Treatment with Caffeic Acid and Resveratrol Alleviates Oxidative Stress Induced Neurotoxicity in Cell and Drosophila Models of Spinocerebellar Ataxia Type3

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a polyglutamine (polyQ) repeat in the protein ataxin-3 which is involved in susceptibility to mild oxidative stress induced neuronal death. Here we show that caffeic acid (CA) and resveratrol (Res) decreased reactive oxygen species (ROS), mutant ataxin-3 and apoptosis and increased autophagy in the pro-oxidant tert-butyl hydroperoxide (tBH)-treated SK-N-SH-MJD78 cells containing mutant ataxin-3. Furthermore, CA and Res improved survival and locomotor activity and decreased mutant ataxin-3 and ROS levels in tBH-treated SCA3 Drosophila. CA and Res also altered p53 and nuclear factor-κB (NF-κB) activation and expression in tBH-treated cell and fly models of SCA3, respectively. Blockade of NF-κB activation annulled the protective effects of CA and Res on apoptosis, ROS, and p53 activation in tBH-treated SK-N-SH-MJD78 cells, which suggests the importance of restoring NF-κB activity by CA and Res. Our findings suggest that CA and Res may be useful in the management of oxidative stress induced neuronal apoptosis in SCA3.


CA and Res inhibit the effects of tBH on oxidative stress, cell viability, and cell apoptosis in SK-N-SH-MJD78 cells. Previous data showed that SK-N-SH-MJD78 cells expressing mutant ataxin-3
with 78 glutamine residues are more sensitive to the effects of oxidative stress on cell viability than are parental SK-N-SH cells 5 . In agreement with previous results, we found that compared with treatment with the vehicle control, treatment with tBH significantly induced ROS production, cytotoxicity, and cell apoptosis in SK-N-SH-MJD78 cells but not in SK-N-SH cells or SK-N-SH-MJD26 cells expressing the normal ataxin-3 containing 26 glutamine residues. Moreover, compared with the vehicle control, tBH dramatically increased the events of mitochondria-mediated cell apoptosis as evidenced by significantly decreased mitochondrial transmembrane potential and Bcl-2 expression. These events occurred concomitantly with an increase in the expression of cytoplasmic cytochrome c and Bax as well as an increase in caspase 3 activity and cleaved caspase 9, caspase 7, caspase 3, and poly ADP-ribose polymerase (PARP) in SK-N-SH-MJD78 cells. Addition of CA and Res blocked the effects of tBH on ROS production, cytotoxicity, and cell apoptosis in SK-N-SH-MJD78 cells ( Fig. 1 and Table 1).
CA and Res weaken the effects of tBH on protein aggregate, mutant ataxin-3, and heat shock protein 27 (Hsp27) levels in SK-N-SH-MJD78 cells. It has been established that the sensitization of apoptosis in neuronal cells with mutant ataxin-3 is associated with increased protein aggregates and decreased Hsp27 expression [33][34][35] . As shown in Fig. 2, compared with SK-N-SH-MJD26 cells, SK-N-SH-MJD78 cells showed increases in mutant ataxin-3 and protein aggregate levels as well as a decrease in Hsp27 expression, and these

CA and Res diminish the effect of tBH on autophagy in SK-N-SH-MJD78 cells. An increase in
autophagy can reduce the expression of mutant ataxin-3 and protein aggregates in neurons, which results in modulation of neurodegeneration in SCA3 mice 35,36 . In SK-N-SH-MJD78 cells, tBH treatment resulted in a decline in autophagy levels as measured by p62, beclin 1, and microtubule-associated protein 1 light chain 3 (LC3)-II protein expression and lysosomotropic agent acridine orange staining. Addition of CA and Res reversed the inhibitory effects of tBH on autophagy levels (Fig. 2).

CA and Res impede the effect of tBH on p53 and NF-κB activation in SK-N-SH-MJD78 cells.
Treatment with tBH significantly induced p53 activation as evidenced by increases in total and phosphorylated p53 expression as well as nuclear p53 expression and p53 transcriptional activity in SK-N-SH-MJD78 cells ( Fig. 3A and C). On the other hand, tBH significantly inhibited NF-κB activation through decreases in IKK-β and IκB-α phosphorylation, IκB-α degradation, nuclear p65 expression, and NF-κB transcriptional activity ( Fig. 3B and C). Treatment with CA and Res abolished the effects of tBH on p53 and NF-κB activation in SK-N-SH-MJD78 cells (Fig. 3).
Modulating NF-κB activity plays a critical role in the beneficial effects of CA and Res on tBH-treated SK-N-SH-MJD78 cells. We further performed transfection experiments with a dominant-negative mutant IκB-α (DNM IκB-α) that is resistant to phosphorylation and degradation of IκB-α and therefore acts as a potent repressor of NF-κB activation 37 . Transfection of DNM IκB-α into SK-N-SH-MJD78 cells blocked the ability of CA and Res to reverse the inhibitory effect of tBH on NF-κB activation ( Fig. 4A and D). Moreover, the effects of CA and Res on the levels of protein aggregates, mutant ataxin-3, and Hsp27 with tBH treatment were negated when SK-N-SH-MJD78 cells were transfected with DNM IκB-α plasmid compared with the wild-type counterpart ( Fig. 4B and D). Furthermore, the protective potency of CA and Res on tBH-induced cell apoptosis, apoptosis-related protein expression, and p53 activation was cancelled in SK-N-SH-MJD78 cells transfected with DNM IκB-α (Fig. 4A,C and D).

CA and Res improve survival rates and climbing activity in tBH-treated ELAV-SCA3tr-Q78
transgenic Drosophila. To further assess the detrimental effects of tBH on SCA3 phenotypes and to test the protective effects of CA and Res, we studied the survival rates and climbing activity of ELAV-SCA3tr-Q78 flies. Compared with ELAV-SCA3tr-Q27 flies, ELAV-SCA3tr-Q78 flies express an ataxin-3 polyQ tract of 78 residues in neurons, which results in reduced survival rates and climbing activity. These reductions are further worsened by tBH treatment. We found that addition of CA and Res enhanced the survival rates and climbing activity in tBH-treated ELAV-SCA3tr-Q78 flies (Fig. 5).

CA and Res ameliorate the effects of tBH treatment in ELAV-SCA3tr-Q78 transgenic
Drosophila. CA and Res decreased ROS production, mutant ataxin-3 expression, the formation of protein aggregates, and augmented Hsp27 expression in tBH-treated ELAV-SCA3tr-Q78 flies. Moreover, CA and Res restrained the effects of tBH on apoptosis-related protein expression, such as of Bax and Bcl-2, as well as expression of p53 and NF-κB in ELAV-SCA3tr-Q78 flies (Fig. 6).

Discussion
Evidence suggests that mutant ataxin-3 is associated with an increase in susceptibility to oxidative stress that has a significant impact on neuronal cell death in SCA3 5, 6, 38 . Our data showed that, in response to tBH treatment, a considerable amount of ROS and apoptotic cell death was observed in SK-N-SH-MJD78 cells but not in SK-N-SH   Overexpression of Hsp27 and Bcl-2 can blunt SK-N-SH-MJD78 cells to staurosporine-induced apoptotic stress, likely because Hsp27 and Bcl-2 serve as antioxidant proteins to protect cells against oxidative stress and subsequent apoptosis 33,34 . Notably, in neuronal and non-neuronal cells, mutant ataxin-3 is causally involved in the down-regulation of Hsp27 and Bcl-2 protein expression by diminishing protein synthesis and mRNA stability, respectively 6,39 . In the fibroblasts and induced pluripotent stem cells from SCA3 patients, the autophagy is impaired and induction of autophagy by rapamycin decreases mutant ataxin-3 expression 40,41 . Overexpression of autophagic beclin-1 protein improves autophagosomal flux and clearance of mutant ataxin-3 respectively in rat primary striatal cells and Neuro-2a cells transfected with mutant ataxin-3 36 . Treatment with temsirolimus, an mTOR inhibitor, can improve motor performance in the transgenic mouse of SCA3 through induction of the autophagy pathway leading to diminution of mutant ataxin-3 and its aggregate levels in neurons 35 . In the present work, our data indicated that compared with the expression in SK-N-SH-MJD26 cells, expression of Hsp27 and Bcl-2, and autophagy levels were reduced and expression of mutant ataxin-3 protein and protein aggregates were increased in SK-N-SH-MJD78 cells. Notably, these changes were much more pronounced in SK-N-SH-MJD78 cells treated with tBH. Our data suggest that CA and Res may through upregulation of the autophagy process decrease the expression of mutant ataxin-3 and protein aggregates, resulting in restoration of the Hsp27 and Bcl-2 protein expression in tBH-treated SK-N-SH-MJD78 cells. A large body of evidence has shown that aberrant activation of the p53 pathway elicits mitochondria-mediated apoptotic neuronal death, which is implicated in the neurodegeneration in several neurological disorders including polyQ diseases 8,11 . Not only in primary cultures of central nervous cells expressing mutant ataxin-3 but also in pontine nuclei of SCA3 transgenic mice, mutant ataxin-3 can increase the expression of total and phosphorylated p53 as well as the transcriptional activity of p53, which is associated with upregulation of Bax and activated caspase-3 and 9 expression and subsequent apoptotic cell death 9,10 . Although activation of NF-κB in acute brain injury is neurotoxic, constitutive activation of NF-κB is required for the maintenance of survival of central neurons 14 . Moreover, impairment of NF-κB activity is involved in the neuronal death in SCA7 and Huntington's disease 17,18,42 . Although the exact effect remains unknown, previous data showed that mutant ataxin-3 can interact with IκB-α   and NF-κB p65 18 . The results of the present study showed that tBH treatment caused activation of the p53 and inactivation of the NF-κB pathway in SK-N-SH-MJD78 cells and that CA and Res could attenuate the effects of tBH by reverting the transcriptional activities of p53 and NF-κB to control levels. Furthermore, when the ability to recover NF-κB activation was impeded by transfection with a DNM IκB-α plasmid, CA and Res could no longer modulate not only p53 activation but also cell apoptosis and its related molecule expression in tBH-treated SK-N-SH-MJD78 cells. Notably, the preventive effect of CA and Res on mutant ataxin-3, protein aggregates, and Hsp27 levels in tBH-treated SK-N-SH-MJD78 cells was reversed when the cells were transfected with the DNM IκB-α plasmid. These data suggest for the first time that the restoration of NF-κB activity is important in the inhibitory effects of CA and Res on p53 activation and cell death in tBH-treated SK-N-SH-MJD78 cells.
It is well established that MJDtr-Q78 transgenic flies, which exhibit tissue-specific expression of ataxin-3 polyQ tracts of 78 residues, have features similar to human SCA3, with late-onset, progressive neurodegeneration and abnormal protein aggregates. Although due to the neuron-specific promoter elav, no remarkable degeneration of adult eye is observed in ELAV-MJDtr-Q78 flies compared with ELAV-MJDtr-Q27 control flies, the ELAV-MJDtr-Q78 flies have a shorter life span and reduced climbing activity 43,44 . The data presented here showed that administration of CA and Res prolonged life span and restored locomotor activity during exposure to tBH in ELAV-MJDtr-Q78 flies. Consistent with the in vitro findings, CA and Res diminished the effects of tBH on ROS production, protein aggregates, and the protein expression of mutant ataxin-3, Hsp27, and apoptotic-related molecules in ELAV-MJDtr-Q78 flies. Moreover, CA and Res reduced the tBH-induced alterations in p53 and p65 expression in ELAV-MJDtr-Q78 flies.
In addition to size of the polyQ expansion in causative proteins, other genetic or environmental factors are also involved in the variability in age at onset of spinocerebellar ataxia 45 . Selective neuronal cell loss is a common feature of polyQ diseases for which until now no established disease-modifying therapy has been available 46 . The data from the present study demonstrated that CA and Res could amend ROS production, mitochondrial-mediated apoptosis, and p53 and NF-κB activation in both tBH-treated Drosophila and human neuroblastoma cell models of SCA3. Notably, we have demonstrated for the first time that CA and Res can restore NF-κB transcriptional activity and that this restoration is associated with decreased neuronal cell death in tBH-treated SK-N-SH-MJD78 cells. Our findings support the beneficial role of CA and Res in diminishing oxidative stress induced neuronal cell death in SCA3. It will be worthy to study the preclinical therapeutic potential of CA and Res in other polyQ diseases. The specific antibodies for hsp27, p53, Bax, Bcl-2, beclin-1, histone H1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cytochrome c, and total and phosphorylated IKK-α/β and IκB-α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against total (pro) and cleaved PARP, caspase-9, caspase-7, and phosphorylated p53 were from Cell Signaling Technology Inc. (Beverly, MA). The specific antibodies for p65, caspase-3, LC3 and β-actin were from BD Biosciences (Boston, MA), Novus Biologicals (Littleton, CO), and MBL International (Wobum, MA), EMD Millipore (Billerica, MA), respectively. Antibodies against p62 and ataxin-3 were obtained from Abcam (Cambridge, MA). The Mitochondria/Cytosol Fractionation Kit and Caspase-3 Colorimetric Assay Kit were from BioVision Inc. (San Francisco, CA, USA) and Millipore (Temecula, CA), respectively.

Materials
Cell culture and treatment. Human neuroblastoma SK-N-SH, SK-N-SH-MJD26, and SK-N-SH-MJD78 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine, 1% penicillin/streptomycin, and 1% nonessential amino acids and containing 10% heat-inactivated fetal bovine serum (Invitrogen Corporation, Carlsbad, CA) at 37 °C under a humidified atmosphere of 5% CO 2 . SK-N-SH-MJD26 and SK-N-SH-MJD78 cells were selected in a culture medium supplemented with 0.1 mg/mL G418 (Invivogen, San Diego, CA). When cells reached 90% confluence, the media were replaced with serum-free media with or without 6 µM tBH plus DMSO vehicle control, 3 µM CA, or Res.
Cell viability assay. The mitochondrial-dependent reduction of 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diph enyltetrazoliumbromide (MTT) to formazan was used to measure cell respiration as an indicator of cell viability. After 48 h, cells were incubated in DMEM containing 0.5 mg/mL MTT for 3 h. The medium was then removed and isopropanol was added to dissolve the formazan. After centrifugation at 5000 × g for 5 min, supernatant from each sample was added to 96-well plates, and the absorbance was read at 570 nm in a VersaMaxTM Tunable Microplate Reader (Molecular Devices Corporation, Sunnyvale, CA).
Protein extraction and Western blot. Protein extracts from transgenic flies and cells were prepared by using RIPA lysis buffer or the Mitochondria/Cytosol Fractionation Kit. The nuclear proteins were extracted by using hypotonic extraction buffer and then hypertonic extraction buffer 47 . Equal amounts of proteins were denatured and separated on SDS-polyacrylamide gels and were then transferred to polyvinylidene difluoride membranes. The blots were incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Immunoreactive protein bands were developed by use of an enhanced chemiluminescence kit (Perkin-Elmer Life Science, Boston, MA) and were visualized by use of a luminescent image analyzer (LAS-1000