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Published in final edited form as: J Neurochem. 2011 Jan 19;116(6):1005–1017. doi: 10.1111/j.1471-4159.2010.07147.x

Methamphetamine oxidatively damages parkin and decreases the activity of 26S proteasome in vivo

Anna Moszczynska 1, Bryan K Yamamoto 1
PMCID: PMC3610410  NIHMSID: NIHMS259081  PMID: 21166679

Abstract

Methamphetamine (METH) is toxic to dopaminergic (DAergic) terminals in animals and humans. An early event in METH neurotoxicity is oxidative stress followed by damage to proteins and lipids. The removal of damaged proteins is accomplished by the ubiquitin-proteasome system (UPS) and the impairment of this system can cause neurodegeneration. Whether dysfunction of the UPS contributes to METH toxicity to DAergic terminals has not been determined. The present investigation examined the effects of METH on functions of parkin and proteasome in rat striatal synaptosomes. METH rapidly modified parkin via conjugation with 4-hydroxy-2-nonenal (4-HNE) to decrease parkin levels and decreased the activity of the 26S proteasome while simultaneously increasing chymotrypsin-like activity and 20S proteasome levels. Prior injections of vitamin E diminished METH-induced changes to parkin and the 26S proteasome as well as long-term decreases in DA and its metabolites’ concentrations in striatal tissue. These results suggest that METH causes lipid peroxidation-mediated damage to parkin and the 26S proteasome. As the changes in parkin and 26S occur before the sustained deficits in DAergic markers, an early loss of UPS function may be important in mediating the long-term degeneration of striatal DAergic terminals via toxic accumulation of parkin substrates and damaged proteins.

Keywords: methamphetamine, toxicity, parkin, proteasome, 4-hydroxy-2-nonenal, dopamine

Introduction

Methamphetamine (METH) is a highly abused psychostimulant drug that, when administered at high doses to experimental animals, causes a selective degeneration of DAergic terminals in the striatum while sparing cell bodies in the substantia nigra pars compacta (SNc) (Hotchkiss & Gibb 1980, Ricaurte et al. 1982). One of early events in METH toxicity is an increase in extracellular DA concentration (Sulzer & Rayport 1990) followed by an overproduction of toxic metabolites of DA oxidation (Wrona et al. 1997), oxidative damage to proteins and lipids (Yamamoto & Zhu 1998, Gluck et al. 2001, Eyerman & Yamamoto 2007), and long-term reductions in DAergic markers in the striatum (Hotchkiss & Gibb 1980, Wagner et al. 1980). Oxidative stress and persistent reductions DAergic markers have also been observed in striatum of human chronic METH users (Wilson et al. 1996, Mirecki et al. 2004, Chang et al. 2007).

The ubiquitin-proteasome system (UPS) is responsible for removal of short-lived regulatory proteins and for degradation of damaged or misfolded proteins. It consists of a 26S proteasome, a large ATP-dependent proteolytic complex, and three classes of enzymes, including E3 ubiquitin-protein ligases such as parkin (EC 6.3.2.-) (Moore 2006), that add polyubiquitin chains to proteins destined for degradation. The 26S is comprised of a catalytic core (20S proteasome) and two regulatory caps (19S proteasomes). The 20S proteasome can act independently of the regulatory caps to degrade oxidized proteins in an ubiquitin- and ATP-independent fashion (Hershko & Ciechanover 1998, Davies 2001).

A decrease in UPS function can lead to toxic accumulation of unwanted proteins and produce damage to DA neurons as evidenced in Parkinson’s disease (Buneeva & Medvedev 2006, Lim 2007). Fornai et al. (2003) linked dysfunction of the striatal proteasome with DA-dependent neuronal loss by showing that an impairment of the proteasome produced a selective DA-dependent toxicity to striatal DAergic terminals, retrograde cell death, and protein aggregation in the SNc in rats. In fact, protein aggregation was observed in the SNc of METH users (Quan et al. 2005), thus indirectly linking METH action with dysfunction of the proteasome in vivo. METH decreased the in vitro activity of 20S proteasome and promoted the formation of ubiquitin- and parkin-positive protein aggregates in DAergic cells (Fornai et al. 2003, Lazzeri et al. 2007). The function of the UPS can be impaired by oxidative damage to any of its components (Shang & Taylor 1995, Bulteau et al. 2001) including the accumulation of damaged parkin in insoluble aggregates (Winklhofer et al. 2003, Chung et al. 2004, LaVoie et al. 2005, Wang et al. 2005, LaVoie et al. 2007). Whether in vivo exposure to METH causes dysfunction of parkin and/or proteasome and contributes to the toxicity to DAergic terminals is unknown.

Based on the very limited and indirect evidence linking METH toxicity to DAergic terminals and damage to the UPS, the present investigation tested the hypothesis that high-dose METH decreases the functions of parkin, 26S proteasome, and 20S proteasome in striatal terminals in vivo. To this end, a rat model of METH toxicity was used to examine parkin and proteasomes in striatal synaptosomes shortly after the administration of METH. Immunoprecipitation of parkin was employed to examine the nature of the damage to parkin. Vitamin E was used to assess the involvement of lipid peroxidation in METH effects on the UPS. The present study demonstrates that METH oxidatively modifies parkin via conjugation with 4-hydroxy-2-nonenal (4-HNE) to decrease parkin levels and the activity of 26S proteasome in striatal synaptosomes.

Materials and Methods

Subjects

Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 175–200 g at the beginning of the experiments were housed two per cage under a 12 h light/dark cycle (lights on from 7:00 A.M. to 7:00 P.M.) in a temperature 21–23°C and humidity-controlled room. Food and water were available ad libitum. Temperature of the rats was measured via a rectal probe digital thermometer (Thermalert TH-8; Physitemp Instruments, Clifton, NJ). All experiments were performed between 7:00 A.M. and 7:00 P.M. and in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC committee of the University of Toledo.

METH and vitamin E administration

D-Methamphetamine hydrochloride and vitamin E were purchased from Sigma-Aldrich (St. Louis, MO). METH (10 mg/kg) or 0.9% saline (1 mL/kg) was administered to rats at 2 hr intervals for a total of four i.p. injections. Vitamin E was administered at a dose of 20 mg/kg per i.p. injection once a day for 4 days and on the day of METH administration, 30 min prior to each METH injection. A similar vitamin E regimen has been shown to block the long-term DA depletions produced by METH (Park et al. 2006).

Preparation of synaptosomes

Crude synaptosomal fractions were prepared from striatal, frontal cortical and cerebellar tissues via differential centrifugation as described previously (Riddle et al. 2002). Briefly, brain areas were dissected, homogenized with a hand glass homogenizer in 0.5 mL of ice-cold 0.32 M sucrose (for western blotting, 20S and 26S assay) or 0.32 M sucrose/5mM deferoxamine mesylate (for immunoprecipitation) and centrifuged (800 × g for 24 min; 4°C) to remove nuclei and large debris (P1). The supernatant (S1) was centrifuged at high speed (22,000 × g for 17 min; 4°C), and the pellet (P2) retained was the crude total synaptosomal fraction. This fraction was re-suspended in ice-cold distilled deionized water (ddH2O) (for western blotting, 20S and 26S assay) or RIPA buffer (50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1.5 M NaCl, 1% Nonidet P-40) containing 5 mM deferoxamine mesylate and protease inhibitor cocktail (for immunoprecipitation). For measurement of 26S activity, water-solubilized synaptosomes were combined (1:1) with proteasome extraction buffer (100 mM Tris-HCl, pH 7.5, 5 mM β-mercaptoethanol, 1 mM EDTA-Na2, 80 mM KCl, 0.5 M sucrose, 10 mM MgCl2, 20% glycerol, 40 µM digitonin, 2 mM ATP, 20 units/mL creatine phosphokinase, 24 mM creatine phosphate dibasic tetrahydrate, and 2 × protease inhibitor cocktail). Total synaptosomes were centrifuged at high speed (22,000 × g for 17 min; 4°C). The resulting supernatant (S3) yielded the crude cytosol-vesicular fraction. For immunoprecipitation experiments, solubilized synaptosomes were briefly (5 s) sonicated on ice and separated into cytosol-vesicular (S3) and membrane (P3) fractions. Synaptosomal fractions were analyzed for protein concentration by the method of Bradford (Bradford 1976) using bovine serum albumin as the standard.

Western blot analysis of parkin and 20S proteasome

Striatal, frontal cortical, and cerebellar synaptosomes were prepared from rats decapitated 1, 24 and 48 hrs after the 4th METH injection and subjected to non-reducing SDS-PAGE, blocking for 1 hr at room temperature (RT) with 5% nonfat dried milk dissolved in TBST (10 mM Tris, 150 mM NaCl, and 0.5% Tween-20), and western blotting with either a monoclonal parkin antibody (1:1,000; 1 hr at RT) (#4211; Cell Signaling Technology, Danfers, MA) or polyclonal antibody cocktail against 20S ‘core’ subunits (α5/α7, β1, β5, β5i, β7) (1:1,000; 1 hr at RT) (PW8155; Enzo Life Sciences, Plymouth Meeting, PA) followed by a horseradish peroxidase (HP)-conjugated secondary antibody and ECL detection. The blots were then stripped using Re-blot Plus reagent (AB1767; Chemicon, Billerica, MA), blocked with non-fat milk, and re-probed using a monoclonal α-tubulin antibody (1:500; 1 hr at RT) (sc-58668; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were again developed using a HP-conjugated secondary antibody and ECL detection. To standardize across blots, each blot contained all experimental groups. Analysis of the membranes was performed using a KODAK Gel Logic 100 imaging system and accompanying software. α-Tubulin was used as a loading control because the levels of α-tubulin in the total synaptosomal fraction were not significantly changed by the METH regimen as compared to saline-treated rats (data not shown). The western blot data were expressed as ratios of parkin or 20S immunoreactivity to α-tubulin immunoreactivity and presented as relative optical density units normalized to saline controls on each gel. This approach normalized differences in the development of the blot and across blots.

Immunoprecipitation of parkin and western blot analysis of parkin, 4-HNE-parkin conjugates and S-nitrosocysteine-parkin conjugates

Striatal and cerebellar synaptosomal fractions were prepared from rats decapitated 1 hr after the 4th METH injection. Protein A/G PLUS agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated in the presence of 2 µL of either a polyclonal parkin antibody (PW9365; Enzo Life Sciences, Plymouth Meeting, PA), parkin peptide corresponding to the epitope of the parkin antibody (PP9364; Enzo Life Sciences, Plymouth Meeting, PA), or RIPA buffer for 4–12 hrs at 4°C, followed by the addition of cytosol-vesicular fractions (300 µg protein) and second incubation (12 hrs at 4°C). Antibody-IgG cross-reactivity was prevented by cross-linking with 20 mM dimethyl-pimelimidate (Sigma-Aldrich, St. Louis, MO). Following immunoprecipitation, parkin protein was dissociated from the beads using SDS Tris-Glycine sample buffer (Bio-Rad, Hercules, CA) and gentle heating (30 min at 37°C), run on 10% Tris–Glycine gels under non-reducing conditions and transferred to PVDF membranes.

The membranes were blocked with 5% nonfat dried milk (1 hr at RT), incubated with either a monoclonal antibody against 4-HNE-protein conjugates (1:500; 12 hrs at 4°C) (MAB3249; R&D Systems, Minneapolis, MN) or a polyclonal antibody against S-nitrosocysteine-protein conjugates (1:500; 1 hr at RT) (N5411; Sigma-Aldrich, St. Louis, MO). The incubation with the S-nitrosocysteine-proteins antibody was preceded by an incubation with alkaline phosphatase-conjugated secondary anti-rabbit antibody (1:3,000; 1 hr at RT) to prevent background staining. The membranes were developed using HP-conjugated secondary antibodies and ECL detection. The blots were stripped, blocked with non-fat milk, and re-probed using a primary monoclonal parkin antibody (1:1,000; 1 hr at RT). Blots were again developed using a HP-conjugated secondary antibody and ECL detection. The increase in 4-HNE antibody immunoreactivity was examined relative to the immunoreactivity of immunoprecipitated parkin in each sample. The data were expressed as the ratio of 4-HNE to parkin immunoreactivity normalized to saline controls. This approach allowed for standardization across all treatment groups and provided a measure of the levels of 4-HNE per immunoprecipitated parkin protein.

Ubiquitination-independent chymotrypsin-like activity of 20S proteasome

Striatal, frontal cortical and cerebellar synaptosomes were prepared from rats decapitated 1, 24, and 48 hrs after the 4th METH injection. Chymotrypsin-like activity of the 20S proteasome was determined in the absence of ATP in a 96-well fluorimetric plate reader (SpectraMAX, Molecular Devices, Sunnyvale, CA) (Ex 350λ, Em 440λ) after the addition of various concentrations (25–400 µM) of the enzyme substrate, a fluorogenic peptide Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY-AMC) (P-802; Sigma-Aldrich, St. Louis, MO), according to Bulteau et al. (2001). Contribution of non-proteasomal proteases in each preparation was assessed by measuring the activity in the presence of epoxomicin (Sigma-Aldrich), a proteasome inhibitor, and subsequently subtracted from the total activity. The assay was validated by determination of linearity with time and protein content, working range, intra- and inter-assay variability, and the effects of different concentrations of epoxomicin. Assays were performed using 20 – 40 µg of cytosolic protein in a total volume of 50 µL. The assay buffer was composed of 25 mM Tris-HCl at pH 7.5, 2 µM epoxomicin in DMSO or DMSO, and the peptide substrate. The reactions were initiated by addition of cytosol-vesicular fractions and monitored at 37°C for 10 min. Chymotrypsin-like activity followed Michaelis-Menten kinetics (not shown). The maximal velocities (Vmax) and Michaelis constant values (Km) were calculated using GraphPad Prism program (GraphPad Software Inc., La Jolla, CA). The standard curve for relative 20S activity quantification was generated using 0-µM AMC (Sigma-Aldrich, St. Louis, MO). The data were expressed as nmols of AMC released/min/mg protein (Vmax) and µM (Km).

Ubiquitination-dependent activity of 26S proteasome

Ubiquitin-dependent proteasome activity was measured using a recombinant protein (hydrolase-insensitive ubiquitin tetramer (tUb4) fused to the reporter enzyme β-glucuronidase (GUS; EC 3.2.1.31)) previously used to assess 26S activity in plant extracts (Kurepa et al. 2008). In our laboratory, the assay was modified and optimized for rat brain tissue, and validated by determination of linearity with time and protein content, intra- and inter-assay variability, and the effects of different concentrations of epoxomicin. tUb4–GUS–His6 (tUb4–GUS) and GUS–His6 (GUS), cloned into pET28b and transformed into Escherichia coli BL21(DE3), were generous gifts from Dr. Smalle (University of Kentucky, Lexington, KY). In our laboratory, both constructs were induced with 1 mM isopropyl 1-thio-β-D-galactopyranoside for 3 h at 37°C, amplified, and purified on Ni-NTA columns under native conditions according to the manufacturer’s protocol (Qiagen, Valencia, CA). Purified proteins were run on a 10% gel, transferred onto PVDF membranes and visualized using MemCode Reversible Protein Stain Kit (Pierce Thermo Scientific, Rockford, IL). As expected, two main bands of molecular weights of ~68 kDa (GUS) and ~102 kDa (tUb4–GUS) were detected. Subsequently, GUS and tUb4–GUS were concentrated by dialysis in 50 mM potassium phosphate buffer, pH 7.0, containing 2 mM MgCl2 and 5% glycerol for 12 hrs at 4°C. The protein concentration was adjusted to 1 ng/µL with dialysis buffer containing 50% glycerol. Both proteins were again subjected to SDS-PAGE, blotted on PVDF membranes and immunodetected using anti-His6 antibody (sc-803, Santa Cruz Biotechnology, Santa Cruz, CA).

Striatal cytosol-vesicular fractions were prepared from rats decapitated 1 hr after the 4th METH injection. For the 26S assay, 10 µL (20–50 µg protein) of cytosol-vesicular fraction was pre-incubated in 90 µL of the GUS-26S assay buffer (50 mM M Tris-HCl, pH 7.5, 2.5 mM β-mercaptoethanol, 0.5 mM mM EDTA–Na2, 40 mM KCl, 0.05 mg/mL BSA, 0.25 M sucrose, 5 mM MgCl2, 10% glycerol, 1 mM ATP, 10 units/mL creatine phosphokinase, and 12 mM creatine phosphate dibasic tetrahydrate) with either DMSO or 20 µM epoxomicin for 15 min at 37°C. Subsequently, 2 ng of purified tUb4–GUS, or GUS, was added at a final concentration of 2 ng, and the reaction was started with 10 µL of 10 mM 4-methylumbelliferyl-β-D-glucuronide (4-MUG; Sigma-Aldrich), a substrate for tUb4–GUS and GUS. After 15 min pre-incubation, reaction kinetics of 4-methylumbelliferone (4-MU) production was followed for 15 min at 37°C in a 96-well fluorimetric plate reader (SpectraMAX, Molecular Devices, Sunnyvale, CA) (Ex 355λ, Em 460λ). The rate of 4-MU production reflects the levels of the tUb4–GUS. The levels of tUb4–GUS depend on 26S activity as it undergoes degradation by 26S proteasome in the absence of its inhibitor, epoxomicin. The production of 4-MU in the absence of epoxomicin is decreased. Therefore, ubiquitin-dependent 26S activity can be expressed as the difference between the reaction rate with and without epoxomicin (26S activity = GUS activity with 26S inhibited - GUS activity with active 26S). Rat brain tissue displayed no detectable endogenous GUS activity (data not shown). The standard curve for relative 26S activity quantification was generated using 0–10 µM 4-MU (Bio-Rad, Hercules, CA). Activity of 26S proteasome was expressed as nmol 4-MU/min/mg protein.

Dopamine content of tissue

One week after METH or saline injections, separate groups of rats were killed by rapid decapitation and the brains were quickly removed and dissected. Whole striata were removed, frozen immediately on dry ice, and stored at −80°C until analysis. The tissue was sonicated in 1 mL of cold 0.1 M perchloric acid and centrifuged at 14,000 × g for 5 min at 4°C. Protein concentrations were determined by the method of Bradford. The supernatant was analyzed for DA using a high-performance liquid chromatography with electrochemical detection. Samples (20 µL) were injected onto a 3-µm C18 reverse-phase column (100 × 2.0 mm; Phenomenex, Torrance, CA). DA was eluted with a mobile phase consisting of 32 mM citric acid, 54.3 mM sodium acetate, 0.074 mM EDTA, 0.215 mM octyl sodium sulfate, and 3% methanol (pH 3.8). Compounds were detected with an LC-4B amperometric detector (BAS Bioanalytical Systems, West Lafayette, IN) with a glassy carbon working electrode maintained at a potential of + 0.670 V relative to an Ag/AgC1 reference electrode. Data were recorded using the EZ Chrom (Scientific Software, Pleasanton, CA) software package. Concentrations were expressed as pg/µg protein.

Data analysis

Data with two treatment groups (METH and saline) were analyzed using Student t-tests. Two-way ANOVA analysis with a Student-Newman-Keuls post-hoc test was used to determine significant differences between groups in experiments with vitamin E pre-treatment. Two-way repeated measures ANOVA analysis followed by the post hoc test was performed on temperature data. All data are expressed as mean ± SEM. Significance was set at p<0.05.

Results

To determine whether high-dose METH causes an early decrease in the levels of parkin protein, the effects of METH on parkin immunoreactivity in total synaptosomal fractions from the striatum, frontal cortex and cerebellum at 1 hr, 24 hrs or 48 hrs after the last injection of the drug were examined. METH significantly decreased parkin/α-tubulin immunoreactivity at 1 hr and 24 hrs (−48%, 0.52 ± 0.17 and −34%, 0.66 ± 0.09, respectively) in the striatum relative to saline-treated controls (1.00 ± 0.11 and 1.00 ± 0.12, respectively) (Figure 1A). In contrast, parkin levels did not differ from the control values at 48 hrs after METH administration (1.07 ± 7 vs. 1.00 ± 14). Parkin/α-tubulin immunoreactivity was not significantly changed either in the frontal cortical (Figure 1B) or cerebellar (Figure 1C) synaptosomes up to 48 hrs after the last METH administration.

Figure 1.

Figure 1

Short-term effects of METH on parkin levels in striatal as compared to frontal cortical and cerebellar synaptosomes. Rats were administered saline (1 mL/kg) or METH (4 × 10 mg/kg. i.p., every 2 hrs) and killed at 1, 24, or 48 hrs after the last injection. Total synaptosomal fractions were prepared from striata, frontal cortices and cerebella. Illustrated are parkin/α-tubulin immunoreactivities normalized to saline control optical densities. Data are expressed as mean ± SEM. (A) In the striatum, METH significantly decreased parkin levels at 1 and 24 hrs after the last drug administration (−48% and −34%, respectively; p<0.05, Student’s t-test; n=5–7/group). (B, C) METH did not change parkin levels in the frontal cortex and cerebellum up to 48 hrs after the last drug administration. * Significantly different from saline controls. The bands shown represent the 52 kDa parkin bands and corresponding 55 kDa α-tubulin bands. Abbreviations: SAL, saline; METH, methamphetamine.

Immunoprecipitation experiments were performed to determine whether METH causes oxidative and/or nitrosative modification of parkin in striatal synaptosomes at 1 hr after the last METH injection. As illustrated in Figure 2A (blots on the left, top panel), less parkin was immunoprecipitated from METH-treated rats as compared to saline-treated controls. No S-nitrosocysteine immunoreactivity was detected on immunoprecipitated parkin (middle panel). Because METH induces lipid peroxidation (Yamamoto & Zhu 1998), subsequent experiments examined whether parkin was modified by the lipid peroxidation by-product, 4-HNE at 1 hr. As demonstrated in Figure 2A (bottom panel), METH increased 4-HNE immunoreactivity of immunoprecipitated striatal parkin as compared to controls. To ascertain the specificity of the bands, either parkin antibody was omitted or parkin peptide was added during immunoprecipitation (Figure 2A, blots on the right). Figure 2B shows the levels of 4-HNE per immunoprecipitated parkin from striatal and cerebellar synaptosomes. As compared to saline-treated controls, METH caused a 40% increase in the levels of 4-HNE-modified parkin in striatal but not cerebellar synaptosomes (1.40 ± 0.12 and 1.05 ± 0.23, respectively).

Figure 2.

Figure 2

Modification of parkin by 4-HNE in striatal synaptosomes in METH-treated rats. Rats were given saline (1 mL/kg) or METH (4 × 10 mg/kg. i.p., every 2 hrs) and killed at 1 hr after the last injection. Parkin was immunoprecipitated from striatal and cerebellar synaptosomes (A) In the striatum, METH decreased the levels of parkin (blots on the left, top panel) and increased the levels of 4-HNE-parkin (bottom panel), but not S-nitrosocysteine-parkin (middle panel) at 1 hr after the last dose of the drug as compared to saline controls. Omission of parkin antibody or addition of parkin peptide corresponding to the epitope of the parkin antibody during immunoprecipitation did not produce bands at 52 kDa neither with antibody against parkin nor with antibody against 4-HNE-protein conjugates (blots on the right). (B) Illustrated are ratios of 4-HNE to parkin immunoreactivity normalized to saline controls. Data are expressed as mean ± SEM. 4-HNE/parkin ratio was augmented by METH in striatal (+40%, p<0.05, Student’s t-test, n=3–6), but not cerebellar, synaptosomes. (C) Incubation of striatal synaptosomes from saline-treated animals with H2O2/Fe2+ (200/10 µM) for 1 hr at 37°C decreased parkin immunoreactivity (blot on the left) and increased 4-HNE immunoreactivity on immunoprecipitated parkin (blot on the right). In samples on the right, the levels of parkin were equalized to show the difference in 4-HNE levels. * Significantly different from saline controls. Abbreviations: SAL, saline; METH, methamphetamine; IP, immunoprecipitation; WB, western blotting; 4-HNE, 4-hydroxy-2-nonenal; STR, striatum; CER, cerebellum; ctrl, control.

To confirm that oxidative stress is responsible for the modification of parkin by 4-HNE and parkin deficit, striatal synaptosomes from control rats were incubated in the presence or absence of H2O2/Fe2+ (200/10 µM) for 1 hr at 37°C and examined for levels of parkin and 4-HNE-parkin conjugates. Parkin levels were lower while 4-HNE-parkin levels were higher in H2O2/Fe2+-treated synaptosomes (Figure 2C). In samples shown on the right, the levels of parkin were equalized (more µg of protein/lane was loaded in H2O2/Fe2+ than in control to equalize the parkin levels) to facilitate the comparison of 4-HNE-parkin levels.

To determine whether attenuation of lipid peroxidation protects parkin against METH, rats were pre-treated with the lipophilic antioxidant vitamin E before saline or METH injections and killed 1 hr after the last injection. Vitamin E attenuated the decrease in parkin levels (Figure 3A) (vehicle + METH vs. vitamin E + METH: 58 ± 6% vs. 91 ± 7% of saline controls) as well as the increase in the levels of 4-HNE-parkin conjugates (Figure 3B) (vehicle + METH vs. vitamin E + METH: 194 ± 22% vs. 158 ± 49% of saline controls). Vitamin E alone had no effect on parkin levels but reduced 4-HNE-parkin conjugates in saline-treated rats (Figure 3B).

Figure 3.

Figure 3

Protection of parkin by vitamin E against METH effects. Rats were administered saline (1 mL/kg) or METH (4 × 10 mg/kg. i.p., every 2 hrs), with or without pre-treatment with vitamin E, and killed at 1 hr after the last injection. Parkin was immunoprecipitated from striatal synaptosomes. Data are expressed as mean ± SEM. Pre-treatment with vitamin E attenuated the METH-induced parkin deficit (vitamin E vs. vehicle: −9% vs. −42%) (A) and increase in 4-HNE-parkin conjugates (vitamin E vs. vehicle: +58% vs. +94%) (B). The bands shown represent the 52 kDa parkin bands and corresponding α-tubulin or 4-HNE bands. (A) There was a significant main effect of the treatment condition (saline or METH) (F(1,20) = 11.652, p<0.01) but not of the pre-treatment condition (vehicle or vitamin E) (F(1,20) = 1.691, p=0.208). However, there was a significant pre-treatment × treatment interaction (F(1,20) = 5.299, p<0.05). (B) There was a significant main effect of the pre-treatment condition (vehicle or vitamin E) (F(1,10) = 14.170, p<0.01). There was no main effect of the treatment condition (saline or METH) (F(1,10) = 3.872, p=0.077) and no significant pre-treatment × treatment interaction (F(1,10) = 1.796, p=0.210). * Significant difference between vehicle + SAL and vehicle + METH, # significant difference between vehicle + METH and vitamin E + METH (p<0.05, two-way ANOVA with Student-Neuman-Keuls post hoc test; n=3–7/group). Abbreviations: SAL, saline; METH, methamphetamine; 4-HNE, 4-hydroxy-2-nonenal.

To assess whether high-dose METH causes a short-term decrease in the activity of the 20S proteasome (catalytic core), rats were treated with METH or saline and decapitated 1 hr, 24 hrs or 48 hrs after the last injection. Kinetic parameters of chymotrypsin-like activity of 20S proteasome were determined in striatal, frontal cortical, and cerebellar synaptosomal cytosol-vesicular fractions from these animals. Chymotrypsin-like activity was chosen for the experiments as the most representative of overall catalytic activity of the proteasome (Kisselev & Goldberg 2005). In contrast to our hypothesis, the Vmax of chymotrypsin-like activity was significantly increased in striatal synaptosomes of METH-treated animals as compared to saline controls at 1 hr after the last dose of the drug (+62%; 0.483 ± 0.05 vs. 0.301 ± 0.027 nmol AMC/min/mg protein) (Figure 4A). METH did not produce significant changes in Vmax of chymotrypsin like activity in either frontal cortical or cerebellar synaptosomes (Figure 4B, C). The Km values in METH-treated rats did not differ from the control values in any brain area (Table 1). The values for the Vmax and Km of synaptosomal chymotrypsin-like activity were similar to those reported previously (Andersson et al. 1999, Farout et al. 2000, Kisselev & Goldberg 2005, Tydlacka et al. 2008, Wang et al. 2008a, Petersen et al. 2010).

Figure 4.

Figure 4

Short-term effects of METH on 20S activity in striatal synaptosomes as compared to frontal cortical and cerebellar synaptosomes. Rats were given saline (1 mL/kg) or METH (4 × 10 mg/kg. i.p., every 2 hrs) and killed at 1, 24, or 48 hrs after the last injection. Synaptosomal fractions were prepared from striata, frontal cortices and cerebella. Illustrated is maximal velocity (Vmax) of chymotrypsin-like activity of 20S proteasome expressed as nmol AMC/min/mg protein. Data are expressed as mean ± SEM. (A) In the striatum, METH increased Vmax of chymotrypsin-like activity at 1 hr after the last drug administration (+62%; p<0.05, Student’s t-test; n=3/group). (B, C) METH did not produce significant changes in Vmax of chymotrypsin like activity in either frontal cortical or cerebellar synaptosomes. * Significantly different from saline controls. Abbreviations: SAL, saline; METH, methamphetamine; AMC, 7-amino-4-methylcoumarin.

Table 1. The effect of METH on apparent Michaelis constant (Km) values of synaptosomal chymotrypsin-like activity of 20S proteasome in saline- and METH-treated rats.

Four injections of saline (1 mL/kg) or METH (10mg/kg) were given every 2 hrs. Rats were killed at 1, 24, or 48 hrs after the last injection. The data are expressed as mean Km value (µM) ± SEM (n=3 per group). METH did not significantly change Km values at 1, 24, or 48 hrs after the last METH administration either in the striatum or frontal cortex or cerebellum.

Time
(hr)
Striatum Frontal cortex Cerebellum

SAL METH SAL METH SAL METH
1 47.7 ± 4.2 44.8 ± 6.4 57.7 ± 1.1 66.5 ± 2.5 76.5 ± 8.3 87.4 ± 7.3
24 59.1 ± 6.1 55.8 ± 5.2 57.7 ± 4.9 62.1 ± 4.5 72.1 ± 6.4 69.0 ± 4.1
48 61.6 ± 11 63.7 ± 3.0 50.5 ± 2.0 56.8 ± 5.9 73.0 ± 4.9 87.9 ± 5.3

To elucidate the cause of the increase in chymotrypsin-like activity, the levels of 20S catalytic core in striatal synaptosomes were examined at 1 hr and 24 hrs after METH administration using a cocktail of antibodies directed against several subunits of 20S proteasome, namely chymotrypsin-like activity-bearing catalytic subunit β5 and its immune counterpart β5i, β1, β7, and α5/α7. Western blotting produced bands at 27–30 kDa, and 24–25 kDa (Figure 5). Densities of these two groups of bands were quantified separately. At 1 hr after METH, there was a statistically significant increase in immunoreactivity of 27–30 kDa bands (+30%; 1.30 ± 0.07 vs. 1.00 ± 0.10). Immunoreactivity of β5 and β5i subunits (24–25 kDa) did not differ between saline- and METH-treated rats. At 24 hrs after METH, 20S proteasome immunoreactivity did not differ from the control values.

Figure 5.

Figure 5

Short-term effect of METH on 20S levels in striatal synaptosomes. Rats were given saline (1 mL/kg) or METH (4 × 10 mg/kg. i.p., every 2 hrs) and killed at 1 or 24 hrs after the last injection. Western blotting with a cocktail of antibodies against several subunits of 20S proteasome produced bands at ~27–30 kDa and ~24–25 kDa. Densities of these two groups of bands were quantified separately. This figure presents 27–30 kDa/α-tubulin immunoreactivities normalized to saline control optical densities. METH significantly increased immunoreactivity of bands concentrated around ~27–30 kDa (+30%; Student’s t-test, p<0.05; mean ± SEM, n=9/group). Immunoreactivities of β5 and β5i subunits (~24–25 kDa) did not differ between saline and METH-treated rats. * Significantly different from saline controls, p<0.05. Abbreviations: SAL, saline; METH, methamphetamine.

Because the activity of 20S proteasome does not reflect the ability of 26S proteasome to degrade ubiquitinated substrates, experiments were designed to examine the effect of METH on ubiquitin- and ATP-dependent activity of 26S proteasome. The 26S activity assay employing ubiquitinated β-glucuronidase (tUb4-GUS) as a 26S proteasome substrate (Kurepa et al. 2008) was modified and optimized for rat brain tissue. Non-ubiquitinated β-glucuronidase (GUS) served as a negative control. Both recombinant proteins were separated by SDS–PAGE, and transferred onto PVDF membrane and immunolabeled with anti-His6 antibody. β-glucuronidase from E.coli has a subunit molecular weight of 68.2 kDa (Jefferson et al. 1986, Kurepa et al. 2008). Figure 6A shows that the difference in molecular weights between tUb4-GUS and GUS corresponds, as expected, to molecular weight of four ubiquitin molecules. To test whether tUb4-GUS can be used as a substrate for rat brain 26S proteasome, the 26S assay was conducted in the presence of tUb4-GUS or GUS. Omission of a proteasome inhibitor, epoxomicin, in the 26S assay mixture containing tUb4-GUS resulted in a decreased reaction rate (RFU/min), indicating degradation of tUb4-GUS by 26S proteasome. Omission of epoxomicin in the assay mixture containing GUS did not change the reaction rate (Figure 6B), indicating that non-ubiquitinated GUS is not a substrate for rat brain 26S proteasome.

Figure 6.

Figure 6

Short-term effect of METH with and without vitamin E pre-treatment on 26S activity in striatal synaptosomes. (A) The difference in molecular weights between purified tUb4-GUS and GUS corresponded to molecular weight of four ubiquitin molecules. (B) Omission of proteasome inhibitor, epoxomicin, from the 26S assay mixture containing tUb4-GUS resulted in decreased reaction rate (RFU/min, expressed s a % of reaction rate in the absence of epoxomicin), indicating degradation of tUb4-GUS by 26S proteasome. Inhibition of the proteasome in the assay mixture containing GUS did not result in changes in the reaction rate, confirming that non-ubiquitinated GUS is not a substrate for 26S proteasome. Rats were given saline (1 mL/kg) or METH (4 × 10 mg/kg. i.p., every 2 hrs), with and without vitamin E pre-treatment, and killed at 1 hr after the last injection. (C) Illustrated is activity of 26S proteasome expressed as nmol 4MU/min/mg protein. Data are expressed as mean ± SEM. METH significantly decreased the activity of 26S proteasome as compared to saline-treated rats (−51%) while pre-treatment with vitamin E blocked the decrease. There was a significant main effect of the pre-treatment condition (vehicle or vitamin E) (F(1,12) = 5.536, p<0.05) but not of the treatment condition (saline or METH) (F(1,12) = 4.221, p=0.062). However, there was a significant pre-treatment × treatment interaction (F(1,12) = 6.919, p<0.05). * Significant difference between vehicle + SAL and vehicle + METH, # significant difference between vehicle + METH and vitamin E + METH (p<0.01, two-way ANOVA with Student-Neuman-Keuls post hoc test; mean ± SEM, n=3–6/group). Abbreviations: SAL, saline; METH, methamphetamine; GUS, β-glucuronidase; 4-MU, 4-methylumbelliferone.

To determine whether high-dose METH causes a short-term decrease in the ability of 26S proteasome to degrade ubiquitinated proteins (i.e. in 26S activity), rats were treated with METH or saline and killed 1 hr after the last injection. In contrast to the 20S proteasome, METH significantly decreased 26S proteasome activity in striatal synaptosomes by 51% as compared to saline-treated rats (2.49 ± 0.44 vs. 5.07 ± 0.59 nmol 4-MU/min/mg protein). Pre-treatment with vitamin E blocked the decrease (Figure 6C).

To assess METH neurotoxicity, DA and DA metabolites (DOPAC and HVA) were measured in METH- and saline-treated rats killed 7 days after METH administration. METH caused significant decreases in striatal concentrations of DA and its metabolites (DA: −60%, 47.9 ± 12.4 vs. 120 ± 10; DOPAC: −40%, 9.66 ± 1.97 vs. 16.1 ± 1.77; HVA: −43%, 4.73 ± 0.41 vs. 8.29 ± 0.69 pg/mg protein), whereas vitamin E blocked these deficits (Figure 7A–C).

Figure 7.

Figure 7

Long-term effect of METH with and without vitamin E pre-treatment on tissue dopamine (DA) and its metabolites concentrations in the striatum. Rats were given saline (1 mL/kg) or METH (4 × 10 mg/kg. i.p., every 2 hrs), with and without vitamin E pre-treatment, and killed 7 days later. Data are expressed as mean ± SEM in pg/mg protein. METH caused significant decreases in tissue content of DA (A) and its two metabolites DOPAC (B) and HVA (C) (−60%, −40%, and −43%, respectively). Pre-treatment with vitamin E blocked these deficits. There was a significant main effect of the pre-treatment condition (vehicle or vitamin E) and treatment condition (saline or METH) for DA and HVA (DA: F(1,15) = 9.395, p<0.01 and F(1,15) = 7.744, p<0.05; HVA: F(1,15) = 8.256, p<0.05 and F(1,15) = 4.565, p<0.05) as well as a significant pre-treatment × treatment interaction for DA (F(1,15) = 6.518, p<0.05). There was a significant main effect of the pre-treatment condition for DOPAC (F(1,15) = 5.155, p<0.05). * Significant difference between vehicle + SAL and vehicle + METH, # significant difference between vehicle + METH and vitamin E + METH (p<0.05, two-way ANOVA with Student-Neuman-Keuls post hoc test; mean ± SEM; n=3–6/group). Abbreviations: SAL, saline; METH, methamphetamine; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid.

Hyperthermia is an important contributing factor in METH toxicity. A decrease in core body temperature decreases METH neurotoxicity whereas an increase in body temperature increases toxicity (Ali et al. 1994, Bowyer et al. 1994). To determine whether vitamin E protects against METH toxicity via a decrease in METH-induced hyperthermia, core body temperature was measured prior to administration of METH and saline and 1 hr after each METH or saline injection. As shown in Figure 8, there were no differences in baseline temperatures between vehicle + saline and vitamin E + saline rats. METH administration to both vehicle- and vitamin E-pre-treated rats significantly increased rectal temperatures over time (up to approximately 40°C as compared to baseline values of approximately 37.5°C). Vitamin E did not significantly reduce core body temperature in METH-administered rats at any time point.

Figure 8.

Figure 8

The effect of METH on core body temperature in the presence and absence of vitamin E. Rats were given saline (1 mL/kg) or METH (4 × 10 mg/kg. i.p., every 2 hrs) with and without vitamin E pre-treatment. Body temperatures (°C) were measured prior to and 1 hr after each METH injection. Values are expressed as mean ± SEM. Arrows indicate each injection of METH. There was no difference in baseline temperatures between vehicle + saline and vitamin E + saline rats. METH administration to both vehicle- and vitamin E-pre-treated rats resulted in significant increases in rectal temperatures over time. Vitamin E did not significantly reduce core body temperature in METH-administered rats at any time point. * Significant difference between vehicle + SAL and vehicle + METH, # significant difference between vitamin E + SAL and vitamin E + METH (p<0.05, two-way ANOVA with repeated measures followed by Student-Neuman-Keuls post hoc test; mean ± SEM; n=6/group). Abbreviations: SAL, saline; METH, methamphetamine.

Discussion

The present study shows that repeated METH administration produces a rapid modification of parkin by 4-HNE, an associated decrease in parkin levels, and a decrease in activity of 26S proteasome with concomitant increase in chymotrypsin-like activity of its catalytic core in vivo. The observed modifications to parkin and decreases in parkin and 26S were attenuated by a lipophilic antioxidant, vitamin E.

Early events in METH neurotoxicity are DA-mediated oxidative stress, hyperthermia, and glutamate (GLU)-mediated nitrosative stress (reviewed in Yamamoto et al. 2010). All of these factors can cause inactivation, insolubility and aggregation of parkin and compromise the protective functions of parkin (Winklhofer et al. 2003, Chung et al. 2004, LaVoie et al. 2005, Wang et al. 2005, LaVoie et al. 2007). Electrophoresis of parkin was performed under non-reducing conditions such that high molecular weight aggregates of parkin would not appear at the molecular weight of the parkin monomer. Therefore, the parkin deficit observed in the current study at 1 hr and 24 hrs after the 4th METH dose (Figure 1A) might reflect an early damage to parkin protein, accompanied by its aggregation and degradation (LaVoie et al. 2007, Schlehe et al. 2008). In addition to damage to parkin by DA-mediated oxidative stress and hyperthermia, a likely explanation for the rapid decrease in parkin immunoreactivity may be its inactivation by 4-HNE (Catala 2009) as 4-HNE immunoreactivity was increased at the same position as the parkin monomer band (~52 kDa) in METH-treated rats (Figure 2). This notion is further supported by the finding that the lipophilic antioxidant vitamin E attenuated both, the parkin deficit and increase in 4-HNE-parkin conjugates, without affecting METH-induced hyperthermia (Figure 3 and 8). Nevertheless, the protection by vitamin E may have been due in part to the reduction in body temperature (last two time points in Figure 8; Park et al. (2006). Lipid peroxidation persists up to 24 hrs after METH in rodents (Gluck et al. 2001, Park et al. 2006). Therefore, the deficit in parkin observed at 24 hrs after METH (Figure 1A) might also be due to METH-induced lipid peroxidation. As the population of DAergic terminals in the striatum is small relative to other nerve terminals; the parkin deficit cannot be entirely associated with DAergic terminals. A decrease in parkin gene expression may occur in neuronal elements other than DA neurons as Nakahara et al. (2003) showed that a neurotoxic dose of METH (40 mg/kg, i.p.) decreased parkin mRNA levels at 1 hr and 2 hrs in the rat striatum, but not in the SNc. At 48 hrs after METH, parkin has been normalized, possibly due to upregulation of its expression and synthesis.

METH triggers production of nitrogen species (RNS) (Anderson & Itzhak 2006, Wang et al. 2008b) via an increase in GLU efflux within the striatum (Nash & Yamamoto 1992). METH-generated RNS can inactivate proteins, including parkin, by S-nitrosylation (Chung et al. 2004). Although S-nitrosylation of parkin was not evident at 1 hr after METH (Figure 2A), parkin S-nitrosylation appears to be a slower process than parkin oxidation (Winklhofer et al. 2003, Chung et al. 2004) and may be evident at later time points.

Available evidence suggests that the 20S proteasome can be damaged and inactivated by oxidative stress (Dasuri et al. 2009) and that inactivation of parkin can also lead to proteasomal inhibition (Hyun et al. 2002, Wang et al. 2005). In contrast to our expectations, chymotrypsin-like activity of 20S proteasome was found to be increased at 1 hr after METH administration (Figure 4A). At the same time, the activity of the 26S proteasome was decreased (Figure 6C). These findings suggest that, at the 1 hr time point, the 20S catalytic core was not oxidatively damaged and may have increased its function in response to METH-induced oxidative stress independently of parkin and 26S proteasome (as functions of both were impaired). This possibility is supported by several lines of evidence. The 20S proteasome is more resistant to oxidative stress than the 26S proteasome and can independently degrade oxidatively modified proteins. Relatively mild oxidative stress can increase proteolysis of these proteins concomitantly with a decrease in 26S proteasome activity (Davies 2001, Kurepa et al. 2008). Exposure of DAergic cells or neurons to mild oxidative stress increased chymotrypsin-like activity (Elkon et al. 2004, Dietrich et al. 2005). In contrast to these and our findings, METH produced a decrease in chymotrypsin-like activity in DAergic cells in vitro (Fornai et al. 2006, Lazzeri et al. 2007). This could have been the result of a more severe oxidative stress produced and/or measured at a later time point. Alternatively, the increase in 20S activity observed in our study could have occurred outside DAergic terminals.

Activity of 20S proteasome can increase in response to oxidative stress through several factors including the dissociation of 19S cap(s) from 26S proteasome (Kurepa et al. 2008), activation of latent 20S by oxidized proteins (Grune et al. 1997, Jariel-Encontre et al. 2008), increase in proteasomal subunit composition/expression (Lee et al. 2004, Singh & Khar 2006, Chondrogianni & Gonos 2007), activation by PA28 or PA200 (Rechsteiner & Hill 2005), activation by intracellular redox status (Kretz-Remy & Arrigo 2003), or formation of immunoproteasome (Ding et al. 2003, Dahlmann 2005, Ethen et al. 2007). Consistent with the increase in 20S activity, we observed a modest increase in cumulative immunoreactivity of a few 20S subunits: α7 (~30 kDa), β1 (~29 kDa), α5 (~28 kDa), and β7 (27 kDa) in METH-treated rats at 1 hr after the last injection (Figure 5). Immunoreactivity of β5 and β5i subunits (~24 and 25 kDa) remained constant. In the study of Ding et al. (2003), mild oxidative stress increased neuronal chymotrypsin-like activity via induction of immunoproteasome subunits (β1i, β2i, and β5i) at 3 hrs but not 1 hr after H2O2 administration. This suggests that in response to METH-induced inflammation (Thomas et al. 2004), mature immunoproteasomes form later than 1 hr after the drug. The β7 and α5 subunits could have increased due to an early METH-triggered pro-apoptotic signal as similar increases, accompanied by marked increase in 20S activity, were observed during apoptosis in vitro (Singh & Khar 2006). Interestingly, an increase in 20S activity and the levels of both α and β subunits were found after exposure of cortical neurons to low, subtoxic concentrations of proteasome inhibitors (Lee et al. 2004). Overall, METH appears to increase chymotrypsin-like activity, in part, via induction of some constitutive catalytic core subunits. The increase in 20S subunit levels was of a lower magnitude than an increase in chymotrypsin-like activity (30% vs. 62%), suggesting additional activation of 20S catalytic core by either PA28 or PA200 or dissociation of 26S. In fact, activation of 20S by PA28 is a likely possibility as PA28 can increase proteasomal activity under low oxidative stress conditions (Osna et al. 2008).

Relatively mild oxidative stress increases proteolysis of oxidized proteins but rapidly and reversibly inactivates enzymes involved in protein ubiquitination and 26S proteasome activity (Davies 2001). Similarly, the activity of 26S proteasome was decreased at 1 hr after METH administration (Figure 6C). The decreased function of 26S proteasome with a concomitant increase in function of 20S proteasome suggests oxidative damage to 19S regulatory cap or dissociation of 19S cap from the 26S proteasome. The 19S is important for 26S activity because it removes ubiquitin chains and unfolds proteins (Liu et al. 2005). Impairment of these functions can limit the rate of protein degradation by 26S proteasome (Dahlmann 2005). In fact, 19S can be damaged by 4-HNE and damage to 19S can inhibit 26S function (Bardag-Gorce et al. 2005). De-ubiquitination of proteins is also achieved by proteasome-associated de-ubiquitinating enzymes (DUBs) (Tai & Schuman 2008). Consequently, the observed decrease in 26S proteasome activity may result from an impairment of proteasome-associated DUBs. In contrast, it is unlikely that the decrease in 26S activity is due to a METH-induced decrease in ATP levels (Chan et al. 1994) because the activity was assayed in the presence of added ATP. The temperature-independent protection by vitamin E (Figure 6C and 8) suggests that lipid peroxidation mediated the damage to the 26S. As with changes in parkin and 20S, the deficit in 26S likely occurred outside DAergic terminals.

The neuroprotective effect of the lipid soluble vitamin E against METH-induced decreases in striatal content of DA and DA metabolites 7 days after drug administration (Figure 7) is in agreement with a previous study indicating that inhibition of lipid peroxidation inhibits the long-term neurotoxic effects of METH in rats (Park et al. 2006). This result indicates that oxidation of lipids contributes to the DAergic toxicity of METH and may be related to the decreases in parkin.

The METH-induced formation of 4-HNE-parkin conjugates and parkin deficit were detected in striatal but not frontal cortical and/or cerebellar synaptosomes (Figures 1A–C and 2B). Similarly, chymotrypsin-like activity of 20S proteasome was increased in striatal but not frontal cortical and cerebellar synaptosomes (Figure 4A–C). A possible explanation for this selectivity is the involvement of DA in the generation of ROS produced by METH (Wrona et al. 1997) as DA concentrations are much higher in the striatum as compared to the frontal cortex and cerebellum. There is growing evidence that METH-induced ROS can also damage other than DAergic striatal components (Yamamoto et al. 2010). Our findings of 30–50% decreases in parkin and 26S activity in striatal synaptosomes suggest that METH compromises the UPS in other synaptosomal populations. Impairments of parkin and 26S outside DAergic terminals might contribute to their loss by inflammation, for example.

METH-induced oxidative damage to parkin and the 26S proteasome may be interdependent events. Parkin is degraded by the UPS (Choi et al. 2000) and a deficit in 26S function might result in parkin aggregation (Junn et al. 2002, Ardley et al. 2003). Alternatively, damaged parkin may lead to inhibition of the proteasome (Hyun et al. 2002, Wang et al. 2005). Regardless of the sequence of events, functional loss of parkin and the 26S proteasome would result in accumulation of parkin substrates and ubiquitinated proteins destined for degradation. Increased protein aggregation could potentiate the decline in UPS function and contribute to METH-induced damage to DAergic terminals. The present investigation did not determine whether parkin and 26S proteasome are causally involved in METH neurotoxicity. However, an efficient UPS system was proven to be critically important in protection of DAergic neurons from degeneration (Fornai et al. 2003, Lo Bianco et al. 2004).

In conclusion, evidence is presented illustrating that repeated METH administrations in vivo causes rapid decreases in parkin protein and 26S proteasome activity via lipid peroxidation-mediated damage. As the rapid changes in parkin and 26S occur well before the sustained deficits in DAergic markers, an early loss of UPS function is an early event that may mediate the long-term damage to striatal DA terminals.

Acknowledgements

This work was supported by NIH grants, DA07606 and DA023085. We are grateful to Dr. Smalle (University of Kentucky, Lexington, KY) for providing E. coli cultures carrying tUb4–GUS–His6 and GUS–His6. Special thanks to Dr. Jasmina Kurepa (University of Kentucky, Lexington, KY) and Mark Pavlyukovskyy for assistance with the 26S proteasome assay.

Abbreviations used

CER

cerebellum

DA

dopamine

DOPAC

3,3-dihydroxyphenylacetic acid

DUBs

de-ubiquitinating enzymes

GLU

glutamate

GUS

β-glucuronidase

4-HNE

4-hydroxy-2-nonenal

HVA

homovanillic acid

Km

Michaelis constant

METH

methamphetamine

4-MU

4-methylumbelliferone

4-MUG

4-methylumbelliferyl-D-glucuronide

RNS

reactive nitrogen species

ROS

reactive oxygen species

SAL

saline

SNc

substantia nigra pars compacta

STR

striatum

Suc-LLVY-AMC

Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin

tUb4

ubiquitin tetramer

UPS

ubiquitin-proteasome system

Vmax

maximal velocity

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