress and neuroprotection of DAergic neurons. They express tyrosine hydroxylase (TH), high affinity dopamine transporter, estrogen receptor-α and -β, neurofilaments, and secrete high levels of dopamine (Kadota et al. 1996; Neilson et al. 2012; Gélinas and Martinoli 2002). In this comprehensive investigation, we outline the roles of RESV in preventing neural parameters of cellular oxidative stress and apoptosis induced by HG exposure in a cellular DAergic system. Our results demonstrate that RESV can modulate the expression and localization of GPR75, and thus might mediate mitochondria pathways of cell stress.
Materials and Methods
Drugs and Chemicals
All reagents and chemicals were purchased from Sigma (St. Louis, MO) unless noted otherwise. Mouse anti-GRP75 (raised against amino acids 525–679 of GRP75 of human origin), rabbit anti-p53 (raised against full length p53 of human origin, for immunofluorescence), rabbit anti-Bcl-2 (raised against a peptide mapping at the N-terminus of Bcl-2 of human origin), rabbit anti-Bax (raised against a peptide mapping near the N-terminus of Bax of mouse origin), mouse anticleaved PARP-1 (poly [ADP-ribose] polymerase, raised against C-terminal purified thymus PARP-1 of calf origin), goat anti-HDAC1 (histone deacetylase 1, raised against amino acids 450 to C-terminus of human HDAC1), and mouse anti-GAPDH (glyceraldehyde 3-phosphatase dehydrogenase, raised against recombinant GAPDH of human origin) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-p53 (raised against amino acids surrounding Ser20 of human p53, for Western blotting) and rabbit anticleaved caspase-3 (raised against amino-terminal residues surrounding Asp175 in human caspase-3) antibodies were purchased from Cell Signaling (Boston, MA). Rabbit anti-VDAC (voltage-dependant anion channel, raised against amino acids 152–169 of VDAC of human origin), mouse anti-TH (raised against rat TH) primary antibodies, and anti-mouse and -rabbit horseradish peroxidise-conjugated secondary antibodies were purchased from Sigma. Anti-mouse Cy3 (cyanine 3)-conjugated secondary antibody was purchased from Medicorp (Montreal, QC, Canada). Goat anti-rabbit FITC (fluorescein isothiocyanate)-conjugated secondary antibody was purchased from Millipore (Temecula, CA).
Cell Culture and Treatments
PC12 cells, obtained from American Type Culture Collection (ATCC, Rockville, MD), were maintained in a humidified environment at 37 °C and 5 % CO2 atmosphere. Cells were grown in Roswell Park Memorial Institute medium 1640 (RPMI 1640) supplemented with 10 % (v/v) heat-inactivated horse serum, 5 % (v/v) heat-inactivated fetal bovine serum (FBS), and gentamicin (50 μg/mL). PC12 cell neuronal differentiation was evoked by administration of nerve growth factor-7S (NGF, 50 ng/mL) in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 1 % FBS for 7 days, as already described (Bournival et al. 2009, 2012). The DMEM containing 1.0 g/L of d-glucose (Sigma D5523) is further called control (CTRL) medium, whereas HG DMEM containing 4.5 g/L of d-glucose (Sigma D7777) is named HG medium. DAergic PC12 cells were incubated with CTRL or HG medium for 96 h, unless stated otherwise. We previously performed lactate dehydrogenase-based cytotoxicity assays to determine the appropriate time of treatment in order to study the apoptotic process in the remaining live cells (Bournival et al. 2012). For the last 24 h of treatment, DAergic PC12 cells were incubated with or without RESV (0.1 μM). RESV concentration was selected after dose response and kinetic studies (Bureau et al. 2008; Bournival et al. 2009). An osmotic control consisting of CTRL medium supplemented with 3.5 g/L of D-mannitol (MANN) was used to rule out a hypertonic effect of HG medium on PC12 cells. Charcoal-stripped serum was used in all experiments to ensure that media were free from steroids. For each experiment, initial seeding density was 30,000 cells/cm2.
Detection of Mitochondrial Superoxide Radical
DAergic PC12 cells were grown and treated on collagen-coated circular glass coverslips (Fisher Scientific, Ottawa, ON, Canada). Intracellular superoxide anion (•O2 −) production was measured with MitoSOX™ Red (Invitrogen, Burlington, ON, Canada), a fluorogenic dye used for the selective detection of superoxide in the mitochondria of live cells. After treating cells with CTRL or HG medium for 3 h with or without RESV, the cells were incubated with MitoSOX™ Red (5 mM) for 10 min at 37 °C. MitoSOX™ Red is rapidly and selectively targeted to the mitochondria. Once in the mitochondria, it is oxidized by superoxide and exhibits red fluorescence. Cells were washed with Hank’s balanced salt solution (HBSS, Invitrogen), and Hoechst 33342 counterstained all nuclei. Cells were fixed in 4 % paraformaldehyde for 6 min at 37 °C. Coverslips were mounted with Molecular Probes’ ProLong® Antifade Kit (Invitrogen). Images were acquired by a Leica SD AF confocal microscope, and analyzed with Leica Application Suite 3.1.3 software (Leica Microsystems, Concord, ON, Canada). To demonstrate MitoSOX™ Red selectivity, a positive control was performed using sodium diethyldithiocarbamate (DDC), a superoxide dismutase (SOD) inhibitor, in CTRL medium.
Immunofluorescence and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay
Apoptotic cells were also detected by both TUNEL (Roche Diagnostics, Laval, QC, Canada) assay and activated caspase-3 immunofluorescence. DAergic PC12 cells were grown and treated on collagen-coated circular glass coverslips. Cells were then fixed in 4 % paraformaldehyde for 15 min at 37 °C, washed with phosphate buffered saline (PBS), and further incubated in a blocking and permeabilizing solution (1 % bovine serum albumin [BSA], 0.18 % fish skin gelatin, 0.1 % Triton-X, and 0.02 % sodium azide) for 30 min at RT. Fixed cells were incubated with polyclonal anticleaved caspase-3 antibody 1:500 in PBS overnight. The slides were washed and treated with Cy3-conjugated secondary antibody diluted 1:500 in PBS for 4 h and then incubated with the TUNEL enzyme and fluorescent dUTP mixture for 1 h at 37 °C. Nuclei were counterstained 4′,6′-diamidino-2-phenylindole (DAPI). Coverslips were mounted with ProLong® Antifade Kit. Images were acquired by a Leica SD AF confocal microscope. DAergic PC12 cells were considered to be apoptotic when they were positive for cleaved caspase-3, and their nuclei were stained with TUNEL. The number of apoptotic DAergic PC12 cells among 300 randomly chosen neuronal was counted on 10 different optical fields from three slides per group, as already reported (Bournival et al. 2009, 2012), with Leica Application Suite 3.1.3 software. In each experiment 50 μM Z-DEVD-FMK (Bachem, Torrance, CA), a cell-permeable caspase-3 inhibitor, was used on DAergic PC12 cells in HG and HG RESV conditions as internal control for caspase-3 activation (Bournival et al. 2009, 2012).
Specific Apoptotic DNA Denaturation Analysis
Specific DNA denaturation in apoptotic cells was assessed with a single-stranded DNA (ssDNA) apoptosis ELISA kit (Chemicon International, Temecula, CA). This procedure is based on the selective denaturation of DNA by formamide in apoptotic cells, but not in necrotic cells (Frankfurt and Krishan 2001). After treatment with CTRL or HG medium with or without RESV, denatured DNA was detected with a monoclonal antibody highly specific to ssDNA and a peroxidase-labeled secondary antibody. The reaction was then stopped with a hydrochloric acid solution, and ssDNA fragmentation was quantified by measuring absorbance at 405 nm with a Multiscan Ascent microplate reader (Thermolab System, Franklin, MA). ssDNA was quantified with reference to CTRL conditions. Absorbance of positive (wells coated with ssDNA) and negative controls (wells treated with S1 nuclease) served as quality control for the ELISA.
DAergic PC12 cells were grown and treated in collagen-coated 6-well plates. Total proteins were extracted using a nuclear extraction kit (Active Motif, Carlsbad, CA). Briefly, cells were washed with a mixture of ice-cold PBS and phosphatase inhibitors, and then harvested in centrifuge tubes. Cell lysis was performed using the supplied buffer, and samples were centrifuged to obtain membrane-free supernatants containing total proteins.
Cytoplasmic-nuclear fractionation was achieved using the nuclear extraction kit. Briefly, cells were washed with a mixture of ice-cold PBS and phosphatase inhibitors, and then harvested in centrifuge tubes. Cytoplasmic membranes were ruptured by treatment with a hypotonic buffer and detergent. Samples were centrifuged to pellet the intact nuclei, and soluble material was preserved as the cytoplasmic fraction. Nuclei were then lysed and conserved in the provided lysis buffer.
Mitochondrial-cytoplasmic fractionation was achieved using a mitochondrial extraction kit (Active Motif, Carlsbad, CA). Cells were washed with a mixture of ice-cold PBS and phosphatase inhibitors, and then harvested in centrifuge tubes. Cells were incubated on ice with isotonic cytosol buffer for 15 min. Cell membranes were ruptured with a pestle homogenizer. Intact cells and nuclei were pelleted after two centrifugations and discarded. Supernatants containing cytoplasm and mitochondria were centrifuged twice to obtain a pellet of mitochondria. The resulting supernatant was preserved as the cytoplasmic fraction. Mitochondria were washed with cytosol buffer and lysed with detergent.
Electrophoresis and Western Blotting Analysis
Protein dosage was performed with a bicinchoninic acid-based sodium dodecyl sulfate (SDS)-compatible Protein Assay Kit (Pierce, Rockfort, IL) for each fraction of every sample. Equal amounts of protein were loaded onto 12 % SDS polyacrylamide gels. After electrophoretic separation, the gels were transferred to polyvinylidene difluoride membranes (0.22 μm pore size, BioRad, Hercules, CA). The blots were blocked for 1 h at room temperature (RT) in Blotto B (1 % nonfat powdered milk, 1 % BSA, 0.05 % Tween 20, 0.5 mg/mL sodium azide, in Tris buffered saline). Dilution of primary anti-GRP75, anti-p53, anti-Bax, anti-Bcl-2, anti-cleaved PARP-1, anti-GAPDH, anti-HDAC1, anti-VDAC, and anti-TH (1:200, 1:200, 1:50, 1:50, 1:1,000, 1:50, 1:50, 1:500, and 1:10,000, respectively) antibodies was prepared in Blotto B. The blots were then incubated with peroxidase-conjugated secondary antibody (1:10,000) in Blotto B for 2 h at RT and finally developed with an enhanced chemiluminescence substrate solution (Haan and Behrmann 2007).
DAergic PC12 cells were grown and treated on collagen-coated circular glass coverslips. Then, they were fixed in 4 % paraformaldehyde for 15 min at 37 °C, washed with PBS, and further incubated in a blocking and permeabilizing solution for 30 min at RT. Fixed cells were incubated with both rabbit anti-p53 antibody 1:100 and mouse anti-GRP75 1:100 in PBS overnight. The slides were washed with and subsequently treated with anti-rabbit Cy3-conjugated and anti-rabbit FITC-conjugated secondary antibodies both diluted 1:500 in PBS for 4 h. Nuclei were counterstained with DAPI. Coverslips were mounted with Molecular Probes’ ProLong® Antifade Kit. Images were acquired by a Leica SD AF confocal microscope. Colocalization was assessed for 100 randomly chosen DAergic PC12 cells on 6 different optical fields from three slides per group with Leica Application Suite 3.1.3 software.
Significant differences between groups were ascertained by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc analysis with the GraphPad Instat program, version 3.06 for Windows (San Diego, CA; www.graphpad.com). All data, analyzed at the 95 % confidence interval, are expressed as mean ± standard error of the mean (SEM) from at least 3 independent experiments. Asterisks indicate statistical differences between the treatment and CTRL condition (***p < 0.001, **p < 0.01, and *p < 0.05); plus signs show statistical differences between the treatment and HG condition (+++ p < 0.001, ++ p < 0.01, and + p < 0.05).
RESV Rescues HG Production of Superoxide
RESV Reduces HG-Induced Apoptosis
We then examined the effect of HG and RESV on later events of the apoptotic cascade leading to DNA fragmentation. Detection of cleaved caspase-3, the terminal effector caspase responsible for late apoptosis-mediated DNA fragmentation (Fan et al. 2005), was conducted by immunofluorescence alongside a TUNEL assay measuring DNA degradation (Fig. 2b, c). In the HG condition, immunofluorescence revealed the presence of cleaved caspase-3 positive cells (Fig. 2c, red signal), while TUNEL assay stained numerous nuclei undergoing DNA fragmentation (Fig. 2c, green signal). Total nuclei were stained with DAPI (Fig. 2c, blue signal). DAergic PC12 cells were considered to be in late apoptosis when they hosted both caspase-3 activation and DNA fragmentation events (Fig. 2c, cells pointed by white arrows). Treatment with RESV for 24 h clearly reduced the presence of apoptotic nuclei as implied by the lower number of DAergic PC12 cells exhibiting both green and red fluorescence. The number of apoptotic cells was also counted (Fig. 2b), as described in the Materials and Methods section. Administration of RESV decreased the number of apoptotic cells compared to the HG condition. MANN medium did not yield a significant rise in apoptotic cells compared to CTRL. To show that caspase-3 activation is a key step in the HG-induced apoptotic pathway, DAergic PC12 cells were pretreated with 50 μM Z-DEVD-FMK, a cell-permeable selective caspase-3 inhibitor, followed by treatment with HG with or without RESV (Fig. 2b, c).
RESV Modulates p53 and GRP75 Subcellular Localization and Colocalization
We previously reported that several natural polyphenols, including the stilbene RESV, exert powerful neuroprotective activity in DAergic PC12 cells against the oxidative burden triggered by the administration of the potent parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in vivo (Blanchet et al. 2008) or its active metabolite 1-methyl-4-phenylpyridinium (MPP+) in vitro (Gagné et al. 2003; Lahaie-Collins et al. 2008; Bournival et al. 2009). Since hyperglycemia has also been listed as a growing risk factor for PD (Hu et al. 2007; Jagota et al. 2012; Sun et al. 2012), we focused our study on the neuroprotective effect of RESV on HG-induced oxidative stress and apoptosis in DAergic PC12 cells with regard to GRP75 and p53 localization.
The Diabetes Control and Complications Trial (1993) together with the U.K. Prospective Diabetes Study (1998) have determined that hyperglycemia is the culprit to blame for tissue damage in type I and type II diabetes. Currently, we know that overproduction of superoxide is the single upstream event leading to the following pathways involved in glucose toxicity (Giacco and Brownlee 2010): (1) increased flux of glucose and other sugars through the polyol pathway; (2) increased intracellular formation of advanced glycation end products (AGEs); (3) increased expression of the receptor for AGEs and its activating ligands; (4) activation of protein kinase C (PKC) isoforms; and (5) overeactivity of the hexosamine pathway. The formation of AGEs and activation of AGE receptors (Shaikh and Nicholson 2008), the activation of PKC (Aoki and Li 2011), and the dysfunction of the polyol pathway (Ahmed et al. 2009) have been identified as contributors in the development of PD. These mechanisms suggest a strong link between neuronal apoptosis observed in PD and hyperglycemic damage in diabetes (Li et al. 2002, 2008; Klein et al. 2004).
In this study, we demonstrated the defensive role of RESV in counteracting cellular distress parameters evoked by HG in DAergic PC12 cells. We tested NGF-differentiated PC12 cells, a known, reliable, and efficient model for the investigation of oxidative stress, apoptosis, and neuroprotection of DAergic neurons (Gélinas and Martinoli 2002; Lahaie-Collins et al. 2008; Bournival et al. 2012). Since oxidative stress is an essential factor in glucose toxicity and in the pathogenesis of PD, we investigated whether RESV protects DAergic PC12 cells by reducing levels of mitochondrial superoxide in HG condition. Our results show that RESV effectively diminishes superoxide production after as early as 3 h suggesting that oxidative damages occurs upstream of apoptosis. In PD, superoxide reacts with iron cations to form hydroxyl radical (Ramasarma 2012), known to exert very deleterious effects on DNA, lipids, and proteins. This ROS can also react with nitric oxide, an important signaling molecule in the brain, to form peroxynitrite, a powerful oxidant shown to play a significant role in protein aggregation pertinent to PD (Danielson and Andersen 2008).
It is currently well known that oxidative stress may cause apoptosis through several pathways: (1) ROS-induced expression or activation of nuclear factor-kappa B (NF-κB) (Gloire et al. 2006); (2) mitochondria-mediated cell apoptosis (Circu and Aw 2010); (3) ROS-mediated DNA damage and p53 activation (Liu and Xu 2011); and (4) stress-activated protein kinases pathway to apoptosis (Johnson and Nakamura 2007). We performed a set of experiments to investigate the apoptotic cascade in DAergic PC12 cells ensuing oxidative stress to further demonstrate the preventive role of RESV. A specific apoptotic DNA denaturation assay demonstrated that RESV significantly prevents apoptosis in cells exposed to HG. We further examined markers of late apoptosis to determine whether the protein cascade leads to terminal events such as the irreversible fragmentation of DNA. RESV in HG clearly reduced the number of apoptotic PC12 cells in comparison to the HG condition alone as shown by the decline in TUNEL and cleaved caspase-3 double-positive cells. Another target of activated caspase-3 is PARP-1, a protein known to participate in the repair of damaged DNA (Wang et al. 2012). Our findings reveal that the PARP-1 protein ratio, full-length versus cleaved, was decreased after HG treatment and was then improved by RESV administration, hence supporting once more the neuroprotective antiapoptotic role of RESV in a HG paradigm. In addition, the Bax and Bcl-2 expression were studied to determine the apoptotic events surrounding the mitochondria. Bax contributes to the leakiness of the outer mitochondrial membrane, while Bcl-2 blocks the permeability transition pore, thus inhibiting mitochondria-mediated programmed cell death (Smith et al. 2008). The rise in the Bax to Bcl-2 ratio is a characteristic feature in apoptosis (Cory and Adams 2002) equally observed in glucose toxicity (Allen et al. 2005) and in several models of PD including human postmortem brains (Vila and Perier 2008). Our data reveal that the Bax/Bcl-2 protein ratio is increased after HG administration, and is decreased by RESV treatment in the HG condition, strongly suggestive of a role for mitochondrial dysfunction in the mechanisms underlying the apoptosis of DAergic neurons in our cellular paradigm of hyperglycemia.
GRP75 has often been linked to PD pathogenesis as reported in studies showing binding properties to PD-associated proteins in the mitochondria (Li et al. 2005; Jin et al. 2005, 2006, 2007; Rakovic et al. 2011) and reduced levels of the protein in postmortem PD brain samples (Jin et al. 2005; Shi et al. 2008; Burbulla et al. 2010). While GRP75 is mainly confined to the outer membrane of mitochondria, several studies have shown that it may bind and sequestrate pro-apoptotic p53 in the cytosol thereby preventing its entry in the nucleus, impeding apoptosis and ultimately promoting p53 degradation by the MDM2 proteasome degradation pathway (Kaul et al. 2001; Wadhwa et al. 2002; Kaul et al. 2005). Such studies were mainly conducted in cancer cells (Kaul et al. 2001, 2005; Wadhwa et al. 2002) or in naive, mitotic PC12 cells (Guo et al. 2009; Li et al. 2011). Our results obtained in post-mitotic PC12 cells, show that HG treatment increases GRP75 expression in the cytoplasm as well as in mitochondria thus suggesting that GRP75 is induced by HG cellular stress. While RESV reduced GRP75 levels in the cytoplasm, it did not ensure a significant effect in diminishing mitochondria GRP75 localization. Apparently, in our cellular paradigm, RESV modulates the subcellular distribution of GRP75 by preventing cytoplasmic levels from rising. RESV may be responsible for quenching HG-induced stress signals that promote the induction of GRP75 in the cytoplasm. Moreover, p53 localization is increased in the nucleus, which points toward a pro-apoptotic effect of HG on DAergic PC12 cells. RESV in HG medium maintains the cellular distribution of p53, which partially accounts for its antiapoptotic properties. Altogether, these results show an increase of GRP75 in the cytoplasm, while p53 levels rise significantly in the nucleus in HG condition, suggesting relatively weak interaction between both markers in post-mitotic cells. Colocalization studies deepened our understanding of the relationship between GRP75 and p53 in our cellular model. We show that GRP75 and p53 have a potential to bind in the cytoplasm, but to a limited extent. Binding in HG condition is significantly enhanced, perhaps due to increased expression of both proteins in the cytoplasm, but still remains limited. We show for the first time that post-mitotic DAergic PC12 cells exert weak binding of GRP75 and p53, which contrasts with findings in nondifferentiated mitotic PC12 cells (Guo et al. 2009; Li et al. 2011). Altogether, our results demonstrate that HG-induced oxidative stress and apoptosis of DAergic PC12 cells can be improved by RESV, sustaining an important role for this naturally occurring polyphenol in diabetes treatment. RESV has been the object of several diabetes studies because of its ability to improve insulin sensitivity, protect pancreatic β cells, and control glycaemia (Milne et al. 2007; Szkudelski and Szkudelska 2011; Lee et al. 2012). Indeed, RESV protects against retinopathy in rats with diabetes (Soufi et al. 2012) and prevents nephropathy in db/db mice by inhibiting lipotoxicity-related apoptosis and oxidative stress in the kidney (Kim et al. 2013). Additional beneficial effects of the stilbene RESV may contribute to alleviate obesity-induced metabolic complications (Rosenow et al. 2012) often related with diabetes. A recent clinical study has found oral administration of RESV to be effective in improving glycaemia in type 2 diabetes mellitus (Bhatt et al. 2012).
Even though RESV is principally metabolized into its glucoronide and sulfate conjugates, recent data show that these metabolites may possess beneficial properties (Delmas et al. 2011). Increased bioavailability due to a synergistic effect with other polyphenols or compounds, such as curcumin or the glycemic control drug metformin, must also be taken into account (Bruckbauer and Zemel 2013; Du et al. 2013). Besides, recent pharmacological advances have improved bioavailability of RESV (for details see Amiot et al. 2013; Neves et al. 2013). Finally, the potential beneficial properties of RESV on human health are broadly displayed in the literature and justify the need to further unravel the powerful cellular role of this dietary polyphenol.
This work was funded by a Natural Sciences and Engineering Research Council (NSERC) Canada Grant to MGM. JR is a NSERC-Vanier student fellow.
- Aoki R, Li YR (2011) α-synuclein promotes neuroprotection through NF-κB-mediated transcriptional regulation of protein kinase Cδ. Sci Signal 4:jc6. doi: 10.1126/scisignal.2002425
- Burbulla LF, Schelling C, Kato H, Rapaport D, Woitalla D, Schiesling C, Schulte C, Sharma M, Illig T, Bauer P, Jung S, Nordheim A, Schöls L, Riess O, Krüger R (2010) Dissecting the role of the mitochondrial chaperone mortalin in Parkinson’s disease: functional impact of disease-related variants on mitochondrial homeostasis. Hum Mol Genet 19:4437–4452. doi: 10.1093/hmg/ddq370 PubMedCrossRefGoogle Scholar
- Kim MY, Lim JH, Youn HH, Hong YA, Yang KS, Park HS, Chung S, Koh SH, Shin SJ, Choi BS, Kim HW, Kim YS, Lee JH, Chang YS, Park CW (2013) RESV prevents renal lipotoxicity and inhibits mesangial cell glucotoxicity in a manner dependent on the AMPK-SIRT1-PGC1α axis in db/db mice. Diabetologia 56:204–217. doi: 10.1007/s00125-012-2747-2 PubMedCrossRefGoogle Scholar
- Lahaie-Collins V, Bournival J, Plouffe M, Carange J, Martinoli MG (2008) Sesamin modulates tyrosine hydroxylase, superoxide dismutase, catalase, inducible NO synthase and interleukin-6 expression in dopaminergic cells under MPP+-induced oxidative stress. Oxid Med Cell Longev 1:54–62PubMedCentralPubMedCrossRefGoogle Scholar
- Li YH, Zhuo YH, Lü L, Chen LY, Huang XH, Zhang JL, Li SY, Wang XG (2008) Caspase-dependent retinal ganglion cell apoptosis in the rat model of acute diabetes. Chin Med J (Engl) 121:2566–2571Google Scholar
- Milne JC, Lambert PD, Schenk S, Carney DP, Smith JJ, Gagne DJ, Jin L, Boss O, Perni RB, Vu CB, Bernis JE, Xie R, Disch JS, Ng PY, Nunes JJ, Lynch AV, Yang H, Galonek H, Isrealian K, Choix W, Iffland A, Lavu S, Medvedik O, Sinclair DA, Olefsky JM, Jirousek MR, Elliott PJ, Westphal CH (2007) Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 450:712–716PubMedCentralPubMedCrossRefGoogle Scholar
- Nelson LE, Valentine RJ, Cacicedo JM, Gauthier MS, Ido Y, Ruderman NB (2012) A novel inverse relationship between metformin-triggered AMPK-SIRT1 signaling and p53 protein abundance in high glucose-exposed HepG2 cells. Am J Physiol Cell Physiol 303:C4–C13. doi: 10.1152/ajpcell.0 0296.2011PubMedCrossRefGoogle Scholar
- Pérez-De La Cruz V, Elinios-Calderón D, Carrillo-Mora P, Silva-Adava D, Konigsberg M, Morán J, Ali SF, Chánez-Cárdenas ME, Pérez-De La Cruz G, Santamariá A (2010) Time-course correlation of early toxic events in three models of striatal damage: modulation by proteases inhibition. Neurochem Int 56:834–842. doi: 10.1016/j.neuint.2010.03.008 PubMedCrossRefGoogle Scholar
- Vignini A, Giulietti A, Nanetti L, Raffaelli F, Giusti L, Mazzanti L, Provinciali L (2013) Alzheimer’s disease and diabetes: new insights and unifying therapies. Curr Diabetes Rev. Epub ahead of printGoogle Scholar
- Wadhwa R, Yaguchi T, Hasan MK, Mitsui Y, Reddel RR, Kaul SC (2002) Hsp70 family member, mot-2/mthsp70/GRP75, binds to the cytoplasmic sequestration domain of the p53 protein. Exp Cell Res 274:246–253Google Scholar
- Wu PF, Xie N, Zhang J–J, Guan X-L, Zhou J, Li LongL-H, Y-L XiongQ-J, Zeng J-H, Fang Wang F, Chen J-G (2012) Resveratrol preconditioning increases methionine sulfoxide reductases A expression and enhances resistance of human neuroblastoma cells to neurotoxins. J Nutr Biochem 24(2013):1070–1077PubMedGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.