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. Author manuscript; available in PMC: 2012 Jan 13.
Published in final edited form as: Neurosci Lett. 2010 Nov 5;488(1):11–16. doi: 10.1016/j.neulet.2010.10.071

Proteomic Insights into the Protective Mechanisms of an In Vitro Oxidative Stress Model of Early Stage Parkinson’s Disease

Brian Bauereis a, William E Haskins a,b,c,d, Richard G LeBaron a, Robert Renthal a,b,e,*
PMCID: PMC3010496  NIHMSID: NIHMS251636  PMID: 21056633

Abstract

Previous studies in Parkinson's disease (PD) models suggest that early events along the path to neurodegeneration involve activation of the ubiquitin-proteasome system (UPS), endoplasmic reticulum-associated degradation (ERAD), and the unfolded protein response (UPR) pathways, in both the sporadic and familial forms of the disease, and thus ER stress may be a common feature. Furthermore, impairments in protein degradation have been linked to oxidative stress as well as pathways associated with ER stress. We hypothesize that oxidative stress is a primary initiator in a multi-factorial cascade driving dopaminergic (DA) neurons towards death in the early stages of the disease. We now report results from proteomic analysis of a rotenone-induced oxidative stress model of PD in the human neuroblastoma cell line, SH-SY5Y. Cells were exposed to sub-micromolar concentrations of rotenone for 48 hours prior to whole cell protein extraction and shotgun proteomic analysis. Evidence for activation of the UPR comes from our observation of up-regulated Binding immunoglobulin Protein (BiP), heat shock proteins, and foldases. We also observed up-regulation of proteins that contribute to the degradation of misfolded or unfolded proteins controlled by the UPS and ERAD pathways. Activation of the UPR may allow neurons to maintain protein homeostasis in the cytosol and ER despite an increase in reactive oxygen species due to oxidative stress, and activation of the UPS and ERAD may further augment clean-up and quality control in the cell.

Keywords: Oxidative stress, Parkinson’s disease, Rotenone, Proteomics, Unfolded protein response

Introduction

Parkinson’s disease (PD) is a complex, progressive neurodegenerative movement disease that results primarily from the death of dopaminergic (DA) neurons in the substantia nigra pars compacta. Although the cause of sporadic PD is unknown, epidemiological studies suggest cooperation with environmental toxins, notably mitochondrial complex I inhibitors and gene mutations [18]. Oxidative stress is a leading theory of the pathogenesis of PD, supported by analysis of postmortem PD brains [33], and by recapitulation of the oxidative damage seen in PD by low-grade, chronic inhibition of complex 1 with rotenone, both in vivo and in vitro [2, 10]. The common feature associated with oxidative stress and subsequent neurodegeneration is endoplasmic reticulum (ER) stress: a disturbance in the ability of the ER to process and/or fold proteins. The resulting accumulation of misfolded proteins will elicit the Unfolded Protein Response (UPR), leading to transcription of chaperones, foldases, ER-associated degradation (ERAD) machinery and antioxidants. Acute activation of the UPR is beneficial in responding to transient stress. However, if ER stress is chronic, or if protective measures are incapable of maintaining ER homeostasis, the UPR activates apoptotic pathways to avoid damage to neighboring cells [32]. Our goal in this study was to produce an early-stage model of PD by using very small doses of the oxidizing toxin rotenone and report on the changes in the whole cell proteome.

Materials & Methods

Cell Culture

Human neuroblastoma cells (SH-SY5Y) were obtained from the American Type Culture Collection (Rockville, MD).

Cell viability

Cell viability was determined by a MTT (3-(4, 5-dimethylthiazol)-2,5diphenyltetrazolium bromide) assay following the manufacture’s guidelines (ATCC catalog # 30-1010K).

Proteasome Activity Assay - Chymotrypsin-like activity

Control and rotenone treated cells were incubated with Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (LLVY-MCA) (obtained from Dr. M. Gaczynska, Univ. of Texas Health Science Center, San Antonio) at 37°C for 1 hour. Fluorescence of released MCA was measured at 460 nm (with 380 nm excitation).

ssDNA Apoptosis

Control and rotenone-treated cells were analyzed for apoptosis using a Millipore ssDNA Apoptosis ELISA kit (APT225) in accordance with the manufacture’s guidelines.

Caspase-3 Activity Assay

Caspase-3 activity was quantified using Ac-DEVD-AMC Caspase -3 fluorogenic substrate (BD Pharmingen) in accordance with the manufacture’s guidelines.

Sample Preparation

Cells were prepared for protein identification by incubation for 48 hours at 37°C with 5nM, 10nM, 20nM, 50nM, and 100nM rotenone, or as a control, vehicle only. Proteins from cell culture monolayers were extracted by adding 500µL buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and 10µL Protease Inhibitor Cocktail (Sigma P8340) to 106 cells. After 30 min lysis at 4°C, cells were centrifuged and the supernatant was stored at −20°C. Total protein was quantified using the Micro BCA kit (Pierce) following manufacture’s protocol. Proteins were carbamidomethylated and precipitated with acetone [37] prior to trypsin digestion (Promega Gold, mass spectrometry grade) (1:20 protein:enzyme). Capillary liquid chromatography-tandem mass spectrometry (LC/MS/MS) was performed with a splitless nanoLC-2D pump (Eksigent), a 50 µm-i.d. column packed with 10 cm of 5 µm-i.d. C18 particles, and a linear ion trap tandem mass spectrometer (LTQ-XLS; ThermoFisher, San Jose, CA), where the top 7 eluting ions were fragmented by collision-induced dissociation (CID). The capillary LC gradient was 2 to 98% 0.1% formic acid/acetonitrile over 60 min at a flow rate of 300 nL/min. Probability-based and error-tolerant protein database searching of MS/MS spectra against the NCBI non-redundant human protein database was performed with a 10-node MASCOT cluster (ver. 2.1). Search criteria included: peak picking with Xcalibur (ver. 2.0.6; ThermoFisher); 1000 ppm precursor ion mass tolerance, 0.8 Da product ion mass tolerance, 3 missed cleavages, trypsin, carbamidomethyl cysteines and oxidized methionines as variable modifications, and an ion score threshold of 20.

Criteria for Protein Identification

Scaffold (version scaffold_2_06_00, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 90.0% probability (Peptide Prophet algorithm [15]). Protein identifications were accepted if they could be established at a greater than 95.0% probability (Protein Prophet algorithm [26]) and contained at least 1 identified peptide.

Results & Discussion

In order to analyze the proteome of early-stage PD, we sought to identify a concentration range of rotenone in which cultured neuroblastoma cells are viable and are not apoptotic after 48 hours. We evaluated the results of three different measurements: cellular viability/mitochondrial activity, apoptosis activity assessed by single-stranded DNA (ssDNA), and apoptosis activity assessed by caspase-3 activity. SH-SY5Y cells were incubated with submicromolar concentrations of rotenone for 48 hours. Mitochondrial activity (MTT assay, Fig.1) was unaffected at a rotenone concentration ≤ 50 nM (p≤ 0.05). However, when the concentration of rotenone reached ≥100 nM, a significant decrease in cell viability was seen (100nM: p=.0001; 100nM-1µM: p=.0006). The ssDNA apoptosis assay showed no significant increase in apoptosis (Fig.2). Furthermore, the caspase-3 activity showed a significant increase at 50 nM and 100 nM (p= .043 and p= .014, respectively) (Fig. 3). Approximately a 2.5 fold increase in activity was seen, which we estimate may only represent 25% of the maximal caspase-3 activation [21]. The increase in caspase-3 activity at 50 nM and 100 nM rotenone does not correlate with an increase in ssDNA apoptosis at the same concentrations, suggesting that compensatory factors were released by the cell to counteract the apoptotic cascade, potentially through UPR activation and subsequent production of anti-apoptotic proteins. Therefore, we chose the unaffected range (5nM, 10nM, 20nM, and 50nM) of rotenone concentrations to look for the initial response of the cells to oxidative stress. This time point and concentration range may represent the earliest phase in the progressive development of Parkinson’s-like neurodegeneration. We also used exposure to 100nM rotenone to examine the proteome in cells displaying decreased viability.

Fig. 1. MTT viability assay.

Fig. 1

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MTT cell viability assay performed on SH-SY5Y cultures pre-incubated with various concentrations of rotenone for 48 hours. Gray bars indicate cells extracted for proteomic analysis (Table 1). Data (mean ± SEM) are expressed as % of control (no rotenone). Asterisk (*) indicates p<0.0001 by ANOVA: 100nM rotenone-treated compared to 50nM; double asterisk (**) indicates p<0.0006 by ANOVA: 250nM-1µM rotenone-treated compared to 5nM–50nM group.

Fig. 2. ssDNA apoptotic assay (ELISA).

Fig. 2

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SH-SY5Y cultures pre-incubated with various concentrations of rotenone for 48 hours. Data (mean ± SEM) expressed as % of control (no rotenone).

Fig. 3. Caspase-3 activity assay.

Fig. 3

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Caspase-3 activity measured on SH-SY5Y cells pre-incubated with various concentrations of rotenone for 48 hours. Data expressed as relative fluorescence units (RFU). Asterisk (*) indicates p=0.043 by ANOVA: 50nM rotenone-treated compared to 5nM–20nM; double asterisk (**) indicates p=0.014 by ANOVA: 100nM rotenone-treated compared to 5nM–50nM rotenone group.

In control SH-SY5Y cells, we identified tryptic peptides from 242 proteins with high probability, including proteins that are related to the UPR and ER stress pathways that are listed in Table 1. Three heat-shock proteins were identified: HSP90 (both the cytosolic HSP90α and ER HSP90β forms), GRP75, and HSP60. GRP75, in concert with HSP60, is thought to participate in the refolding of proteins translocated into the mitochondria [17, 20]. Additionally, some components involved in the UPS system were identified: the ubiquitin carboxyl-terminal hydrolase enzyme, and a ubiquitin E1 activating enzyme. Both proteins are vital to the function of the UPS and both have been found to be dysfunctional in moderate to late stages of PD [23]. Finally, glutathione S-transferase (GST), a major player in the detoxification and protection against oxidizing toxins, was identified in the control group [6]. This constitutive expression of stress-related proteins in dopaminergic neurons suggests that these neurons have adapted to the endogenous stress associated with dopamine metabolism and subsequent production of reactive oxygen species (ROS) [1]. In other words, neurons may rely on their constitutive levels of HSPs as a preventative mechanism of defense against protein misfolding induced by stressful factors or those that are associated with neurodegenerative diseases [4].

Table 1. Identified proteins related to UPR/ER stress pathways following treatment with rotenone.

Proteins listed with International Protein Index (IPI) [16], MASCOT score, and function. Protein not described in text listed with references to PD and neurodegeneration.

Rotenone 48 hrs Function Protein Accession Numbers Mascot Score
Control chaperone/foldase HSP90-beta [5,14] IPI00414676 422
chaperone/foldase HSP90-alpha isoform 2 [5,14] IPI00382470 288
chaperone/foldase GRP75 IPI00007765 90
chaperone/foldase HSP60, mitochondrial IPI00784154 151
chaperone/foldase TCP1 T-complex subunit 3 isoform b [38] IPI00290770 67
chaperone/foldase TCP1 T-complex protein 1 subunit alpha [38] IPI00290566 75
chaperone/foldase TCP1 T-complex protein 1 subunit beta [38] IPI00297779 202
chaperone/foldase Peptidyl-prolyl cis-trans isomerase A IPI00419585 40
UPS system Ubiquitin carboxyl-terminal hydrolase isozyme IPI00018352 192
UPS system Ubiquitin-like modifier-activating enzyme 1 IPI00645078 127
antioxidant Glutathione S-transferase IPI00219757 132
5nM chaperone/foldase HSP90-beta [6,15] IP00I414676 155
chaperone/foldase HSP60, mitochondria IPI00784154 101
chaperone/foldase HSP75, mitochondria IPI0030275 65
chaperone/foldase TCP1 T-complex protein 1 subunit zeta [38] IPI00027626 53
chaperone/foldase Peptidyl-prolyl cis-trans isomerase A IPI00419585 63
chaperone/foldase Prefoldin subunit 5 IPI00015361 93
UPS system Ubiquitin-like modifier activating enzyme 1 IPI00552452 33
UPS system Proteasome subunit beta type 3 IPI00028004 88
antioxidant Glutathione S-Transferase IPI00219757 34
antioxidant Peroxiredoxin-2 [9] IPI00027350 31
mitochondrial maintenance Prohibitin-2 [22] IPI00027252 59
modulates oxidative stress Protein DJ-1 IPI00298547 40
10nM chaperone/foldase HSP90-beta [5,14] IPI00414676 241
chaperone/foldase HSP90AA1 isoform 1 [5,14] IPI00784295 77
chaperone/foldase HSP90AB4P [5,14] IPI00555565 49
chaperone/foldase BiP IPI00003362 97
chaperone/foldase HSP70 protein 6 [25] IPI00339269 45
chaperone/foldase HSC71 isoform 1[25] IPI00003865 130
chaperone/foldase GRP75 IPI00007765 53
chaperone/foldase HSP60, mitochondrial IPI00784154 29
chaperone/foldase HSP90 co-chaperone Cdc37 IPI00013122 70
chaperone/foldase HSP70/HSP90 organizing protein [4] IPI00013894 29
chaperone/foldase TCP1 T-complex protein 1 subunit beta [38] IPI00297779 116
chaperone/foldase Peptidyl-prolyl cis-trans isomerase A [36] IPI00419585 44
chaperone/foldase Protein disulfide-isomerase A6 isoform 2 [36] IPI00299571 89
chaperone/foldase Protein disulfide-isomerase A3 [36] IPI00025252 38
chaperone/foldase Prefoldin subunit 6 IPI00005657 46
UPS system Ubiquitin carboxyl-terminal hydrolase isozyme IPI00018352 51
UPS system Ubiquitin-like modifier-activating enzyme 1 IPI00645078 46
UPS system Ubiquitin-conjugating enzyme E2 IPI00003949 57
UPS system 26S proteasome subunit S10B protease IPI0021926 43
UPS system 26S proteasome subunit B5 isoform 3 IPI00383971 29
antioxidant Glutathione S-transferase IPI00219757 152
antioxidant Superoxide dismutase IPI00218733 58
modulates oxidative stress Protein DJ-1 IPI00298547 62
20nM chaperone/foldase HSP90-beta [5,14] IPI00414676 347
chaperone/foldase HSP90-alpha isoform 1 [5,14] IPI00784295 107
chaperone/foldase HSP90B1 endoplasmin/GRP94 [5,14] IPI00027230 73
chaperone/foldase BiP IPI00003362 77
chaperone/foldase HSC71 isoform 1 [25] IPI00003865 329
chaperone/foldase HSP70 protein 7 [25] IPI00011134 109
chaperone/foldase GRP75 IPI00007765 58
chaperone/foldase HSP60, mitochondrial IPI00784154 258
chaperone/foldase HSP27 [24,28] IPI00025512 31
chaperone/foldase TCP1 T-complex subunit 3 isoform b [38] IPI00290770 63
chaperone/foldase TCP1 T-complex protein 1 subunit alpha [38] IPI00290566 91
chaperone/foldase TCP1 T-complex protein 1 subunit beta [38] IPI00297779 52
chaperone/foldase TCP1 T-complex protein 1 subunit delta [38] IPI00302927 72
chaperone/foldase TCP1 T-complex protein 1 subunit epsilon [38] IPI00010720 110
chaperone/foldase Peptidyl-prolyl cis-trans isomerase A IPI00419585 109
chaperone/foldase Peptidyl-prolyl cis-trans isomerase B IPI00646304 60
chaperone/foldase Protein disulfide-isomerase A6 isoform 2 [36] IPI00299571 73
chaperone/foldase Protein disulfide-isomerase A3 [36] IPI00025252 70
chaperone/foldase Calreticulin IPI00020599 38
UPS system Ubiquitin-conjugating enzyme E2 IPI00003949 69
UPS system Proteasome subunit alpha type-1 IPI00016832 49
UPS system Proteasome subunit alpha type-7 IPI00024175 40
antioxidant Glutathione S-transferase IPI00246975 41
antioxidant Superoxide dismutase IPI00218733 92
antioxidant Peroxiredoxin-1 [9] IPI00000874 48
antioxidant Peroxiredoxin-6 [9] IPI00220301 122
mitochondrial maintenance Prohibitin-1 [22] IPI00017334 59
mitochondrial maintenance Prohibitin-2 [22] IPI00027252 78
microtubule remodeling Stathmin IP00479997 104
microtubule remodeling Stathmin-2 IPI00218667 49
microtubule remodeling Stathmin-4 isoform 1 IPI00006575 41
export misfolded proteins Transitional ER ATPase IPI00022774 38
antinflammatory Cytosolic Phosolipase A2 IPI00384577 54
50nM chaperone/foldase HSP90-beta [5,14] IPI00414676 266
chaperone/foldase HSP90-alpha isoform 2 [5,14] IPI00382470 173
chaperone/foldase HSP90B1 endoplasmin/GRP94 [5,14] IPI00027230 32
chaperone/foldase BiP IPI00003362 71
chaperone/foldase HSP75, mitochondrial IPI00030275 77
chaperone/foldase HSP70 protein 1 [25] IPI00304925 30
chaperone/foldase HSP70 protein 6 [25] IPI00339269 23
chaperone/foldase HSC71 isoform 1 [25] IPI00003865 103
chaperone/foldase HSP60, mitochondrial IPI00784154 182
chaperone/foldase HSP10, mitochondrial [11] IPI00220362 78
chaperone/foldase TCP1 T-complex protein 1 subunit alpha [38] IPI00290566 45
chaperone/foldase TCP1 T-complex protein 1 subunit 3 isoform c [38] IPI00552715 54
chaperone/foldase TCP1 T-complex protein 1 subunit zeta [38] IPI00027626 34
chaperone/foldase Peptidyl-prolyl cis-trans isomerase A IPI00419585 36
chaperone/foldase Peptidyl-prolyl cis-trans isomerase B IPI00646304 52
chaperone/foldase ERp57 [7] IPI0025252 51
chaperone/foldase Protein disulfide isomerase A5 [36] IPI0031479 51
UPS system Ubiquitin carboxyl-terminal hydrolase isozyme L1 IPI00018352 62
UPS system E3 Ubiquitin ligase UBR5 IPI00026320 35
UPS system E3 Ubiquitin ligase MARCH6 IPI00105518 29
UPS system Proteasome subunit alpha type-5 IPI00291922 23
antioxidant Glutathione S-transferase IPI00219757 60
antioxidant Thioredoxin IPI00216298 71
mitochondrial maintenance Prohibitin-1 [22] IPI00017334 38
mitochondrial maintenance Prohibitin-2 [22] IPI00027252 54
microtubule remodeling Stathmin IP00479997 37
neurite growth Neuron Navigator 2 isoform 1 IPI00217052 27
antinflammatory Cytosolic Phosolipase A2 IPI00384577 62
mitochondrial apoptosis Porin/VDAC1 IPI00216308 48
mitochondrial apoptosis Porin/VDAC2 IPI00241145 54
100nM chaperone/foldase HSP90-beta [5,14] IPI00414676 170
chaperone/foldase HSP90-alpha isoform 2 [5,14] IPI00382470 52
chaperone/foldase HSP90AB2P [5,14] IPI00455599 49
chaperone/foldase HSP90B1 endoplasmin/GRP94 [5,14] IPI00027230 59
chaperone/foldase HSP90 ATPase activator [5,14] IPI00030706 53
chaperone/foldase BiP IPI00003362 101
chaperone/foldase HSP70 protein 1 [25] IPI00304925 93
chaperone/foldase HSP70 protein 4 [25] IPI00002966 53
chaperone/foldase HSP70 protein 6 [25] IPI00339269 93
chaperone/foldase HSC71 isoform 1 [25] IPI00003865 236
chaperone/foldase GRP75 IPI00007765 76
chaperone/foldase HSP60 IPI00917575 235
chaperone/foldase HSP70/HSP90 organizing protein [3] IPI00013894 33
chaperone/foldase TCP1 T-complex protein 1 subunit beta [38] IPI00297779 94
chaperone/foldase TCP1 T-complex protein 1 subunit delta [38] IPI00302927 67
chaperone/foldase TCP1 T-complex protein 1 subunit epsilon [38] IPI00010720 109
chaperone/foldase Peptidyl-prolyl cis-trans isomerase B IPI00646304 72
chaperone/foldase Protein disulfide-isomerase A3 [36] IPI00025252 59
chaperone/foldase Protein disulfide-isomerase A6 isoform 2 [36] IPI00299571 107
chaperone/foldase Calnexin IPI00020984 52
UPS system Ubiquitin carboxyl-terminal hydrolase isozyme L1 IPI00018352 56
UPS system Ubiquitin-like modifier-activating enzyme 1 IPI00645078 38
UPS system Proteasome subunit beta type-1 IPI00025019 30
UPS system Proteasome subunit beta type-6 IPI00000811 44
antioxidant Glutathione S-transferase IPI00219757 80
antioxidant Thioredoxin IPI00216298 57
microtubule remodeling Stathmin IP00479997 77
microtubule remodeling Stathmin-2 IPI00218667 65
mitochondrial maintenance Prohibitin-1 [22] IPI00017334 60
mitochondrial maintenance Prohibitin-2 [22] IPI00027252 70
export misfolded proteins Transitional ER ATPase IPI00022774 46
suppress apoptosis Apoptosis Inhibitor 5 isoform 2 IPI00554742 32
pro-apoptosis Cytochrome c IPI00465315 34
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At 5nM, 10nM, 20nM, and 50nM concentrations of rotenone we identified tryptic peptides from 545 proteins with high probability, including proteins that are related to survival, growth, and protection (Table 1). Of great interest, at 5nM and 10nM, DJ-1 was identified. DJ-1 is classically associated with PD by deletion and point mutations shown to be responsible for the onset of familial PD [34]. We also detected BiP, starting at 10nM and continuing through 100nM. The expression of BiP indicates the activation of the UPR [32]. BiP can act to protect cells from oxidative stress. This stress can cause partial unfolding or aggregation of proteins. However, BiP can temporarily bind to hydrophobic residues exposed by ROS, thereby allowing the protein to potentially refold and/or preventing protein aggregation [25].

UPR activation has been shown to increase the expression of multiple chaperones, foldases, and components of the UPS and ERAD system [32]. This increase is clearly evident in Table 1. A recent immunohistochemistry study showed an increase in ER-resident chaperones in PD brain [35], suggesting that our results are relevant to human pathology. To validate an increase in UPS and ERAD components, we assayed the chymotrypsin-like activity of the 20S proteasome (Figure 4). From 20nM to 100nM rotenone, a significant increase in the activity of the chymotrypsin-like activity is seen (p= .000012) compared to the lower concentrations of rotenone and control. This suggests that the UPR has increased the expression of proteasome components necessary to handle the increased burden of misfolded/unfolded proteins due to the increase in oxidative stress. Also, of interest is the identification of the transitional ER ATPase at the 20nM and 100nM group. This protein regulates E3 ligase activity and may be required for export of misfolded proteins from the ER to be degraded by the proteasome. Of further importance, we identified two, components of the mitochondrial permeability transition pore (mPTP): Voltage-Dependant Anion Channel (VADC) 1 and 2. It has been shown recently that VDAC1 acts as a mitochondrial target of Parkin-mediated poly-ubiquitin chains and is therefore necessary for PINK1/Parkin-directed autophagy of damaged mitochondria [8]. Specifically it has been shown that with a decrease in mitochondrial membrane potential, Parkin translocates to the mitochondria in response to ROS. So with VDAC1 acting as a mitochondrial target of Parkin-mediated poly-ubiquitin chains, a Parkin-dependent mitophagic clearance could arrest the release of pro-apoptotic factors from damaged mitochondria [8]. So an increase in VDAC1 can aid in the removal of damaged mitochondria by assisting damaged mitochondria into forming autophagosomes.

Fig. 4. Proteasome activity Assay.

Fig. 4

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Fluorogenic substrate measured chymotrypsin-like activity in 20S proteasomes from SH-SY5Y cells exposed to various concentrations of rotenone for 48 hours. Data expressed as relative fluorescence units (RFU). Asterisk (*) indicates p=0.000012 by ANOVA: 20nM–100nM rotenone-treated group compared to control-10nM group.

At higher rotenone concentrations, we found several proteins that are involved in cytoskeletal remodeling and development of neurites (Stathmin1, Stathmin2, Neuron Navigator 2), as well as maintaining mitochondrial homeostasis (Prohibitin1, Prohibitin2) [27, 19, 30].

The UPR activation and its related products are able to keep the neurons healthy enough to sustain viability and overcome the induced stress up to a point. It has been clearly shown in past studies that with sustained UPR activation, apoptosis occurs [32, 13, 18]. Previous studies showed activation of the UPR after oxidative stress was induced in neuronal cells [12, 31] using high toxin concentrations that resulted in 50% cell death within 24-hours. We observed similar effects with 100nM rotenone, which significantly decreased cell viability (Fig. 1), increased caspase-3 activity (Fig. 3), and increased cytochrome c (Table 1). We believe this marks a threshold of defense that the neurons are capable of producing.

Our results indicate that SH-SY5Y cells activate the UPR acutely at rotenone doses that the cells can survive. As the concentration of rotenone increased so did the expression of proteins necessary to handle the accumulation of misfolded or unfolded proteins (Table 1). Much information exists about neurodegenerative disorders in the moderate to late stages of the diseases, where most of the outward symptoms manifest themselves. However, an understanding of the early stages of the progression towards apoptosis in PD can greatly assist research toward preventative therapies.

In conclusion, our whole cell proteomic analysis identified proteins that are involved in the protective UPR and ER stress pathways in dopaminergic neurons subjected to oxidative stress. Our results clearly show that these cells have the ability to overcome low-level oxidative damage, which might resemble the initial biochemical events of PD. However, with higher levels of stress, or prolonged stress, the protective pathways are insufficient to prevent the activation of apoptosis. The question remains whether the UPR and ER pathways can be harnessed and manipulated to provide a new early intervention strategy for treatment of PD and other neurodegenerative diseases.

Acknowledgments

We thank the UTSA Proteomics Core (supported by NIH G12 RR013646), and Dr. Maria Gaczynska for assistance.

Footnotes

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