Rotenone induces oxidative stress and dopaminergic neuron damage in organotypic substantia nigra cultures

Molecular Brain Research 134 (2005) 109 – 118 www.elsevier.com/locate/molbrainres

Research report

Rotenone induces oxidative stress and dopaminergic neuron damage in organotypic substantia nigra cultures Claudia M. Testaa,b,*, Todd B. Sherer a,b, J. Timothy Greenamyrea,b,c a

Center for Neurodegenerative Disease, Emory University, Atlanta, GA 30322, USA b Department of Neurology, Emory University, Atlanta, GA 30322, USA c Department of Pharmacology, Emory University, Atlanta, GA 30322, USA Accepted 4 November 2004 Available online 6 January 2005

Abstract Rotenone, a pesticide and complex I inhibitor, causes nigrostriatal degeneration similar to Parkinson disease pathology in a chronic, systemic, in vivo rodent model [M. Alam, W.J. Schmidt, Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats, Behav. Brain Res. 136 (2002) 317–324; R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson’s disease, Nat. Neurosci. 3 (2000) 1301–1306; S.M. Fleming, C. Zhu, P.O. Fernagut, A. Mehta, C.D. DiCarlo, R.L. Seaman, M.F. Chesselet, Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone, Exp. Neurol. 187 (2004) 418–429; T.B. Sherer, J.H. Kim, R. Betarbet, J.T. Greenamyre, Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation, Exp. Neurol. 179 (2003) 9–16.]. To better investigate the role of mitochondria and complex I inhibition in chronic, progressive neurodegenerative disease, we developed methods for long-term culture of rodent postnatal midbrain organotypic slices. Chronic complex I inhibition over weeks by low dose (10–50 nM) rotenone in this system lead to dose- and time-dependent destruction of substantia nigra pars compacta neuron processes, morphologic changes, some neuronal loss, and decreased tyrosine hydroxylase (TH) protein levels. Chronic complex I inhibition also caused oxidative damage to proteins, measured by protein carbonyl levels. This oxidative damage was blocked by the antioxidant a-tocopherol (vitamin E). At the same time, a-tocopherol also blocked rotenone-induced reductions in TH protein and TH immunohistochemical changes. Thus, oxidative damage is a primary mechanism of mitochondrial toxicity in intact dopaminergic neurons. The organotypic culture system allows close study of this and other interacting mechanisms over a prolonged time period in mature dopaminergic neurons with intact processes, surrounding glia, and synaptic connections. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Parkinson’s Keywords: Mitochondria; Complex I; Organotypic; a-Tocopherol; Parkinson disease; Antioxidant

1. Introduction Parkinson disease (PD) is a slowly progressive neurodegenerative disorder marked by relatively selective loss of * Corresponding author. Center for Neurodegenerative Disease, Department of Neurology, Emory University, Whitehead Biomed Research Building, 505F, 615 Michael Street, Atlanta, GA 30322, USA. Fax: +1 404 727 3728. E-mail address: [email protected] (C.M. Testa). 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.11.007

substantia nigra pars compacta (SNpc) dopaminergic neurons [26], along with the appearance of Lewy body intraneuronal inclusions and dystrophic neurites in SNpc and other affected areas [9,41]. Less than 10% of PD cases are clearly familial [13,18]. The cause of sporadic PD is unknown, but it likely involves a combination of genetic predisposition and long-term environmental exposures [22,58]. Epidemiological studies suggest that pesticide exposure can increase PD risk [21,42,59]. Many pesticides inhibit mitochondrial function. There is increasing evidence

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that mitochondrial dysfunction may be a key factor underlying specific neuronal loss in neurodegenerative disorders, particularly PD [5,22]. Investigation of mitochondrial dysfunction in PD gained momentum with the finding that a synthetic opiate contaminant, MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces acute, permanent parkinsonism via its active metabolite, MPP+ (1-methyl-4-phenyl-2,3,-dihydropyridinium), a mitochondrial complex I inhibitor [36,38]. The specific effect of MPTP on dopaminergic neurons, however, stems from its selective uptake into dopaminergic cells via the dopamine transporter [31]. In contrast, studies of idiopathic PD point to a systemic complex I impairment, with defects seen in platelets, muscle, and brain [10,24,37,39,45], implying that a systemic mitochondrial defect can lead to relatively selective neuronal damage. Indeed, chronic systemic exposure of rats to the pesticide rotenone, a classic mitochondrial complex I inhibitor, reproduces many features of PD, including levodopa-responsive motor deficits, nigrostriatal dopaminergic pathway degeneration, and intraneuronal inclusions [1,2,6,15,27,48]. This in vivo model therefore ties together systemic pesticide exposure, widespread complex I inhibition, and PD-like pathology. Rotenone models are now being used to investigate the mechanisms whereby dopaminergic neurons are injured in this chronic, systemic process. Complex I inhibition has several potentially damaging consequences. One possible result of complex I inhibition is increased formation of reactive oxygen species (ROS), creating oxidative damage within the cell. Oxidative stress has been implicated in PD [63]. Increased oxidative damage to lipids [14,32], DNA [44,62], and proteins [16] has been observed in PD SNpc, along with decreased levels of reduced glutathione [49]. Oxidative damage, rather than a bioenergetic defect, is also seen in the in vivo rotenone model [4,47]. The in vivo rotenone model is relevant to human PD pathophysiology as it reproduces key features of PD in a mature, intact adult mammalian brain with all of its inherent connections and cell–cell interactions. On the other hand, it is labor-intensive, expensive, and variable [15,27,48,65]. Dissociated cell culture systems are more easily manipulated, but these systems employ isolated, often non-neuronal cell types to investigate changes over a limited (hours to few days) time frame. While some relevant studies of complex I inhibition in dissociated cell culture employ mature dopaminergic neurons [52], the majority use immature cells. Organotypic dsliceT culture models represent a useful intermediate tool for studying chronic, progressive cell damage [17]. Slice cultures are simplified and flexible compared to in vivo models, yet still make use of mature neurons, remain viable in cultures for weeks to months, and maintain substantial neuron–neuron and neuronal–glial interactions [30,43]. Here, we use an organotypic slice culture system to investigate how mitochondrial dysfunction can lead to SNpc cell damage

in pathologic conditions such as PD. Rotenone was tested on intact postnatal neurons in this system for its chronic injury, rather than acute toxicity, effects. The system was then used to examine oxidative damage as a potential mechanism of chronic dopaminergic neuronal injury. Low concentrations of rotenone over weeks resulted in slow loss of dopaminergic cell processes, changes in cell morphology, and decreased tyrosine hydroxylase (TH) protein, along with increased oxidative damage to proteins. The antioxidant a-tocopherol protected slices from oxidative damage, while simultaneously preventing SNpc morphologic damage and TH protein loss. Thus, oxidative damage is an important mechanism underlying dopaminergic cell damage. Chronic organotypic slice cultures provide a useful model system for investigating mechanisms of neurodegenerative disease.

2. Materials and methods 2.1. Organotypic slice cultures The protocol was modified from Stoppini et al. [57]. All animal use was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and was approved by Emory University Institutional Animal Care and Use Committee. All care was taken to minimize pain or discomfort. Postnatal day 10 (P10) Lewis rat pups were fully anesthetized with isoflurane. Brains were rapidly removed and transferred to cold sterile chopping buffer (in mM, 110 sucrose, 60 NaCl, 3 KCl, 1.25 NaH2PO4, 28 NaHCO3, 5 d-glucose, 0.5 CaCl2, 7 MgCl2, 0.6 ascorbate) in a sterile hood work area. Brains were rapidly blocked, glued to the slicer chuck (OTS-4000 tissue slicer, FHC Inc, Maine), and transferred to the slicer basin filled with cold, oxygenated chopping buffer. Sections were cut to 300 Am and transferred to a sterile petri dish of cold dissection medium [Gey’s balanced salt solution (Sigma, Saint Louis, Missouri) with 0.5% glucose and 3 mM KCl]. Coronal sections at the level of the substantia nigra were chosen. Sections were inspected and cut into slices under a dissection microscope. During this procedure, the midbrain was isolated and then cut into two hemispheres, as detailed below. Slices therefore included the SNpc, surrounding cells, other midbrain nuclei, and local connections, not a full nigrostriatal pathway. After inspection, each slice was transferred onto a Millicell-CM membrane insert (Millipore, Billreka, Massachusetts) set in a 6-well plate on 1 ml of OPTI-MEM (GibcoBRL, Carlsbad, California)-based serum-containing medium. Plates were kept in a 37 8C tissue culture incubator. After 2–4 days, medium was changed to Neurobasal (GibcoBRL)-based serum-free medium with B-27 (GibcoBRL) and l-glutamine without antibiotics or antimitotics. Medium was then changed three times a week. Slices were grown for 10 days prior to drug treatments.

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2.2. Drug treatments

2.4. Protein extraction

For drug treatments, coronal sections at the level of the SNpc were cut in half, separating the two hemispheres. The two half sections were then used as a matched pair of slices, one receiving drug, and the other used as an internal control. Three sets of matched slices from one rat were grown in each 6-well plate, one slice per well. Matched slice sets were treated with rotenone vehicle (100% ethanol) versus rotenone alone, rotenone and a-tocopherol (dl-a-tocopherol, Avacado Research Chemicals, UK), or rotenone and a-tocopherol vehicle dimethyl sulfoxide (DMSO, Sigma). Some slice pairs received a-tocopherol versus DMSO, or DMSO versus no treatment. Drug treatments lasted 1–6 weeks. Fresh drugs were added to the medium at every medium change. At the end of treatments, matched pairs were processed in parallel, then analyzed for change in the drug-exposed slices compared to the matched vehicle slices. Immunohistochemistry sections were compared directly. For protein analysis, percent change of the treated sample from its matched control was generated for each pair; these numbers were then averaged per experimental condition (see Protein sample analysis section).

Slices were rinsed in phosphate buffered saline (PBS), cut out of membrane insert frames, combined with protein extraction reagent (Pierce, Illinois), protease inhibitors (Sigma), and 1% h-metcaptoethanol, and homogenized in the cold. Tissue homogenate was centrifuged at 10,500 rpm for 5 min at 4 8C. Supernatant containing protein extract was stored at 80 8C. Total protein concentration was determined with a Molecular Devices Spectromax plate reader system.

2.3. Immunohistochemistry For immunohistochemistry experiments, entire tissue culture plates, i.e., three slice pairs per rat, were fixed and stained. For protein carbonyl and Western blot experiments (see below), some complete plates were fixed. The majority was processed for protein extraction; for those plates, one slice pair per rat, at least two rats per condition, was fixed to allow direct comparison of immunohistochemistry and protein results. Slices were fixed with cold 4% paraformaldehyde, rinsed in PBS and stored at 4 8C in PBS. Slices were subsequently thoroughly rinsed with Tris buffered saline (TBS), treated with 3% H2O2 for 10 min, gently detached from their membrane inserts, and rinsed again with TBS. They were then incubated in 10% normal goat serum in TBS with 0.5% Triton X-100 for 10 min, and transferred to the same solution with primary antibody (mouse anti-rat monoclonal to tyrosine hydroxylase, Chemicon, Temecular, CA) overnight. Slices were then rinsed in TBS, incubated in biotinylated goat anti-mouse secondary antibody (1:200, Jackson ImmunoResearch, West Grove, Pennsylvania), followed by avidin–biotin complex method detection (ABC elite kit, Vector Laboratories, Burlingame, California). The final product was visualized with 3,3V-diaminobenzidine tetrachloride (DAB, Sigma). Sections were examined and photographed under brightfield microscopy with Axiovision (Zeiss, Oberkochen, Germany) software. All images presented were shot on the same day, under identical light and camera conditions, using a 10 objective. Digital images were processed identically with Adobe Photoshop to assemble montages.

2.5. Protein sample analysis To generate sufficient protein for analysis, two to three identically treated slices from one rat (one tissue culture plate) were pooled per protein extraction. The matched vehicle control slices were pooled the same way; paired treated and control protein samples were then processed in parallel. If two slice pairs from one rat were used for protein, the third pair was fixed and processed for immunohistochemistry as above. Results for each treated sample were compared to its matched control sample to generate a percent change from control for each rat. Protein samples were analyzed for both protein carbonyls and TH protein levels via Western blot (see below) whenever possible. However, some samples had protein concentrations too low for protein carbonyl analysis, or total protein levels too low to generate samples for both analyses; then, either Western blot or protein carbonyl measurements were done. In all cases, only matched pairs in which both treated and control samples from the same rat could be analyzed together were used; treated or control samples were never run in isolation. 2.6. Protein carbonyl detection The Oxyblot Protein Oxidation Detection Kit (Serologicals, Atlanta, Georgia) was used according to the manufacturer’s protocol on dot blot membranes. Protein carbonyl detection was done within 1 week of sample collection. Briefly, 10 Ag total protein per sample was mixed with an equal volume of 12% SDS and two volumes of 1 2,4dinitrophenylhydrazine (DNPH), which derivatizes protein carbonyls to 2,4-dinitrophenylhydrazone (DNP). After the reaction was stopped with the provided neutralization solution, 2 or 3 Ag total protein was spotted onto the dot blot membrane (same total protein amount used for all dots in one experiment). Dot blots were made in duplicate and analyzed as averages of the duplicates whenever both membranes were available. Protein was cross-linked to an Immobulink-P membrane (Millipore) with a UV Stratalinker (Stratagene, La Jolla, California). DNP was detected with the rabbit anti-DNP primary antibody and goat antirabbit IgG secondary antibody as provided in the oxyblot kit. After immunostaining as per the oxyblot kit, signal was

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detected using Kodak Digital Science 1D image analysis software version 3.0.2. The same-sized circle was used to capture net intensity for all dots in one experiment. 2.7. Western blot analysis 5 Ag total protein per lane of extracted protein samples underwent gel electrophoresis on a 12% polyacrylamide gel at 200 V for 40 min, then transferred to an Immobilonk-P membrane (Millipore) in a BioRad wet buffer (Licor Biosciences, Lincoln, Nebraska). Membranes underwent double label immunohistochemistry using primary antibodies for TH (1:2000, mouse anti-rat monoclonal, Chemicon) and MAP kinase p44/42 (1:1000, mouse anti-rat monoclonal, Cell Signalling Technology). Signal detection was with IRdye 800-conjugated goat anti-rabbit (Rockland, Gilbertsville, Pennsylvania) and Alexa Fluro 680-conjugated goat anti-mouse (Molecular Probes, Oregon) secondary antibodies. Signal was detected on an Odyssey Imager (Licor Biosciences). Protein from each sample was run simultaneously on two different blots; duplicate blots were averaged whenever both were available. TH signal was normalized to MAP kinase, detected on the same blots. 2.8. Statistical analysis A two-tailed, paired Student’s t test for independent samples was used for statistical analysis. Significance was set at P V 0.05.

3. Results 3.1. Rotenone affects intact substantia nigra dopaminergic neurons Matched pairs of slices were treated over weeks with low concentrations of rotenone or vehicle (100% ethanol), then fixed and stained with tyrosine hydroxylase (TH) immunohistochemistry. SNpc neurons were identified by size and location compared to other TH-positive cells. With increasing rotenone dose and exposure duration, TH-positive SNpc neurons showed progressive loss of processes compared to matched vehicle-treated slices (Fig. 1). Eventually, there was apparent loss of TH-positive neurons compared to a general neuronal marker (NeuN), although this was not quantified. Exposure to 5 nM rotenone for up to 4 weeks had little effect (data not shown). A rotenone concentration of 10 nM had little to no effect on TH-positive neurons at 1 or 2 weeks, but resulted in a mild loss of processes at 4 weeks (Fig. 1GH), and minimal further morphologic changes at 6 weeks (data not shown). 20 nM rotenone was consistently toxic at 2 weeks, but had much less effect at 1 week (Figs. 1C–F). 20 nM at 4 weeks caused severe cell loss (data not shown). Similar to 20 nM over 2 weeks, 50 nM at 1 week led to distinct loss of cell processes and morphologic changes, with

Fig. 1. TH immunoreactivity changes after chronic rotenone treatment. Matched pairs of slices (AB, CD, EF, GH) were exposed to vehicle (A, C, E, G) versus 10 to 50 nM rotenone for 1–4 weeks. Each pair features two slices, one from each hemisphere of the same rat brain section, cultured and treated in the same experiment. (B) 50 nM rotenone exposure over 1 week results in loss of TH-immunoreactive processes with only mild cell loss. (D) In contrast, slices exposed to 20 nM rotenone over 1 week show only mildly decreased processes compared to control. (F) The effect of 20 nM rotenone exposure for 2 weeks resembles that of 50 nM  1 week treatment. (H) Slices exposed to 10 nM rotenone start to show mild loss of processes after 4 weeks. Scale bar = 100 Am.

only mild cell loss (Fig. 1AB). Thus, TH immunoreactivity changes progressed with increasing time at a given rotenone dose, or increasing doses over the same duration. Chronic rotenone treatments can therefore be biased towards sick neurons with decreasing processes and ongoing damage, rather than acute or overwhelming cell loss. In subsequent experiments, TH immunohistochemistry was used to confirm damage or protection of SNpc neurons. 3.2. Rotenone induces oxidative damage in organotypic midbrain slice cultures ROS and other free radicals can damage all types of macromolecules, including proteins and lipids. One com-

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mon form of oxidative damage to proteins is protein carbonyl formation. Protein sulfhydryl groups can react directly with ROS, or with lipid peroxidation breakdown products generated by lipid oxidation, to produce protein carbonyls [55]. Protein carbonyl levels can therefore be used as an index of oxidative stress and resulting oxidative damage. To examine oxidative stress as a mechanism of rotenone-induced injury, protein carbonyl measurements were performed on matched sample pairs, as detailed in Materials and methods (see especially Drug treatments and Protein sample analysis sections). Briefly, pairs of rotenonetreated and control samples underwent dot blot analysis for protein carbonyls. Protein carbonyl levels were then compared between rotenone versus vehicle conditions within each matched pair; each pair comprised slices from one rat. To address mechanisms of chronic neuronal injury prior to actual cell death, rotenone doses were chosen based on production of chronic injury and loss of processes, rather than apparent cell loss (see section above). Chronic exposure to the complex I inhibitor rotenone resulted in oxidative damage, as indicated by increased levels of protein carbonyls (Figs. 2A and 3A). Oxidative damage increased with rotenone dose and exposure time. The change compared to vehicle-treated control samples was significant ( P b 0.002) for both 50 nM rotenone at 1 week exposure, and 20 nM rotenone at 2 weeks exposure. No significant change in protein carbonyls was seen after 1 week of 20 nM rotenone, or 2 to 4 weeks of 10 nM rotenone (data not shown). 3.3. a-Tocopherol protects slices from rotenone-induced oxidative damage To further test the role of oxidative damage in rotenoneinduced neuronal injury, slices were treated with 100 AM or 10 AM of the antioxidant a-tocopherol along with rotenone. Slices were treated in parallel with rotenone-only groups, in the same experiments as above. Protein samples were then analyzed via dot blots for protein carbonyl levels, as described above. a-Tocopherol 100 AM rescued slices from oxidative damage induced by 50 nM rotenone for 1 week (Fig. 2A). The 100-AM concentration of a-tocopherol also significantly attenuated protein carbonyl levels when added to 20 nM rotenone for 2 weeks (Fig. 3A). Addition of 10 AM a-tocopherol with either rotenone dose was ineffective (data not shown). The a-tocopherol vehicle DMSO, at the same concentration used for 100 AM a-tocopherol treatments, did not significantly alter oxidative damage by either rotenone dose (data not shown). 3.4. Rotenone-induced oxidative damage to organotypic slices is associated with damage to TH-positive neurons Protein carbonyl measurements indicate oxidative damage to the slice as a whole. We therefore used TH immunohistochemisty and Western blotting to confirm

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Fig. 2. Chronic rotenone exposure concurrently induces oxidative damage, decreases TH protein levels, and damages TH-positive neurons. aTocopherol significantly reduces rotenone-induced oxidative damage in slices, while also preserving TH protein levels, and restoring neuronal morphology. (A) Protein carbonyls as a measurement of oxidative damage by rotenone. 50 nM rotenone (R) over 1 week results in a significant increase of protein carbonyls over untreated (vehicle only) matched controls. 100 AM a-tocopherol (aT) added with rotenone affords significant protection from oxidative damage. Solid black bars, rotenone exposure alone. Gray bars, rotenone plus 100 AM a-tocopherol. *P b 0.02; **P b 0.002. Data are shown as percent change treated samples compared to matched controls, means F SEM of samples from 14 rats per condition, run in 7 independent experiments. (B) Treatment with 50 nM rotenone for 1 week results in a significant decrease in TH protein compared to matched control levels. Samples are the same experimental groups, in most cases the identical protein samples, presented in panel (A). Thus, 50 nM  1 week rotenone exposure decreases TH protein levels along with increasing oxidative damage. Addition of 100 AM a-tocopherol with rotenone significantly preserves TH protein levels, as well as preventing protein carbonyl formation. #P b 0.01, ##P b 0.001. Data are shown as percent change treated samples compared to matched controls, means F SEM of slice pairs from 9 to 12 rats, run in 5 independent experiments. (C–F) TH immunohistochemistry confirms damage versus protection of SNpc neurons. (CD, EF) Matched pairs of slices taken from the same rats and treated in the same experiments as adjacent slices analyzed for oxidative damage (A) and TH protein levels (B), were immunostained for TH. (C, E) TH-immunoreactive SNpc neurons in vehicle-treated control slices appear normal. (D) Rotenone at 50 nM for 1 week damages dopaminergic neurons. TH-positive SNpc neurons exhibit prominent loss of processes, as well as some cell body shrinkage, and minimal cell loss. Staining changes correlate with reduced TH protein (B) and increased protein carbonyl formation (A) in slices from the same experiments. (F) In contrast, SNpc neurons exposed to 100 AM a-tocopherol with 50 nM rotenone over 1 week retain much of their normal appearance, in concert with preserved TH protein (B) and reduced protein carbonyl levels (A). Scale bar = 100 Am.

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damage to dopaminergic neurons under the same conditions that increased oxidative stress. Samples from the same drug treatment experiments analyzed for protein carbonyl formation were also used for Western blot detection of TH protein levels, as detailed in Materials and methods (see Protein sample analysis section). In all possible cases, the exact same protein samples run for protein carbonyl levels were also used for Western blotting. TH protein levels decreased in rotenoneexposed slices compared to untreated slices, after 50 nM rotenone for 1 week (Fig. 2B) or 20 nM over 2 weeks (Fig. 3B). The change in TH compared to matched vehicle-treated controls was significant for both rotenone doses ( P b 0.001 for 50 nM  1 week, P b 0.02 for 20 nM  2 weeks). TH protein levels were therefore relatively depressed in slices with oxidative damage. TH immunohistochemistry on slices from the same rats and same tissue culture plates used for protein carbonyl and Western blot analyses confirmed damage to TH-positive SNpc neurons after both 50nM  1 week (Fig. 2CD) and 20 nM  2 weeks (Fig. 3CD) rotenone doses, consistent with the oxidative damage results. TH immunoreactivity changes were similar to those seen in initial immunohistochemisty experiments (Fig. 1).

Fig. 3. The effects of 20 nM rotenone over 2 weeks, shown here, are similar to those of 50 nM  1 week (Fig. 1). Oxidative damage, TH protein levels, and TH immunohistochemistry change in tandem with chronic low-dose rotenone exposure. At this lower dose, longer duration rotenone treatment, a-tocopherol again significantly attenuates rotenone-induced oxidative damage while protecting dopaminergic neurons. (A) Protein carbonyls as a measurement of oxidative damage. Exposure of slices to 20 nM rotenone (R) over 2 weeks results in a significant increase of protein carbonyls over untreated (vehicle only) matched controls. 100 AM a-tocopherol (aT) added with rotenone affords significant protection from oxidative damage. Solid black bars, rotenone exposure alone. Grey bars, rotenone plus 100 AM a-tocopherol. **P b 0.002; P b 0.005. Data are shown as percent change treated samples compared to matched controls, means F SEM of samples from 13 to 14 rats per condition, run in 5 independent experiments. (B) Treatment with 20 nM rotenone for 2 weeks results in significantly decreased TH protein levels compared to matched controls. Samples are the same experimental groups, in most cases the identical protein samples, presented in panel (A). Thus 20 nM  2 weeks rotenone exposure decreases TH protein levels along with increasing oxidative damage. Addition of 100 AM a-tocopherol with rotenone significantly preserves TH protein levels, as well as preventing protein carbonyl formation. *P b 0.02; P b 0.05. Data are shown as percent change treated samples compared to matched controls, means F SEM of slice pairs from 12 to 17 rats, run in 5 independent experiments. (C–F) TH immunohistochemistry confirms damage versus protection of SNpc neurons. (CD, EF) Matched pairs of slices taken from the same rats, and treated in the same experiments as adjacent slices analyzed for oxidative damage (A) and TH protein levels (B), were immunostained for TH. (C, E) TH-immunoreactive SNpc neurons in vehicle-treated control slices appear normal. (D) Rotenone at 20 nM for 2 weeks markedly reduces TH-positive SNpc processes; changes are similar to those seen with 50 nM  1 week (Fig. 1CD) Staining changes correlate with reduced TH protein and increased protein carbonyl formation. (F) In contrast, SNpc neurons exposed to 100 AM a-tocopherol with 20 nM rotenone over 2 weeks retain much of their normal appearance, in concert with preserved TH protein (B) and reduced protein carbonyl levels (A). Scale bar = 100 Am.

3.5. a-Tocopherol protects dopaminergic neurons from rotenone-induced damage a-Tocopherol given with rotenone attenuated the rotenone-induced protein carbonyl increases. If oxidative stress contributes to dopaminergic neuron damage, then we expect a-tocopherol to also protect dopaminergic cells, concomitant with decreasing overall oxidative damage. To test this, a-tocopherol plus rotenone-treated protein samples from the same group of experiments analyzed for protein carbonyl formation were also used in Western blot detection of TH protein levels, as detailed above. a-Tocopherol at 100 AM significantly preserved TH protein levels along with protecting slices from oxidative damage, for both 50 nM rotenone for 1 week (Fig. 2B) and 20 nM over 2 weeks (Fig. 3B) doses. For both rotenone doses, TH levels after rotenone plus a-tocopherol were not significantly different compared to matched vehicle-treated controls (Figs. 2B and 3B). DMSO (a-tocopherol vehicle) added with rotenone did not significantly change the decrease in TH protein levels produced by either 50 nM or 20 nM rotenone doses alone (data not shown). TH immunohistochemistry on slices from the same rats, adjacent to the slices used for protein samples, confirmed protection of SNpc cells and processes by 100 AM a-tocopherol added with rotenone (Figs. 2EF and 3EF). 100AM a-tocopherol alone or DMSO alone for 1 to 2 weeks did not alter TH-positive SNpc neurons in slice culture compared to untreated controls (data not shown). 10 AM a-tocopherol added with rotenone did not improve TH immunoreactivity over rotenone alone

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pairs at either rotenone dose (data not shown). Thus, the antioxidant a-tocopherol prevented rotenone-induced oxidative damage, TH protein level changes, and SNpc neuronal injury.

4. Discussion Chronic organotypic slice cultures are a good model system for investigation of long-term low-level mitochondrial injury, potentially a key contributor to PD pathophysiology. With chronic exposure to the mitochondrial complex I inhibitor rotenone, we observed destruction of TH-positive SNpc neuron processes, morphologic changes, and decreased TH protein levels in rat slices. Complex I inhibition can damage human dopaminergic neurons, as demonstrated by acute MPTP toxicity [36]. The MPTP product MPP+ harms neurons by inhibiting complex I [38]; however, the selective effect of MPP+ on dopaminergic neurons is created by its active transport into these cells [31]. In contrast to this specifically targeted toxin, there is evidence for systemic complex I impairment in PD [10,24,37,39,45]. Mechanisms of mitochondrial dysfunction and neuronal injury in PD must then account for both widespread decreases in complex I activity and relatively selective neuronal injury. Rotenone, a highly lipophilic organic pesticide, readily crosses the blood–brain barrier and cell membranes without specific, active transport. Systemically administered rotenone acts on complex I throughout the brain, not solely in dopaminergic neurons [6]. Even so, systemic rotenone exposure in rats [1,6,65] and primates [23] leads to particularly prominent SNpc degeneration. The in vivo rotenone model demonstrates that a widespread mild complex I defect can have a greater pathologic impact on specific neurons. However, in vivo rotenone effects can vary [15,27]; for example, about half of the rats that survive the full chronic rotenone infusion course develop SNpc degeneration [48,65]. Chronic organotypic cultures allow extended low-dose rotenone exposure of late postnatal SNpc neurons in a controlled, reproducible system. Damage to dopaminergic neurons can be directly visualized with TH immunohistochemistry, and measured with TH protein levels. We therefore used this simplified but consistent format to explore the connection between complex I inhibition and oxidative stress. Complex I inhibition has several potential functional consequences, including decreased ATP production, altered calcium handling, and oxidative damage. Complex I is the first of four multisubunit protein complexes in the mitochondrial electron transport chain responsible for converting energy from cellular metabolism into the proton gradient used by complex V to generate ATP. Electron transport chain dsyfunction could lead to loss of the proton gradient, impaired ATP production, and a bioenergetic defect. However, in dissociated culture systems, rotenone does

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not kill cells via ATP depletion [47,64]. Instead, rotenone increases oxidative stress [47,64]. A moderate level of complex I inhibition, as postulated for PD, causes significant increases in oxygen-based free radicals (reactive oxygen species, ROS) [51]. Mitochondria are the major source of ROS in nearly all cell types. Electrons are normally transferred forward through complex I via tightly constrained ubiquinone intermediates, with a small bleakQ of partially reduced semiquinone species able to interact with molecules outside the electron transport chain and generate free radicals [8]. Molecular oxygen, an avid electron acceptor consumed in normal mitochondrial functioning, is readily available to pick up bleakedQ electrons. ROS are thus generated at a low rate during mitochondrial respiration [11]. The electron leak is upstream of the rotenone and MPP+ binding area, so partial complex I inhibition by these compounds can create a back up of electrons, increasing the electron leak, ROS levels, and oxidative stress [3,25,35]. Although ROS act in normal cell signaling, excess ROS attack mitochondrial and cellular components. The results of increased ROS are seen in PD substantia nigra: oxidative damage to lipids [14,32,61], proteins [16], and DNA [44,62], along with depletion of antioxidant defenses such as reduced glutathione [40,49]. Increased oxidative stress is therefore implicated in PD pathology. Mitochondrial toxin models demonstrate the link between complex I inhibition and oxidative damage. MPTP induces oxidative stress in mouse brain [54]. Oxidative damage is a primary functional consequence of rotenone in dissociated cell systems [46,64] and in vivo [47]. We show here that chronic rotenone exposure causes oxidative damage in slices in a dose- and time-dependent manner. Damage to dopaminergic neurons in slices correlates with increased protein oxidation, and decreases with protection from oxidative stress. Oxidative damage as a mechanism of rotenone-induced neuronal injury was confirmed in the slice culture system by concurrent rescue of oxidative stress levels, TH protein levels, and TH immunohistochemisry changes with atocopherol. a-Tocopherol is the most extensively studied of the vitamin E compounds, a group of tocopherols and tocotrienols that provides the major lipid-based antioxidant activity in mammalian cells [56]. In addition to its protective effect in slice cultures, a-tocopherol decreases MPP+ and rotenone-induced oxidative stress and cell death in dissociated cell culture systems [20,47]. Despite its efficacy in vitro, a-tocopherol has not performed well in PD clinical trials. Potential reasons include slow absorption and poor CNS penetration [60]. Its lipophilic profile and potential binding to nonspecific surfaces, both natural and artificial, may drive its effective concentration level up in vitro as well as in vivo, as seen in this and other studies [20,47]. Even so, the organotypic slice experiments presented here demonstrate that a-tocopherol can effectively rescue postnatal dopaminergic neurons in their native, in situ context from a complex I inhibitor. This system therefore provides a tool

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for investigating mechanisms of toxicity and neuroprotection without in vivo limitations such as absorption or liver metabolism. Antioxidants vary widely in their actions: some scavenge particular ROS while others react more generally; some change from anti- to pro-oxidant activities within physiologic range; and many have other roles unrelated to reduction/oxidation. Future studies will take advantage of this model system to detail antioxidant and other effects of potential therapeutics. Oxidative stress may interact with other pathophysiologic mechanisms, including genetic lesions, to create PD pathology [22]. For example, oxidative stress may contribute the to a-synuclein aggregation seen in PD and mitochondrial dysfunction models. a-Synuclein damaged by free radicals is more prone to aggregation [53]. In vivo rotenone infusion generates both increased insoluble protein carbonyls and increased a-synuclein aggregation [6,7,27]. Oxidative modification of a-synuclein in PD brain can lead to protein aggregation [19]. Conversely, elevated a-synuclein in vitro can make cells more sensitive to oxidative stress [28,33,34]. Increased levels of normal a-synuclein can cause PD [12,29,50]; the combination of high asynuclein levels with oxidative stress may partially explain this phenomenon. Transgenic mice provide valuable asynuclein overexpression and familial PD-associated mutation models. Mice rapidly metabolize rotenone; however, rotenone consistently injures mouse SNpc neurons in slices (our own observations), making use of transgenic mouse tissue sources possible in this system. Studies underway combine exogenous toxins with genetic lesions to investigate the basic interactions between a-synuclein and other disease-causing proteins with mitochondrial dysfunction. Better understanding of basic neuronal injury pathways and neuroprotective mechanisms may help design new therapeutic agents with both targeted mechanisms of action and improved in vivo activity.

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This work was supported by NS044267 and a Cotzias Fellowship from the American Parkinson Disease Association (CMT); Michael J. Fox Foundation Fellowship (TBS); ES12068 and the Picower Foundation (JTG). The authors wish to thank Christine Marstellar and Michael Bryant for their excellent technical assistance.

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