ER stress response plays an important role in aggregation of α-synuclein
© Jiang et al; licensee BioMed Central Ltd. 2010
Received: 13 August 2010
Accepted: 13 December 2010
Published: 13 December 2010
Accumulation of filamentous α-synuclein as Lewy bodies is a hallmark of Parkinson's disease. To identify the mechanisms involved in α-synuclein assembly and determine whether the assemblies are cytotoxic, we developed a cell model (3D5) that inducibly expresses wild-type human α-synuclein and forms inclusions that reproduce many morphological and biochemical characteristics of Lewy bodies. In the present study, we evaluated the effects of several histone deacetylase inhibitors on α-synuclein aggregation in 3D5 cells and primary neuronal cultures. These drugs have been demonstrated to protect cells transiently overexpressing α-synuclein from its toxicity.
Contrary to transient transfectants, the drug treatment did not benefit 3D5 cells and primary cultures. The treated were less viable and contained more α-synuclein oligomers, active caspases 3 and 9, as well as ER stress markers than non-treated counterparts. The drug-treated, induced-3D5 cells, or primary cultures from transgenic mice overexpressing (<2 fold) α-synuclein, displayed more α-synuclein oligomers and ER stress markers than non-induced or non-transgenic counterparts. Similar effects were demonstrated in cultures treated with tunicamycin, an ER stressor. These effects were blocked by co-treatment with salubrinal, an ER stress inhibitor. In comparison, co-treatment with a pan caspase inhibitor protected cells from demise but did not reduce α-synuclein oligomer accumulation.
Our results indicate that an increase of wild-type α-synuclein can elicit ER stress response and sensitize cells to further insults. Most importantly, an increase of ER stress response can promote the aggregation of wild type α-synuclein.
Parkinson's disease (PD) and related disorders known as synucleinopathies are characterized by abnormal accumulation of α-synuclein (α-Syn). Such filamentous aggregates in neuronal perikarya and processes are referred to as Lewy bodies and Lewy neurites, respectively [1, 2]. Most synucleinopathies are sporadic, but some are linked to missense mutation or multiplications of the gene encoding α-Syn [3–8].
In order to understand how and why α-Syn accumulation affects neuronal survival, several experimental models have been generated. In some cell-based studies overexpression of α-Syn did not affect cell survival but sensitized the cultures (mutant α-Syn in particular) to insults [9, 10]. In others, the results were different. Infection of primary mesencephalic neuronal cultures with lentiviral constructs containing A53T mutant α-Syn led to reduced cell viability . Transient co-expression of mutant α-Syn and synphilin-1 in H4 cells was reported to cause formation of cytoplasmic α-Syn inclusions and cytotoxicity. These changes were alleviated by treatment with Agk2, a histone deacetylase (HDAC) inhibitor . Transient expression of wild-type α-Syn in the nuclei of SH-SY5Y cells, but not cytoplasm, was reported to cause α-Syn aggregation and cytoxicity that can be prevented by a 48-hour (h) treatment with 10 mM sodium butyrate (SB) . It remains unknown whether benefits comparable to those observed in transient transfectants are attainable in stable transfectants overexpressing α-Syn and whether comparable outcomes can be achieved with exposure to other HDAC inhibitors, since there are multiple classes of HDAC. Some of these issues were examined in the present studies using cell cultures, referred to as 3D5, of human neuroblastoma BE2-M17D cells overexpressing wild-type human α-Syn . The overexpression of human α-Syn in 3D5 cultures is regulated by the tetracycline-off (TetOff) inducible mechanism. These cells develop neuritic processes and express neuronal markers in response to all-trans-retinoic acid (RA) treatment . 3D5 cells that have been treated with RA for 10 days (ds) followed by 28 ds of induced α-Syn expression are capable of forming α-Syn inclusions that reproduce many morphological and biochemical characteristics of Lewy bodies . Similar α-Syn inclusions were not readily detected in 3D5 cultures after 14 ds of induced α-Syn expression, although the cultures were biochemically demonstrated to contain α-Syn oligomers.
It has been well documented that acetylation and deacetylation of histone proteins play important roles in the epigenetic regulation of transcription and other cellular functions [15, 16]. Deacetylation of histone proteins leads to the silencing of gene expression. At least four major classes of HDACs have been identified in mammalian cells [17–21] and several HDAC inhibitors have been developed. Some HDAC inhibitors are more specific to certain subclasses of HDAC than others [17, 18]. For example, fatty acid derivatives such as sodium butyrate (SB) and valproic acid (VPA) are capable of inhibiting class I (HDAC8 in particular) and class II HDAC . In comparison, Agk2 is more specific to sirtuin 2, a member of class III HDAC .
In this study we used RA-differentiated 3D5 cultures, with or without induced α-Syn expression, and evaluated their responses to SB, VPA or Agk2 exposure. We began with cultures that have 10 ds of induced α-Syn expression. The goal was to establish background information on the drug dosage and duration of treatment and use the information in a subsequent cell-based study with an extended duration (e.g 28 ds) of α-Syn overexpression. We unexpectedly found that SB or other HDAC inhibitors treatment of 3D5 cells with induced α-Syn expression caused an increase of α-Syn aggregate accumulation in a time- and dose-dependent manner. Such changes were not detected in the SB-treated, non-induced cultures or in 3D5 cells treated with sodium acetate (SA). More important, 3D5 cultures with α-Syn induction were less viable than those without the induction when exposed to HDAC inhibitors. Similar results were obtained using primary neuronal cultures generated from α-Syn transgenic (TG) mice and their non-transgenic (NT) littermates. The TG mice express wild-type human α-Syn at levels much lower than that displayed by the TetOff induced 3D5 cultures. These findings prompted us to examine whether α-Syn aggregation is closely associated with apoptosis or upstream pathways.
SB exposure causes accumulation of α-Syn oligomers and activation of caspase 3 in a dose- and time-dependent manner
Cultures with SB exposure accumulate α-Syn oligomers with different solubility
Morphological assessment of cultures and SKI samples
SB exposure does not affect the level of α-Syn in 3D5 cells with induced α-Syn expression
To demonstrate further that SB treatment did not affect oligomeric α-Syn accumulation via an increase of α-Syn synthesis, we analyzed 3D5 cells that have been exposed to SB, SB plus Tet (2 μg/ml, for blocking of α-Syn induction), SB plus cycloheximide (CHX, 20 μM, for inhibition of protein synthesis), CHX or Tet for 36 hs by Western blotting. The analysis showed the co-treated cultures contained only slightly fewer α-Syn oligomers than those treated with SB, but much more than those treated with CHX or Tet (Additional file 3). The results support the possibility that oligomer accumulation in SB-treated cells is unlikely caused by an increase of α-Syn expression.
Caspase inhibitor treatment has no affect on α-Syn oligomer accumulation
We next determined whether the SB-treated cultures differed from non-treated in terms of apoptosis and whether apoptosis per se is responsible for accumulation of α-Syn oligomers (Figure 7B). The SB-treated cultures were found to contain far more active forms of capases 3, caspase 9 and cleaved PARP than the non-treated, indicating the treated underwent apoptosis. In cells co-treated with SB and CI, the amount of both active capases 3 and 9 was reduced, whereas the level of α-Syn oligomers was not, indicating α-Syn aggregation is likely an upstream event of caspase activation.
Exposure of α-Syn overexpressing 3D5 cultures to SB or other HDAC inhibitors induces ER stress response leading to accumulation of α-Syn oligomers and apoptosis
To determine whether ER stress plays a role in α-Syn oligomer accumulation, we used a known ER stress inducer, tunicamycin (TM). Similar to SB-treated cells, the TM-treated cultures displayed higher levels of α-Syn oligomer than their non-treated counterparts (Figure 8B). Moreover, the increase was impeded by co-treatment with Sal, thus establishing a close link between ER stress and α-Syn oligomer accumulation.
To verify the effects of drug treatment on ER stress, lysates from α-Syn expressing 3D5 cells (marked as α-Syn+ in Figure 8), treated with or without SB, were probed with antibodies to several markers relevant to ER stress response (i.e. GRP78, p-EIF2α, XBP-1, ATF6α and CHOP) and with antibodies to GAPDH to serve as loading control (Figure 8B). The SB exposure caused an increase in the ratio of p-EIF2α to total EIF2α and that of CHOP and other ER stress markers to GAPDH (Additional file 4, compared SB to Con). α-Syn+ cultures co-treated with Sal and SB displayed lower levels of CHOP (a molecule involved in ER stress-induced apoptosis), XBP-1 (Additional file 4), but higher levels of p-EIF2α and GRP78 when compared to the SB-treated. The results are consistent with those reported previously, indicating that Sal is an inhibitor of EIF2α dephosphorylation and that such inhibition leads to an increase in molecular chaperones such as GRP78 and a decrease in CHOP [37–39]. Studies of 3D5 cultures exposed to other HDAC inhibitors (i.e. Agk2, VPA) yielded comparable results (Additional files 5 & 6).
Overexpression of α-Syn elicits ER stress response
Previous studies reported that induced expression of A53T mutant α-Syn led to PC12 cell death and that the toxicity is mediated via ER stress and mitochondrial cell death pathways . However, it was unknown whether induced expression of wild-type α-Syn in 3D5 cells also elicits an ER stress response. We investigated this issue by probing 3D5 cell lysates with or without induced α-Syn expression for ER stress markers. The induced cultures (α-Syn+) displayed a higher ratio of either GRP78 to GAPDH or p-EIF2α to total EIF2α than their non-induced (α-Syn-) counterparts, indicating the overexpression of wild-type α-Syn per se can cause an ER stress response (Figure 8B & Additional file 4). It is worth noting that without drug treatment neither α-Syn- nor α-Syn+ cultures had detectable amounts of CHOP, XBP-1 or ATF6α.
Induced expression of α-Syn sensitizes 3D5 cultures to SB or TM toxicity via ER stress response
Cultures from α-Syn+ and α-Syn- were either treated with SB, TM or not for 36 hrs followed by cell viability assay (Figure 8A). At this time point cell death occurred in both cultures with drug treatment. The drug exposure significantly caused more cell loss in the α-Syn+ cultures than their α-Syn- counterparts. This difference in drug response was significantly reduced in cultures exposed to SB plus Sal or to TM plus Sal.
Similar to what we observed in α-Syn+ cultures, exposure of α-Syn- cultures to SB, VPA, Agk2 or TM caused an increase in ER stress response (Figure 8B, Additional files 4 & 5). However, the drug treatment was less effective in altering the level of most ER stress markers and activation of caspase 3 in α-Syn- than α-Syn+ cultures. Together, the results indicate that α-Syn overexpression elicits ER stress response leading to the sensitization of 3D5 cultures to insults.
α-Syn- cultures co-treated with Sal and SB or other aforementioned agents also led to increase in the level of EIF2α phosphorylation and that of GRP78. However, the amount of ATF6 was not affected. The result is consistent with a recent report in which Sal treatment did not affect ATF6 ER stress pathway .
ER stress response, α-Syn aggregation and apoptosis in primary neuronal cultures exposed to SB and other HDAC inhibitors
To determine whether the effects of HDAC inhibitors treatment on α-Syn aggregation and ER stress response are unique to 3D5 cultures, we next compared primary cultures from α-Syn PAC mice (also referred to as TG mice) to those of non-transgenic (NT) littermates for their responses to the drug exposure, focusing first on cell viability. The TG mice expressed low levels of wild-type human α-Syn and did not have α-Syn aggregates or neurodegeneration at any age (up to 24 months).
Treatment of primary neuronal cultures with SB also resulted in an ER stress response and activation of caspase 3 (Figure 10). Importantly, such response was more intense in the SB-treated TG cultures than similarly treated NT controls and less intense with Sal co-treatment (Additional file 8).
Many studies have been devoted to identifying the mechanisms involved in α-Syn assembly and determine whether the accumulation of α-Syn assemblies is cytotoxic or protective. Although the results are far from conclusive, it is reasonable to suggest that the physicochemical property of α-Syn oligomers determines whether they are cytotoxic and what relation the resultant cytotoxicity has to aggregate formation. In this regard, the 3D5 transfectant is of great value for identification of pathways that modulate the risk of α-Syn-mediated cytotoxicity and agents capable of inhibiting accumulation of α-Syn and its assemblies, due to its ability to accumulate α-Syn assemblies without compromising cell survival .
A number of agents have been tested in previous cell-based studies and shown to reduce α-Syn aggregation and its toxicity [11, 12, 42, 43]. In the present study, we investigated the effect of SB, Val or Agk2 treatment on α-Syn assembly/accumulation in 3D5 cultures. We demonstrated that exposure to each of these drugs reduced the viability of differentiated, α-Syn induced 3D5 cells and enhanced α-Syn oligomer accumulation, and that overexpression of wild-type α-Syn alone is not cytotoxic. This is in contrast to what was observed in previous studies , in which overexpression of α-Syn in the nuclei, but not cytoplasm, of neuroblastoma cells, SH-SY5Y, resulted in cell death. Treatment of SH-SY5Y cultures with 10 mM SB for 48 hs was shown to protect cells from toxicity caused by nuclear accumulation of α-Syn, but did not adversely affect those with cytoplasmic α-Syn accumulation. Treatment of H4 cells overexpressing A53T α-Syn with Agk2 was shown to reduce cell death caused by cytoplasmic α-Syn aggregates. In contrast, treatment of α-Syn overexpressing 3D5 cells with Agk2 caused cell death. The discrepancy may reflect differences in cell type, as supported by the results from our analysis of non-induced 3D5 cultures and non-transfected SH-SY5Y cells (Additional file 9). Cell death was noted in 3D5 cultures within 24 hs of SB (10 mM) treatment. In comparison, cell death was not observed up to 48 hrs after treatment of SH-SY5Y cells, indicating they are less sensitive than 3D5 cells to SB cytotoxicity. Therefore, to evaluate whether drug treatment has beneficial effects or not in cell-based studies, it is important to examine more than one cell type. For this reason, we included primary neuronal cultures in the present studies. This also allowed us to substantiate the pathobiological relevance of findings from cell-line based studies.
We showed that exposing 3D5 and primary cultures to HDAC inhibitors caused an accumulation of α-Syn oligomers, an increase in ER stress response and a decrease in cell survival. In this regard, several recent studies have demonstrated the induction of ER stress response in cell cultures following treatment with different HDAC inhibitors [30–32]. In our studies, such stress response was greater for cultures with α-Syn overexpression than those without. We considered that the difference is at least in part, if not entirely, due to differences in the basal level of ER stress markers. This is in light of our results that (i) α-Syn overexpression alone, regardless of whether it led to the assembly of filamentous α-Syn  or not, elicited ER stress response, (ii) inhibition of EIF2α dephosphorylation via Sal treatment protected cultures from undergoing apoptosis caused by HDAC inhibitors exposure, (iii) treatment of cultures with ER stress inducer TM also resulted in α-Syn oligomer accumulation as in the HDAC inhibitors treated, and (iv) such accumulation was reduced by co-treatment of cultures with Sal. It has been reported that overexpression of A53T mutant α-Syn or wild-type α-Syn phosphorylated at serine 129 caused ER stress and cell death in PC12 or SH-SY5Y cells [40, 44], and that expression of human α-Syn in yeast cells led to blocking of ER-Golgi traffic . Recently, induced α-Syn expression was shown to increase the vulnerability of PC12 cells to stressors such as thapsigargin, TM and 1-methyl-4-phenyl-pyrisinium (MPP+) . We also have data demonstrating differences between the induced and non-induced 3D5 cultures in sensitivity to neurotoxin treatment (i.e. MPP+, rotenone, 6-hydroxydopamine(6-OHDA), paraquat) (Additional file 10). Other investigators have also provided evidence supporting the important role of ER stress response in PD pathogenesis. For example: (i) activation of ER stress response was detected in dopaminergic neurons in the substantia nigra of postmortem PD cases , and (ii) PD animal or cell models generated via exposure to neurotoxins displayed ER stress response [48, 49].
We do not know how HDAC inhibitor treatment leads to ER stress in neuronal cells. However, recent studies of non-neuronal cells demonstrated that (i) exposure of breast cancer cells to panobinostat (a pan HDAC inhibitor) led to deacetylation of GRP78, dissociation of GRP78 from PERK and increase the levels of pEIF2α, ATF4 and CHOP , (ii) exposure of colorectal carcinoma cells to tricostatin A (a pan-HDAC inhibitor) affected the binding of HDAC to GRP78 promotor  and (iii) the C-terminal region of RTN-C protein, an ER membrane protein, can interact with HDAC8 in vitro .
Our studies showed that 3D5 and primary cultures have different sensitivities to HDAC inhibitor treatment. When the same dosage of SB or VPA was used to treat these cultures, it required a longer duration of incubation for primary cultures to display cytotoxicity. In contrast, 3D5 cells were less sensitive than primary cultures to Agk2 exposure. The cause(s) of such differences in drug sensitivity remains unknown. We noted that a previous study has demonstrated a protective effect of Agk2 treatment on rat primary neuronal cultures overexpressing A53T α-Syn via lentiviral infection of constructs containing mutant α-Syn . The discrepancy between the previous study and our primary neuronal culture study may be due to differences in experimental paradigm. Low levels of overexpressing wild-type human α-Syn in mice had no impact on the viability of mouse primary neuronal cultures. In comparison, overexpression of A53T α-Syn by viral infection reduced cell survival.
We showed that 3D5 and primary cultures treated with HDAC inhibitors have different quantity/quality of α-Syn oligomers. Most α-Syn oligomers appearing in primary neuronal cultures are dimers and relatively soluble, whereas those in 3D5 cells are larger oligomers with different solubility (ranging from buffer-extractable to sarkosyl-insoluble). The distinction may reflect difference in α-Syn expression levels. This is supported by our findings that the level of α-Syn in TetOff induced 3D5 cells is far greater than that expressed in primary neuronal cultures from TG mice (based on equal sample loading (Additional file 11). Moreover, it has been reported that a critical concentration of α-Syn is required to initiate α-Syn assembly in test tubes, and that formation of dimer is an early step of α-Syn assembly . The difference between cell types in α-Syn oligomers production may also reflect the rate of α-Syn assembly. In this regard, several molecules have been shown to promote α-Syn assembly in vitro and in vivo [52–56]. It is possible that the amount/type of endogenous inducer for α-Syn assembly may vary in different cell types.
Our finding that an overexpression of wild-type human α-Syn in both 3D5 and primary cultures can elicit ER stress response is noteworthy. According to some studies, the accumulation of α-Syn in the human brain elevates significantly during aging [57, 58]. There is also ample evidence that the unfold protein response is compromised with aging . Therefore, there is a potential risk of using HDAC inhibitors as therapeutics for elderly subjects with neurodegenerative disorders .
Materials and methods
Conditional transfectant 3D5
Alpha-Syn transfectant 3D5 was derived from a human neuroblastoma cell line (BE2-M17D) and characterized previously [13, 14]. The transfectant expresses wild-type human α-Syn and displays neuronal phenotypes upon TetOff induction and incubation with RA . Cultures of 3D5 were maintained in DMEM/10% fetal bovine serum with 2 μg/mL Tet (referred to as non-induced or α-Syn-) or without (referred to as induced or α-Syn+) at 37°C and 5% CO2. Those intended for biochemical analysis were seeded at 0.5 × 106 cells/plate (100 × 20 mm, BD Biosciences, San Jose, CA) or 1.5 × 105 cells/well in 6-well plates. For immunofluorescence or spectrophotometric assay, 3D5 cells were seeded at 2 × 104 cells/well on coverslips in 24-well plates and 1 × 104 in 48-well plates (Bellco Glass Inc, Vineland, NJ), respectively. Media were replaced the next day with Neurobasal medium (Invitrogen, Carlsbad, CA), 2% B-27 supplement (antioxidant free, Invitrogen), 2 mM L-glutamine (Sigma) and 10 μM RA (Sigma-Aldrich, St Louis, MO). After 10 ds of differentiation and induced α-Syn overexpession, cells were treated with HDAC inhibitors [SB (sigma), VPA (Sigma) and Agk2 (EMD Biosciences, La Jolla, CA)], a derivative of short-chain fatty acid [sodium acetate (SA)], an endoplasmic reticulum (ER) stress inducer [tunicamycin (TM, Sigma)], an inhibitor of phosphatases that act on the eukaryotic translation factor 2 subunit alpha (EIF2α) [salubrinal (Sal, Tocris Bioscience, Ellisville, Missouri)], a protein synthesis inhibitor [cycoheximide (CHX, Sigma), or a pan caspase inhibitor (CI) [Z-VAD (OMe)-FMK (EMD Biosciences)]. The cultures were treated with different drug concentrations for durations described in the Result section. Some cultures were treated with two of the aforementioned drugs or with SB and tetracycline (2 μg/ml, for blocking α-Syn induction).
Primary neuronal cultures were prepared from hippocampal and cortical brain tissues of postnatal (day 1) mice (see below) with or without transgenic expression of wild-type human α-Syn, according to a protocol reported previously . They were plated at 6 ~ 7 × 105 cells per well on poly-D-lysine (Sigma) coated 6-well plate and maintained for 7 ds before treatment in the absence or presence of different drugs for different durations.
All animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) and were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996.
Alpha-Syn PAC transgenic mice
Mice were provided by Dr. Farrer's Laboratory and will be described in detail elsewhere. Briefly, transgenic mice were generated using a P1 artificial Chromosome (PAC) RP-1 27 M07, which was previously identified to contain the entire wild-type human α-Syn gene and regulatory sequences PAC DNA . Purified DNA was injected into FVB/N (Taconic, Germantown, NY) fertilized oocytes and transplanted into pseudo-pregnant ICR (Harlan, Indianapolis, IN) female mice. Transgenic founders were genotyped by PCR and analyzed for gene expression by quantitative RT-PCR and western blotting. The founder line with the highest expression was expanded and maintained on FVB/N background (Taconic). Human protein expression in 6 month-old mice is approximately 2 fold of endogenous murine α-Syn. The expression level is highest in the hippocampus followed by cortex, olfactory tubercle and striatum.
To detect α-Syn, we used monoclonal antibody Synuclein-1 (BD Biosciences) and polyclonal antibody Ab98 . For other proteins we used rabbit polyclonal antibodies to total and cleaved Caspase-3, Caspase 9 and PARP (Cell Signaling, Danvers, MA), phosphorylated EIF2α (pEIF2α, Invitrogen), GADD153/CHOP10 (Sigma), XBP-1 and ATF6α (Santa Cruz Biotechnology, Santa Cruz, CA). We also used monoclonal antibodies to GRP78 (BD Biosciences), total EIF2α (T-EIF2α, Cell Signaling) and GAPDH (1D4, Covance Research Products, Berkeley, CA). The binding of primary antibody to protein of interest was detected using peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Chemicon, Temecula, CA) and Alexa594 or Alexa488 goat anti-mouse antibodies (Invitrogen).
Western blot analysis
Cell cultures were harvested and centrifuged at 200 × g for 15 min. The pellet was resuspended in a lysis buffer [20 mM MES, pH 6.8; 80 mM NaCl, 1 mM MgCl2, 2 mM EGTA, 10 mM NaH2PO4, 20 mM NaF, phenylmethylsulfonyl fluoride (PMSF, 1 μg/ml and leupeptin, 10 μg/mL], homogenized and centrifuged at 180 × g for 15 min at 4°C. The cell lystates were mixed with Tricine-SDS sample buffer (Invitrogen) and 2% β-mercaptoethanol, boiled for 5 min and resolved by SDS-PAGE using 10-20% Tris/Tricine gel (Bio-rad, Hercules, CA). Precision plus protein standards (Bio-Rad) were included as references. Proteins separated by SDS-PAGE were transferred onto nitrocellulose and processed for immunolabeling for proteins of interest. Membranes probed with GAPDH antibody were used as loading controls. Immunoreactivity was visualized with enhanced chemiluminescence (ECL plus, Amersham Pharmacia Biotech, Buckinghamshire, UK) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL) and analyzed as before .
Fractionation Studies of Culture Specimens
Cell lysates were fractionated to derive buffer extractable (SN1), 5 M NaCl/1% sarkosyl-soluble (SN2) and 1% sarkosyl-insoluble fractions (SKI) as reported . The SKI fraction was finally resuspended in 50 mM Tris (pH 8.0). All fractions and lysates were analyzed by Western blotting. A portion of the SKI sample from TetOff induced cultures with or without SB treatment and corresponding sample from cultures without α-Syn induction were analyzed further (see below).
Electron and immunoelectron microscopies
Sakosyl-insoluble samples were processed for electron and immunoelectron microscopies using protocols described previously . Cultures with or without SB treatment were processed as before  for electron microscopy.
Cells grown on coverslips were rinsed with PBS, fixed in 4% paraformaldehyde and permeabilized with 0.1 M Tris-buffered saline (TBS, pH 7.6) containing 0.5% triton X-100 for 5 min. They were subsequently blocked with 3% goat serum in TBS, incubated with antibody Synuclein-1 in TBS containing 1% goat serum overnight at 4°C then incubated with Alexa594 goat anti-mouse secondary antibody for 1 h. For Thioflavin S staining the coverslips were immersed in 0.0005% thioflavin S/PBS/formalin and washed 3 times with 70% ethanol and TBS. After immuno- and thiofavin S staining, the coverslips were stained with DAPI (Invitrogen) for 10 min to locate the nuclei and evaluated by confocal fluorescence microscopy (Zeiss LSM 510; Carl Zeiss MicroImaging, Thornwood, NY).
The viability of 3D5 cells was assessed by incubation of cultures with 2 μM calcein AM (Invitrogen), according to the manufacturer's recommendation. Fluorescence emitted from live cells was measured at 495 nm (excitation)/530 nm (emission) using a Cary Eclipse Fluorometer and Spectra Max M2 and Soft Max Protein 4.6 software (Molecular Devices, Sunnyvale, CA). All measurements were performed in triplicate. Viability of primary neuronal cultures was assessed by counting cells in live cultures. We randomly captured the image of cultures at 10-15 fields (200×). Each image was evaluated independently by two-individuals for intact cells to derive the number of cells in all fields as well as the average cell counts per field. After image capturing, the cultures were processed for Western blot analysis.
Data from at least 3 sets of independent experiments were analyzed by one-way Anova with Dunnett's post hoc test or Student's t test to determine statistical significance.
List of Abbreviations
buffer-soluble, SN2: salt plus sarkosyl-extractable
cultures with induced α-Syn expression
cultures without induced α-Syn expression
tetracycline-off, TL: total lysates
We thank Mei Yue (Mayo Clinic) for assistance in animal experiments. This study was supported by the National Institute of Health (P50-NS40256), the Mayo Foundation (Yen) and the Smith Fellowship Awards from Mayo Clinic (Jiang).
- Cookson MR: The biochemistry of Parkinson's disease. Annu Rev Biochem. 2005, 74: 29-52. 10.1146/annurev.biochem.74.082803.133400.PubMedView ArticleGoogle Scholar
- Lippa CF, Duda JE, Grossman M, Hurtig HI, Aarsland D, Boeve BF, Brooks DJ, Dickson DW, Dubois B, Emre M, Fahn S, Farmer JM, Galasko D, Galvin JE, Goetz CG, Growdon JH, Gwinn-Hardy KA, Hardy J, Heutink P, Iwatsubo T, Kosaka K, Lee VM, Leverenz JB, Masliah E, McKeith IG, Nussbaum RL, Olanow CW, Ravina BM, Singleton AB, Tanner CM, Trojanowski JQ, Wszolek ZK: DLB and PDD boundary issues: diagnosis, treatment, molecular pathology, and biomarkers. Neurology. 2007, 68: 812-819. 10.1212/01.wnl.0000256715.13907.d3.PubMedView ArticleGoogle Scholar
- Chartier-Harlin MC, Kachergus J, Roumier C, Mouroux V, Douay X, Lincoln S, Levecque C, Larvor L: Alpha-synuclein locus duplication as a cause of familial Parkinson's disease. Lancet. 2004, 364: 1167-1169. 10.1016/S0140-6736(04)17103-1.PubMedView ArticleGoogle Scholar
- Ibáñez P, Bonnet AM, Débarges B, Lohmann E, Tison F, Pollak P, Agid Y, Dürr A, Brice A: Causal relation between alpha-synuclein gene duplication and familial Parkinson's disease. Lancet. 2004, 364: 1169-1171.PubMedView ArticleGoogle Scholar
- Krüger R, Kuhn W, Müller T, Woitalla D, Graeber M, Kösel S, Przuntek H: Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat Genet. 1998, 18: 106-108.PubMedView ArticleGoogle Scholar
- Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B: Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science. 1997, 276: 2045-2047. 10.1126/science.276.5321.2045.PubMedView ArticleGoogle Scholar
- Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Hulihan M, Peuralinna T, Dutra A, Nussbaum R, Lincoln S: Alpha-Synuclein locus triplication causes Parkinson's disease. Science. 2003, 302: 841-10.1126/science.1090278.PubMedView ArticleGoogle Scholar
- Zarranz JJ, Alegre J, Gómez-Esteban JC, Lezcano E, Ros R, Ampuero I, Vidal L, Hoenicka J: The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann Neurol. 2004, 55: 164-173. 10.1002/ana.10795.PubMedView ArticleGoogle Scholar
- Jiang H, Wu YC, Nakamura M, Liang Y, Tanaka Y, Holmes S, Dawson VL, Dawson TM, Ross CA, Smith WW: Parkinson's disease genetic mutations increase cell susceptibility to stress: mutant alpha-synuclein enhances H2O2- and Sin-1-induced cell death. Neurobiol Aging. 2007, 28: 1709-1717. 10.1016/j.neurobiolaging.2006.07.017.PubMedView ArticleGoogle Scholar
- Yu WH, Matsuoka Y, Sziráki I, Hashim A, Lafrancois J, Sershen H, Duff KE: Increased dopaminergic neuron sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in transgenic mice expressing mutant A53T alpha-synuclein. Neurochem Res. 2008, 33: 902-911. 10.1007/s11064-007-9533-4.PubMedView ArticleGoogle Scholar
- Outeiro TF, Kontopoulos E, Altmann SM, Kufareva I, Strathearn KE, Amore AM, Volk CB, Maxwell MM, Rochet JC, McLean PJ: Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson's disease. Science. 2007, 317: 516-519. 10.1126/science.1143780.PubMedView ArticleGoogle Scholar
- Kontopoulos E, Parvin JD, Feany MB: Alpha-synuclein acts in the nucleus to inhibit histone acetylation and promote neurotoxicity. Hum Mol Genet. 2006, 15: 3012-3023. 10.1093/hmg/ddl243.PubMedView ArticleGoogle Scholar
- Takahashi M, Ko LW, Kulathingal J, Jiang P, Sevlever D, Yen SH: Oxidative stress-induced phosphorylation, degradation and aggregation of alpha-synuclein are linked to upregulated CK2 and cathepsin D. Eur J Neurosci. 2007, 26: 863-874. 10.1111/j.1460-9568.2007.05736.x.PubMedView ArticleGoogle Scholar
- Ko LW, Ko HH, Lin WL, Kulathingal JG, Yen SH: Aggregates assembled from overexpression of wild-type alpha-synuclein are not toxic to human neuronal cells. J Neuropathol Exp Neurol. 2008, 67: 1084-1096. 10.1097/NEN.0b013e31818c3618.PubMedPubMed CentralView ArticleGoogle Scholar
- Lee KK, Workman JL: Histone acetyltransferase complexes: one size doesn't fit all. Nat Rev Mol Cell Biol. 2007, 8: 284-295. 10.1038/nrm2145.PubMedView ArticleGoogle Scholar
- Kouzarides T: Chromatin modifications and their function. Cell. 2007, 128: 693-705. 10.1016/j.cell.2007.02.005.PubMedView ArticleGoogle Scholar
- Carey N, La Thangue NB: Histone deacetylase inhibitors: gathering pace. Curr Opin Pharmacol. 2006, 6: 369-375. 10.1016/j.coph.2006.03.010.PubMedView ArticleGoogle Scholar
- McLaughlin F, La Thangue NB: Histone deacetylase inhibitors open new doors in cancer therapy. Biochem Pharmacol. 2004, 68: 1139-1144. 10.1016/j.bcp.2004.05.034.PubMedView ArticleGoogle Scholar
- de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB: Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J. 2003, 370: 737-749. 10.1042/BJ20021321.PubMedPubMed CentralView ArticleGoogle Scholar
- Taunton J, Hassig CA, Schreiber SL: A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science. 1996, 272: 408-411. 10.1126/science.272.5260.408.PubMedView ArticleGoogle Scholar
- Rundlett SE, Carmen AA, Kobayashi R, Bavykin S, Turner BM, Grunstein M: HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription. Proc Natl Acad Sci USA. 1996, 93: 14503-14508. 10.1073/pnas.93.25.14503.PubMedPubMed CentralView ArticleGoogle Scholar
- Oh M, Choi IK, Kwon HJ: Inhibition of histone deacetylase1 induces autophagy. Biochem Biophys Res Commun. 2008, 369: 1179-83. 10.1016/j.bbrc.2008.03.019.PubMedView ArticleGoogle Scholar
- Boutillier AL, Trinh E, Loeffler JP: Selective E2F-dependent gene transcription is controlled by histone deacetylase activity during neuronal apoptosis. J Neurochem. 2003, 84: 814-828. 10.1046/j.1471-4159.2003.01581.x.PubMedView ArticleGoogle Scholar
- Hofmanová J, Vaculová A, Lojek A, Kozubík A: Interaction of polyunsaturated fatty acids and sodium butyrate during apoptosis in HT-29 human colon adenocarcinoma cells. Eur J Nutr. 2005, 44: 40-51.PubMedView ArticleGoogle Scholar
- Louis M, Rosato RR, Brault L, Osbild S, Battaglia E, Yang XH, Grant S, Bagrel D: The histone deacetylase inhibitor sodium butyrate induces breast cancer cell apoptosis through diverse cytotoxic actions including glutathione depletion and oxidative stress. Int J Oncol. 2004, 25: 1701-1711.PubMedGoogle Scholar
- Liu Q, Shimoyama T, Suzuki K, Umeda T, Nakaji S, Sugawara K: Effect of sodium butyrate on reactive oxygen species generation by human neutrophils. Scand J Gastroenterol. 2001, 36: 744-50. 10.1080/003655201300192012.PubMedView ArticleGoogle Scholar
- Giardina C, Boulares H, Inan MS: NSAIDs and butyrate sensitize a human colorectal cancer cell line to TNF-alpha and Fas ligation: the role of reactive oxygen species. Biochim Biophys Acta. 1999, 1448: 425-438. 10.1016/S0167-4889(98)00156-6.PubMedView ArticleGoogle Scholar
- Rosato RR, Wang Z, Gopalkrishnan RV, Fisher PB, Grant S: Evidence of a functional role for the cyclin-dependent kinase-inhibitor p21WAF1/CIP1/MDA6 in promoting differentiation and preventing mitochondrial dysfunction and apoptosis induced by sodium butyrate in human myelomonocytic leukemia cells (U937). Int J Oncol. 2001, 19: 181-191.PubMedGoogle Scholar
- Emanuele S, D'Anneo A, Bellavia G, Vassallo B, Lauricella M, De Blasio A, Vento R, Tesoriere G: Sodium butyrate induces apoptosis in human hepatoma cells by a mitochondria/caspase pathway, associated with degradation of beta-catenin, pRb and Bcl-XL. Eur J Cancer. 2004, 40: 1441-1452. 10.1016/j.ejca.2004.01.039.PubMedView ArticleGoogle Scholar
- Baueister P, Dong D, Fu Y, Lee AS: Transcriptional induction of GRP78/BiP by histone deacetylase inhibitors and resistance to histone deacetypase inhibitor-induced apoptosis. Mol Cancer Ther. 2009, 8: 1086-1093. 10.1158/1535-7163.MCT-08-1166.View ArticleGoogle Scholar
- Suzuki M, Endo M, Shinohara F, Echigo S, Rikiishi H: Enhancement of cisplatin cytotoxicity by SAHA involved endoplasmic reticulum stress-mediated apoptosis in oral squamous cell carcinoma cells. Cancer Chemother Pharmacol. 2009, 64: 1115-1122. 10.1007/s00280-009-0969-x.PubMedView ArticleGoogle Scholar
- Rao R, Nalluri S, Kolhe R, Yang Y, Fiskus W, Chen J, Ha K, Buckley KM, Balusu R, Coothankandaswamy V, Joshi A, Atadja P, Bhalla KN: Treatment with panobionstat induces glucose-regulated protein 78 acetylation and endoplamic reticulum stress in breast cancer cells. Mol Cancer Ther. 2010, 9: 942-952. 10.1158/1535-7163.MCT-09-0988.PubMedView ArticleGoogle Scholar
- Montilla-López P, Muñoz-Agueda MC, Feijóo López M, Muñoz-Castañeda JR, Bujalance-Arenas I, Túnez-Fiñana I: Comparison of melatonin versus vitamin C on oxidative stress and antioxidant enzyme activity in Alzheimer's disease induced by okadaic acid in neuroblastoma cells. Eur J Pharmacol. 2002, 451: 237-243.PubMedView ArticleGoogle Scholar
- Murphy MG: Membrane fatty acids, lipid peroxidation and adenylate cyclase activity in cultured neural cells. Biochem Biophys Res Commun. 1985, 132: 757-763. 10.1016/0006-291X(85)91197-0.PubMedView ArticleGoogle Scholar
- André N, Braguer D, Brasseur G, Gonçalves A, Lemesle-Meunier D, Guise S, Jordan MA, Briand C: Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells. Cancer Res. 2000, 60: 5349-5353.PubMedGoogle Scholar
- Boyce M, Bryant KF, Jousse C, Long K, Harding HP, Scheuner D, Kaufman RJ, Ma D, Coen DM, Ron D, Yuan J: A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science. 2005, 307: 935-939. 10.1126/science.1101902.PubMedView ArticleGoogle Scholar
- Lee do Y, Lee KS, Lee HJ, Kim do H, Noh YH, Yu K, Jung HY, Lee SH, Lee JY, Youn YC, Jeong Y, Kim DK, Lee WB, Kim SS: Activation of PERK signaling attenuates Abeta-mediated ER stress. PLoS One. 2010, 5: e10489-10.1371/journal.pone.0010489.PubMedView ArticleGoogle Scholar
- Pae HO, Jeong SO, Jeong GS, Kim KM, Kim HS, Kim SA, Kim YC, Kang SD, Kim BN, Chung HT: Curcumin induces pro-apoptotic endoplasmic reticulum stress in human leukemia HL-60 cells. Biochem Biophys Res Commun. 2007, 353: 1040-1045. 10.1016/j.bbrc.2006.12.133.PubMedView ArticleGoogle Scholar
- Zhu Y, Fenik P, Zhan G, Sanfillipo-Cohn B, Naidoo N, Veasey SC: Eif-2a protects brainstem motoneurons in a murine model of sleep apnea. J Neurosci. 2008, 28: 2168-2178. 10.1523/JNEUROSCI.5232-07.2008.PubMedView ArticleGoogle Scholar
- Smith WW, Jiang H, Pei Z, Tanaka Y, Morita H, Sawa A, Dawson VL, Dawson TM, Ross CA: Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutant alpha-synuclein-induced toxicity. Hum Mol Genet. 2005, 14: 3801-3811. 10.1093/hmg/ddi396.PubMedView ArticleGoogle Scholar
- Ladriere L, Igoillo-Esteve M, Cunha DA, Brion JP, Bugliani M, Marchetti P, Eizirik DL, Conp M: Enhanced signaling dowstream of ribonucleic acid-activated protein kinase-like endoplasmic reticulm kinase potentiates lipotoxic endoplasmic reticulum stress in human islets. J Clin Endocrinol Metab. 2010, 95: 1442-1449. 10.1210/jc.2009-2322.PubMedView ArticleGoogle Scholar
- Hillmer AS, Putcha P, Levin J, Högen T, Hyman BT, Kretzschmar H, McLean PJ, Giese A: Converse modulation of toxic alpha-synuclein oligomers in living cells by N'-benzylidene-benzohydrazide derivates and ferric iron. Biochem Biophys Res Commun. 2010, 391: 461-466. 10.1016/j.bbrc.2009.11.080.PubMedPubMed CentralView ArticleGoogle Scholar
- Putcha P, Danzer KM, Kranich LR, Scott A, Silinski M, Mabbett S, Hicks CD, Veal JM, Steed PM, Hyman BT, McLean PJ: Brain-permeable small-molecule inhibitors of Hsp90 prevent alpha-synuclein oligomer formation and rescue alpha-synuclein-induced toxicity. J Pharmacol Exp Ther. 2010, 332: 849-857. 10.1124/jpet.109.158436.PubMedPubMed CentralView ArticleGoogle Scholar
- Sugeno N, Takeda A, Hasegawa T, Kobayashi M, Kikuchi A, Mori F, Wakabayashi K, Itoyama Y: Serine 129 phosphorylation of alpha-synuclein induces unfolded protein response-mediated cell death. J Biol Chem. 2008, 283: 23179-88. 10.1074/jbc.M802223200.PubMedView ArticleGoogle Scholar
- Cooper AA, Gitler AD, Cashikar A, Haynes CM, Hill KJ, Bhullar B, Liu K, Xu K, Strathearn KE, Liu F, Cao S, Caldwell KA, Caldwell GA, Marsischky G, Kolodner RD, Labaer J, Rochet JC, Bonini NM, Lindquist S: Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models. Science. 2006, 313: 324-328. 10.1126/science.1129462.PubMedPubMed CentralView ArticleGoogle Scholar
- Ito S, Nakaso K, Imamura K, Takeshima T, Nakashima K: Endogenous catecholamine enhances the dysfunction of unfolded protein response and alpha-synuclein oligomerization in PC12 cells overexpressing human alpha-synuclein. Neurosci Res. 2010, 66: 124-130. 10.1016/j.neures.2009.10.005.PubMedView ArticleGoogle Scholar
- Hoozemans JJ, van Haastert ES, Eikelenboom P, de Vos RA, Rozemuller JM, Scheper W: Activation of the unfolded protein response in Parkinson's disease. Biochem Biophys Res Commun. 2007, 354: 707-711. 10.1016/j.bbrc.2007.01.043.PubMedView ArticleGoogle Scholar
- Holtz WA, O'Malley KL: Parkinsonian Mimetics Induce Aspects of Unfolded Protein Response in Death of Dopaminergic Neurons. J Biol Chem. 2003, 278: 19367-19377. 10.1074/jbc.M211821200.PubMedView ArticleGoogle Scholar
- Ryu EJ, Harding HP, Angelastro JM, Vitolo OV, Ron D, Greene LA: Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease. J Neurosci. 2002, 22: 10690-10698.PubMedGoogle Scholar
- Nepravishta R, Bellomaria A, Polizio F, Paci M, Melino S: Reticulon RTN1-C(CT) peptide: a potential nuclease and inhibitor of histone deacetylase enzymes. Biochemistry. 2010, 49: 252-258. 10.1021/bi9012676.PubMedView ArticleGoogle Scholar
- Krishnan S, Chi EY, Wood SJ, Kendrick BS, Li C, Garzon-Rodriguez W, Wypych J, Randolph TW, Narhi LO, Biere AL, Citron M, Carpenter JF: Oxidative dimer formation is the critical rate-limiting step for Parkinson's disease alpha-synuclein fibrillogenesis. Biochemistry. 2003, 42: 829-837. 10.1021/bi026528t.PubMedView ArticleGoogle Scholar
- McLean PJ, Kawamata H, Hyman BT: Alpha-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons. Neuroscience. 2001, 104: 901-912. 10.1016/S0306-4522(01)00113-0.PubMedView ArticleGoogle Scholar
- Junn E, Ronchetti RD, Quezado MM, Kim SY, Mouradian MM: Tissue transglutaminase-induced aggregation of alpha-synuclein: Implications for Lewy body formation in Parkinson's disease and dementia with Lewy bodies. Proc Natl Acad Sci USA. 2003, 100: 2047-2052. 10.1073/pnas.0438021100.PubMedPubMed CentralView ArticleGoogle Scholar
- Brandt I, Gérard M, Sergeant K, Devreese B, Baekelandt V, Augustyns K, Scharpé S, Engelborghs Y, Lambeir AM: Prolyl oligopeptidase stimulates the aggregation of alpha-synuclein. Peptides. 2008, 29: 1472-1478. 10.1016/j.peptides.2008.05.005.PubMedView ArticleGoogle Scholar
- Ryo A, Togo T, Nakai T, Hirai A, Nishi M, Yamaguchi A, Suzuki K, Hirayasu Y: Prolyl-isomerase Pin1 accumulates in lewy bodies of parkinson disease and facilitates formation of alpha-synuclein inclusions. J Biol Chem. 2006, 281: 4117-4125. 10.1074/jbc.M507026200.PubMedView ArticleGoogle Scholar
- Gerard M, Debyser Z, Desender L, Kahle PJ, Baert J, Baekelandt V, Engelborghs Y: The aggregation of alpha-synuclein is stimulated by FK506 binding proteins as shown by fluorescence correlation spectroscopy. FASEB J. 2006, 20: 524-526.PubMedGoogle Scholar
- Brighina L, Prigione A, Begni B, Galbussera A, Andreoni S, Piolti R, Ferrarese C: Lymphomonocyte alpha-synuclein levels in aging and in Parkinson disease. Neurobiol Aging. 2010, 31: 884-885. 10.1016/j.neurobiolaging.2008.06.010.PubMedView ArticleGoogle Scholar
- Li W, Lesuisse C, Xu Y, Troncoso JC, Price DL, Lee MK: Stabilization of alpha-synuclein protein with aging and familial parkinson's disease-linked A53T mutation. J Neurosci. 2004, 24: 7400-7409. 10.1523/JNEUROSCI.1370-04.2004.PubMedView ArticleGoogle Scholar
- Naidoo N: ER and aging-Protein folding and the ER stress response. Ageing Res Rev. 2009, 8: 150-159. 10.1016/j.arr.2009.03.001.PubMedView ArticleGoogle Scholar
- Hahnen E, Hauke J, Tränkle C, Eyüpoglu IY, Wirth B, Blümcke I: Histone deacetylase inhibitors: possible implications for neurodegenerative disorders. Expert Opin Investig Drugs. 2008, 17: 169-184. 10.1517/13543722.214.171.124.PubMedView ArticleGoogle Scholar
- Kivell BM, McDonald FJ, Miller JH: Method for serum-free culture of late fetal and early postnatal rat brainstem neurons. Brain Res Brain Res Protoc. 2001, 6: 91-99. 10.1016/S1385-299X(00)00037-4.PubMedView ArticleGoogle Scholar
- Touchman JW, Dehejia A, Chiba-Falek O, Cabin DE, Schwartz JR, Orrison BM, Polymeropoulos MH, Nussbaum RL: Human and mouse alpha-synuclein genes: comparative genomic sequence analysis and identification of a novel gene regulatory element. Genome Res. 2001, 11: 78-86. 10.1101/gr.165801.PubMedPubMed CentralView ArticleGoogle Scholar
- Dickson DW, Liu W, Hardy J, Farrer M, Mehta N, Uitti R, Mark M, Zimmerman T, Golbe L, Sage J, Sima A, D'Amato C, Albin R, Gilman S, Yen SH: Widespread alterations of alpha-synuclein in multiple system atrophy. Am J Pathol. 1999, 155: 1241-1251.PubMedPubMed CentralView ArticleGoogle Scholar
- Jiang P, Ko LW, Jansen KR, Golde TE, Yen SH: Using leucine zipper to facilitate alpha-synuclein assembly. FASEB J. 2008, 22: 3165-3174. 10.1096/fj.08-108365.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<url>http://creativecommons.org/licenses/by/2.0</url>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.