Increased Striatal mRNA and Protein Levels of the Immunophilin

Increased Striatal mRNA and Protein Levels of the Immunophilin FKBP-12 in Experimental Parkinson’s Disease and Identification of FKBP-12-Binding Proteins Anna Nilsson,†,‡,# Karl Sko1 ld,†,‡,# Benita Sjo1 gren,§ Marcus Svensson,†,‡ Johan Pierson,†,| Xiaoqun Zhang,§ Richard M. Caprioli,⊥ Jos Buijs,O Bjo1 rn Persson,O Per Svenningsson,§ and Per E. Andre´ n*,†,‡ Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, P.O. Box 583 Biomedical Centre, SE-75123 Uppsala, Sweden, Department of Pharmaceutical Biosciences, Uppsala University, P.O. Box 591 Biomedical Centre, SE-751 24 Uppsala, Sweden, Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm, Sweden, Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, P.O. Box 574 Biomedical Centre, SE-751 23 Uppsala, Sweden, Mass Spectrometry Research Center, Vanderbilt University School of Medicine, 465 21 Avenue South, Room 9160 C, Nashville, Tennessee 37232-8575, and Biacore AB, Rapsgatan 7, 75450 Uppsala, Sweden Received April 3, 2007

FKBP-12, a 12 kDa FK506-binding protein (neuroimmunophilin), acts as a receptor for the immunosuppressant drug FK506. Neuroimmunophilins, including FKBP-12, are abundant in the brain and have been shown to be involved in reversing neuronal degeneration and preventing cell death. In this report, we have utilized several analytical techniques, such as in situ hybridization, Western blotting, twodimensional gel electrophoresis, and liquid chromatography electrospray tandem mass spectrometry to study the transcriptional expression as well as protein levels of FKBP-12 in the unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease. The FKBP-12 protein was also detected directly on brain tissue sections using mass spectrometry profiling. We found increased levels of FKBP12 mRNA and protein in the dorsal and middle part of the 6-OHDA lesioned striatum. Thus, these studies clearly demonstrate that FKBP-12 is increased in the brain of a common animal model of Parkinson’s disease (PD). Additionally, we have identified potential binding partners to FKBP-12 that may be implicated in the pathophysiology of Parkinson’s disease, such as alpha-enolase, 14-3-3 zeta/delta, pyruvate kinase isozymes, and heat shock protein 70, using surface plasmon resonance sensor technology in combination with mass spectrometry. In conclusion, these data strongly suggests that FKBP-12 is altered in an experimental model of PD. Keywords: Parkinson’s disease • proteomics • protein interactions • 6-OHDA • striatum • mass spectrometry

Parkinson’s disease (PD) is a common degenerative neurological disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) that innervate the striatum and, to a lesser extent, other basal ganglia nuclei.1 The cause of neuronal death underlying PD is unknown, but appears to involve multiple factors acting together, including

aging, genetic susceptibility, and toxic environmental exposures.2 The neurodegeneration may be related to multiple causes such as mitochondrial dysfunction, oxidative stress, excitotoxicity, apoptosis, inflammation, and proteasome failure.3 The finding of mutations in the genes coding for R-synuclein, parkin, DJ-1, and ubiquitin C-terminal hydrolase L1 in familiar PD indicate that malfunction of the ubiquitinproteasome system is a common final pathway of neurodegeneration.4,5

* To whom correspondence should be addressed: Dr. Per E. Andre´n, Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, Box 583 Biomedical Centre, SE-75123 Uppsala, Sweden. Tel., +46 18 471 7206; fax, +46 18 471 4422. E-mail: [email protected]. † Laboratory for Biological and Medical Mass Spectrometry, Uppsala University. ‡ Department of Pharmaceutical Biosciences, Uppsala University. # Equal contribution to this work. § Department of Physiology and Pharmacology, Karolinska Institute. | Department of Medicinal Chemistry, Uppsala University. ⊥ Vanderbilt University School of Medicine. O Biacore AB.

The clinical characteristics of PD are tremor, bradykinesia, and rigidity.6 At present, only symptomatic treatments are efficient. Such treatments are directed at replacing dopaminergic neurotransmission by administration of L-3,4-dihydroxyphenylalanine (L-DOPA), the precursor of dopamine, and other drugs that stimulate dopaminergic neurotransmission. However, while the disease progresses, the L-DOPA therapy often results in the emergence of motor fluctuations (‘on-off’ phenomenon), abnormal involuntary movements (dyskinesia),

Introduction

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FKBP-12 and Parkinson’s Disease

and psychiatric complications, which are major limitations to replacement therapy.7 Although there is effective symptomatic treatment for PD, there are no proven neuroprotective or neurorestorative therapies.7 However, compounds that bind to immunophilins are a promising new class of drugs for the treatment of neurodegenerative diseases. The immunophilins are chaperone proteins with peptidyl-prolyl cis-trans isomerase activity, but they also have other diverse functions, including regulation of mitochondrial permeability transition pores, stabilization and control of ion channels, and facilitation of protein-protein interaction, which have only been partially characterized.8 The immunophilin protein FKBP-12, the 12-kDa FK506binding protein, is a ubiquitous protein that acts as a receptor for the immunosuppressant drug FK506.9 The FK506 class of neuroimmunophilin compounds has been shown to elicit both neuroprotection and neuroregeneration in neurotoxic chemical models of PD.10 Further, FK506 has also been shown to enhance nerve regeneration in a variety of experimental situations including nerve crush11 and transection, and spinal cord injury.12,13 FKBP-12 interacts with subunits of two intracellular calcium release channels, the inositol 1,4,5-triphosphate receptor and the ryanodine receptor, as well as the transforming growth factor β (TGF-β) type I receptor.14 FKBP-12 inhibits basal signaling of these three receptors. It also regulates the Pglycoprotein multidrug transporter.15 In the presence of FK506, FKBP-12 binds the regulatory B subunit of the calciumdependent phosphatase calcineurin (CaN), blocking substrate access to the catalytic site of the A subunit.16 In the present study, we have investigated the mRNA and protein levels of FKBP-12 in the same tissue sample in an animal model of PD, that is, the unilateral 6-hydroxydopamine (6-OHDA) lesion model. 6-OHDA induces oxidative stress and/ or apoptosis in the nigro-striatal dopaminergic pathway.17 An important step in the pathway leading to apoptosis is the increase in intracellular calcium.18 FKBP-12 is involved in the calcium homeostasis, for example, through the interaction with the ryanodine receptor.19 A range of different techniques, such as in situ hybridization, Western blotting, two-dimensional gel electrophoresis (2D-GE) in combination with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) peptide mass fingerprinting, and capillary liquid chromatography electrospray ionization tandem MS (nanoLC-ESI-MS/MS) were used. MALDI MS profiling was performed directly on brain tissue sections. We searched for protein interaction partners of FKBP-12 using surface plasmon resonance (SPR) sensor chip technology in combination with mass spectrometry. This is a relatively new methodology, allowing the detection and characterization of molecular interactions, that has been applied to identify protein interaction partners present in brain extract.20 Our study demonstrates a significant increase of the expression of the transcriptional and protein level of FKBP-12 in the 6-OHDA denervated striatum of the brain.

Materials and Methods Animals. Male Sprague Dawley rats (n ) 12) (B&K, Sollentuna, Sweden) weighing between 190 and 290 g at the beginning of the study were used in all experiments. The animals were housed together for 7 days to acclimatize and were maintained in a temperature (20-24 °C) and humidity (5070%) controlled environment with a 12-h light/dark cycle. Food (Lactamin R3) and water were available ad libitum. All animal

research articles procedures were approved by the local animal ethics committee and carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC). Unilateral 6-OHDA Lesions. The animals were anesthetized by giving ketamine (100 mg/kg, Ketalar, Parke-Davis, Morris Plains, NJ)/xylazine (5 mg/kg, Rompun vet, Bayer, Gothenburg, Sweden) intraperitoneally (ip) and placed in a stereotactic instrument. To maximize the selective dopamine depletion by 6-OHDA, the noradrenaline uptake inhibitor desipramine (25 mg/kg, Sigma-Aldrich, St. Louis, MO) and monoamine oxidase-B inhibitor pargyline (5 mg/kg, Research Biochemicals International, Natick, MA) were co-injected ip 30 min prior to surgery. A few drops of lidocaine (40 mg/mL, Xylocain, AstraZeneca, So¨derta¨lje, Sweden) were used for local anesthesia when the incision was made to expose the skull. A small hole was drilled to allow implantation of a thin cannula into the brain. The neurotoxin 6-OHDA (6-OHDA HBr with ascorbic acid, Research Biochemicals International) was dissolved in saline to a concentration of 5 mg/mL and intracerebrally infused into the right medial forebrain bundle. Stereotaxic coordinates from bregma for the medial forebrain bundle were anterior-posterior, -2.8 mm; medial-lateral, + 2.0 mm; and depth from dura, 8.3 mm.21 All surgeries were performed under aseptic conditions. The 6-OHDA solution (2.5 µL) was infused during 5 min, and the probe was left in the brain for another 5 min before it was removed. The animals were maintained at 37 °C and monitored for any distress during the postoperative period. Drug Treatment and Behavioral Assessments. Administration of a dopamine receptor agonist to animals with a unilateral 6-OHDA lesion of the nigro-striatal pathway results in contralateral rotations, which might be used as a measurement of the degree of lesioning.22 Therefore, the response to the dopamine receptor agonist apomorphine (Apoteksbolaget, Stockholm, Sweden) was tested once at a concentration of 0.5 mg/kg subcutaneously (sc) 21 days after the 6-OHDA injection to verify the effectiveness of the lesion. Behavioral assessments were performed by putting the rats into individual plastic observation cages, where they were allowed to habituate for 30 min before the apomorphine injection. The animals were videotaped, and the videotapes were evaluated by trained observers. Only animals showing more than 1.67 contralateral rotations per min after apomorphine injections during the 60 min observation period were used in the experiments. A washout period of 7 days was allowed after the apomorphine injection. The animals were then sacrificed by decapitation. Their brains were rapidly dissected, frozen on dry ice, and stored in a freezer (-80 °C). The time period from decapitation until the brain was frozen on dry ice was approximately 90 s. An additional evaluation of the efficacy of the lesion was performed postmortem, by measuring the levels of the dopamine transporter on sections made from the animals (see ref 23). For this purpose, sections were incubated with 3 nM of the selective dopamine transporter ligand [3H] 2-β-(4-carbomethoxy3-β-(4-flurophenyl) tropane (CFT; 87.0 Ci/mmol; DuPont, NEN, Stockholm, Sweden). To further evaluate the efficacy of the lesion, the levels of tyrosine hydroxylase mRNA and protein in dopaminergic neurons were measured.23 Two-Dimensional Gel Electrophoresis. Brain tissue dissected from the striatum of the 6-OHDA-treated and the untreated side of the brain was suspended to a concentration of 10 mg brain tissue/mL sample buffer. The sample buffer Journal of Proteome Research • Vol. 6, No. 10, 2007 3953

research articles consisted of 40 mM Tris base, 7 M Urea, 2 M thiourea, 4% CHAPS, 10 mM 1,4-dithioerythritol, and 1 mM EDTA. The suspension was sonicated for approximately 30 s to disrupt the cells and centrifuged at 2000g for 20 min at room temperature to sediment cell debris and undissolved material. The resulting extract was immediately frozen and stored at -80 °C. Three different animals were used to confirm the differences found in the gels. Approximately 1 mg of the samples was applied at the anodic end of the immobilized pH gradient (IPG) strips (pH 6-11) (GE Healthcare, Uppsala, Sweden). The proteins were focused at 500 V for 1 min, after which the voltage was gradually increased to 3500 V for 1.5 h and kept constant at 3500 V for 14 h. The second-dimension separation was performed using 14.0% polyacrylamide gels (GE Healthcare). The gels were fixed (40% methanol and 10% acetic acid) for 1 h at room temperature. The proteins were visualized using silver staining24 or colloidal Coomassie blue staining.25 The resulting gels from the untreated and the 6-OHDAlesioned side of the brain were scanned (Imagescanner, GE Healthcare), and the spots were compared for differences (ImageMaster, GE Healthcare). Selected spots were automatically picked (Ettan gel spotpicker, GE Healthcare) and transferred to a microtiter plate. The proteins within the gel plugs were enzymatically digested with 20 µL of 50 mg/mL trypsin and thereafter extracted using 40 µL of 50% acetonitrile/0.1% trifluoroacetic acid (TFA). The extracted peptides were desalted using C18 packed micropipette tips (ZipTip, Millipore, Billerica, MA). The samples were applied onto a MALDI target of stainless steel using the drieddroplet method.26 A saturated solution of R-cyano-4-hydroxycinnamic acid in acetonitrile/water 50:50 and 0.5% TFA was used as matrix. Peptide mass fingerprinting was performed using a reflectron MALDI time-of-flight (TOF) mass spectrometer (Ettan, GE Healthcare). The mass spectrometer was used in positive mode. Mass spectra were internally calibrated using trypsin autodigest peptides as internal standards. All peptide masses were matched against theoretical peptide masses of proteins in a database. Identification was performed using the Profound V4.10 (www.proteometrics.com) and Mascot (www.matrixscience.com) search engines. In Situ Hybridization. In situ hybridization experiments were carried out as previously described.27 Briefly, 35S-labeled anti-sense and sense cRNA probes were prepared by in vitro transcription from cDNA clones corresponding to nucleotides 245-568 of the rat FKBP-12 gene. The transcription was performed from 50-100 ng of linearized plasmid using [35S]UTP (>1000 Ci/mmol; DuPont NEN) and T3 or T7 RNA polymerases. The probes were purified on Sephadex G50 column and precipitated in sodium acetate (0.1 vol)/absolute ethanol (2.5 vol). Cryostat sections were postfixed in 4% PFA for 5 min at room temperature, rinsed twice in 4× sodium chloride-sodium citrate buffer (SSC), and placed into 0.25% acetic anhydride in 0.1 M triethanolamine/4× SSC (pH 8) for 10 min at room temperature. After dehydration in graded alcohols, the sections were hybridized overnight at 55 °C with 106 cpm of 35S-labeled probe in 50 µL of hybridization solution (20 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl, 50% formamide, 10% dextran sulfate, 1× Denhardt’s, 250 µg/mL yeast tRNA, 100 µg/mL salmon sperm DNA, 0.1% SDS, and 0.1% sodium thiosulphate). The slides were washed in 4× SSC (5 min, four times), RNAse A (20 µg/mL) (20 min, at 37 °C), 2× SSC (5 min, twice), 1× SSC (5 min), 0.5× SSC (5 min) at room temperature, and rinsed in 0.1× SSC at 65 °C (30 min, twice) (all washes contained 1 mM DTT), before being dehydrated in graded 3954

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alcohols. The slides were then exposed on X-ray films for 2-14 days. The films were quantified by densitometry, using National Institutes of Health IMAGE 1.61 software. Data were analyzed by Student’s t test with significance defined as p < 0.05. Western Blotting. Frozen tissue samples (striatum) were sonicated in 1% SDS, and boiled for 10 min. Small aliquots of the homogenate were retained for protein determination by the BCA protein assay method (Pierce, Rockford, IL) using bovine serum albumin as a standard. Equal amounts of protein (30 µg) were loaded onto 16% acrylamide gels, and the proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (0.2 µm) (Schleicher & Schuell, Keene, NH).28 The membranes were immunoblotted using monoclonal antibodies against FKBP-12 (BD Biosciences, Mountain View, CA); 1:1000 dilution) and tyrosine hydroxylase (TH) (Chemicon, Temecula, CA, 1.5000 dilution). Antibody binding was revealed by incubation with goat anti-mouse horseradish peroxidase-linked IgG (1:750 dilution) (Pierce, Stockholm, Sweden) and the ECL immunoblotting detection system (GE Healthcare). Antibody binding was detected by enhanced chemiluminescence (GE Healthcare) and quantified by densitometry, using National Institutes of Health IMAGE 1.61 software. Data were analyzed by Student’s t test with significance defined as p < 0.05. Profiling MALDI MS Directly on Brain Tissue Sections. Tissue sections from rat brains were cut in 12 µm sections with a microtome (Leica CM 3000, Leica Microsystems AG, Wetzlar, Germany) at -15 °C and placed on flat stainless steel MALDI target plates kept at -15 °C, and then immediately moved onto dry ice.29,30 The tissue sections on the MALDI target plates were then placed in a desiccator for approximately 1 h in room temperature until dry and then transferred to a cold room (4 °C). A pipet was used to deposit 0.1 µL of freshly prepared matrix solution, i.e., sinapinic acid, (3,5-dimethoxy-4-hydroxycinnaminic acid, Sigma-Aldrich) in a mixture of 2-propanol/ water (1:1) containing 0.1% TFA and 20 mM N-octylglucoside (octyl β-D-glucopyranoside, Fluka, Buchs Switzerland)) to specific brain regions of the tissue sections.29,30 The samples were analyzed using a MALDI-TOF mass spectrometer (Voyager DE-STR, Applied Biosystems, Framingham, MA). The instrument was operated in positive linear mode under optimized delayed extraction conditions. Mass spectra were randomly acquired within each matrix spot (averaging 1 mm in diameter) using an average of 500 laser shots for each brain location and processed as previously described.29,30 The mass spectra were calibrated using m/z peaks obtained from the tissue corresponding to thymosin β-10 (m/z 4964), ubiquitin (m/z 8566), and hemoglobin R-chain (m/z 15 200). These proteins were abundant in all mass spectra acquired from the brain tissue. The results from the intact and dopaminedenervated hemispheres from each examined brain region were evaluated by paired Student’s t test. P-values less than 0.05 were considered significant. Protein identification was performed as described previously.31 FKBP-12 Interacting Proteins. Cytosolic Protein Preparation. One rat and one mouse (B&K, Sollentuna, Sweden) were sacrificed by decapitation and their brains rapidly dissected out and frozen on dry ice. The brains were then treated separately. Each brain was sonicated in 1 mL/200 mg ice-cold 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.7 µL/mL Protease inhibitor cocktail III (Sigma-Aldrich, Stockholm, Sweden) on ice. Cell debris was removed by centrifugation for 20 min at 2900g, 4 °C. Cytosolic proteins were isolated by centrifugation,


and membraneous proteins were removed by centrifugation for 45 min at 29 000g, 4 °C. Protein concentration of the resulting cytosolic protein solution was determined by a BCA quantification kit (Pierce). The solution was diluted in 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl to 0.5 mg/mL and frozen in aliquots. Cleavage of Recombinant FKBP-12-GST Protein. FKBP-GST protein (purchased from Abnova Corp., Taiwan) was available in 380 µL of buffer (50 mM Tris-HCl and 10 mM reduced glutation, pH 8, C ) 0.34 µg/µL). The protein solution was concentrated by reducing the volume to 55 µL on a filter device (Microcon YM-3, Millipore, Bedford, MA) with a cutoff limit of 3 kDa. A total of 5.5 µL of 10× cleavage buffer (50 mM TrisHCl, 1.5 M NaCl, 10 mM EDTA, and 10 mM DTT) and 1.2 µL of PreScission Protease (GE Healthcare) was added to the sample, and cleavage was performed for 16 h at 4 °C. The cleaved product was separated on a gelfiltration column (Superdex 75, GE Healthcare) using a SMART system with a flow rate of 20 µL/min, and the eluate was monitored by UV and collected in 50 µL fractions. The protein fractions were verified by Western blotting, as described above, using a primary rabbit antibody against GST (1:1000; Clontech, New York, NY) and a primary mouse antibody against FKBP-12 (1: 1000; Transduction Laboratories, BD Biosciences, Stockholm, Sweden). Immobilization of FKBP-12 on a Biacore Sensor Chip. All experiments were carried out using Biacore 3000 SPR sensor with control software version 4.0, carboxymethylated sensor chips (CM5), and with HBS-N (10 mM HEPES and 150 mM NaCl, pH 7.4) as running buffer (Biacore, Uppsala, Sweden). Flow rates during immobilization and ligand fishing experiments were 10 µL/min unless stated otherwise. For ligand fishing experiments with cytosolic mouse brain extract, FKBP-12 was immobilized on all four flow cells of the internal fluidic system. Briefly, the sensor chip surface was activated by equal amounts of 1-ethyl-3-(3-diaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer’s recommendation. A total of 44 µL of FKBP-12 (pH adjusted to ∼4.5) was then injected with a flow of 5 µL/min. The surface was deactivated using 1 M ethanolamine, and noncovalently bound FKBP-12 was removed with 10 5-µL 1% acetic acid (HAc) pulses. For ligand fishing experiments with cytosolic rat proteins, FKBP-12 was immobilized in the single, large area flow cell of the external Surface Prep unit. Briefly, the sensor chip surface was activated with 70 µL of 1:1 solution of EDC/NHS. FKBP12 was then injected (pH adjusted to ∼4.5) followed by deactivation with 1 M ethanolamine and washing by 10 5-µL pulses of 50 mM NaOH to remove any noncovalently bound FKBP-12. Surface Characterization. To quantify immobilization levels and activity FKBP-12 immobilized in the surface prep unit, the sensor chip was docked in the instrument before and after the immobilization procedure. Immobilization levels were obtained by monitoring the SPR response increase upon immobilization. The immobilization quality was tested using an antibody against FKBP-12 (Transduction Laboratories, BD Biosciences, Stockholm, Sweden). The antibody was diluted 400 times and injected at a flow rate of 30 µL/min, while 0.25% TFA was used to remove bound antibody. Binding and Recovery of FKBP-12 Binding Proteins from Mouse. Cytosolic mouse brain proteins were thawed, and octylβ-D-glucopyranoside (OGP; Sigma-Aldrich) was added to a final

research articles concentration of 50 mM. The binding and recovery of cytosolic proteins were carried out in all four flow cells with a userdefined method for 12 or 15 cycles as follows: 40 µL of cytosolic brain extract was injected. The flow system, excluding the flow cells, was then washed with 50 mM NaOH and 2% HAc/50 mM OGP, and HBS-N/10 mM OGP. The flow cells were shortly flushed with HBS-N/10 mM OGP followed by incubation of a recovery solution (0.25% TFA) in the flow cell area for 30 s to release proteins that bound to FKBP-12. The recovery solution was eluted and collected in a protein LoBind tube (Eppendorf AG, Hamburg, Germany). Finally, the flow system was washed once more with the same procedure as used between brain extract injection and protein recovery. To control for unspecific interactions, the exact same setup was used with a nonimmobilized sensor chip (blank chip). Binding and Recovery of FKBP-12 Binding Proteins from Rat. Cytosolic rat brain proteins were thawed and centrifuged for 10 min at 17 000g, 4 °C, to remove remaining residues. The supernatant was diluted to 0.25 mg/mL in 50 mM Tris-HCl, pH7.4, and 150 mM NaCl, and OGP was added to a final concentration of 50 mM. The binding and recovery of cytosolic proteins were performed in the surface prep unit for either 6 or 8 cycles as follows: system wash followed by injection of 40 µL of cytosolic brain extract, needle wash with 2% HAc/50 mM OGP, and a rinse of the sensor chip with 50 mM ammonium bicarbonate (NH4HCO3), pH8.0. The bound material was eluted using 0.25% TFA and collected in a protein LoBind tube. The advantage of this unit is that the sensor chip surface is increased 4-fold, rendering larger amounts of proteins in each cycle. Furthermore, the risk of detecting nonspecific binders is significantly reduced, due to the decrease in injection and recovery channels. The drawback is that the monitoring capacity is lost. To control for unspecific interactions, the exact same setup was used with a nonimmobilized sensor chip (blank chip). Trypsination-Resuspension. Sequencing-grade modified trypsin was purchased from Promega (Promega Corp., Madison, WI) and diluted to a concentration of 0.02 µg/µL in 0.1 mM HCl. A total of 5 µL of trypsin and 45 µL of NH4HCO3, pH 8.0, was added to the recovered protein solution and left for enzymatic cleavage overnight at room temperature. The peptide solution was then lyophilized and resuspended in 8 µL of 0.25% HAc before MS analysis. MS Identification of FKBP-12 Binding Proteins. The peptide mixture was analyzed on a nanoLC system (Ettan MDLC, GE Healthcare) coupled with an electrospray linear ion trap(LTQ) or an LTQ-FT (Fourier Transform) MS (Thermo Electron, San Jose´, CA) mass spectrometer. The sample was injected and desalted on a precolumn (300 µm inner diameter (i.d.) × 5 mm, C18 PepMapT, 5 µm, 100 Å, LC Packings, Amsterdam, The Netherlands) at a flow rate of 10 µL/min for 10 min. A 15-cm fused silica emitter with a 75 µm i.d. and a 375 µm outer diameter (Proxeon Biosystems; Odense, Denmark) was used as the analytical column. The emitter was packed in-house with a slurry of reverse-phased Reprosil-Pur C18-AQ 3-µm resin (Dr. Maisch GmbH; Ammerbuch-Entringen, Germany) dispersed in methanol using a pressurized packing devise (Proxeon Biosystems; Odense, Denmark). The mobile phases used were Buffer A (0.25% HAc in water) and Buffer B (84% acetonitrile (ACN) and 0.25% HAc in water). The peptide samples were separated during a 40 min gradient from 3 to 60% Buffer B, and the MS data was collected in a data-dependent manner. The acquisition continuously switches between full MS scan (m/z 300Journal of Proteome Research • Vol. 6, No. 10, 2007 3955

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Figure 1. (A) Microphotography showing the efficacy of the 6-OHDA nigro-striatal lesion. The binding of the dopamine transporter ligand, 3H-CFT, is strongly decreased in the 6-OHDA-lesioned hemisphere when compared to the unlesioned hemisphere. (B) Western blots showing elevated levels of FKBP-12 protein in striatum in the 6-OHDA-denervated hemisphere. (C and D) Histograms showing the quantification of FKBP-12 and TH protein levels in the intact and the denervated hemisphere. * p < 0.05, Student’s t test.

2000), zoom scan (most intense peak in full scan), and MS/ MS scan (most intense peak in zoom scan) where the most intense peak can be picked twice in a time window of 40 s and is then put on an exclusion list during a 150 s period. The MS/ MS data was converted into a combined mgf-file. When using the FTICR instrument, the survey scan was performed in the FT at a resolution of 100 000, and MS/MS were performed on the four most abundant ions in the LTQ. The mgf-files were searched against UniProtKB-Mus musculus and Rattus norvegicus using either Mascot or X!Tandem, with the following settings: no fixed modifications, potential oxidation on methionine, a precursor MS window of (2 Da, and fragment mass accuracy of 0.7 Da.

Results Behavioral and Biochemical Assessments of the 6-OHDA Lesioning. The extent of the 6-OHDA-induced dopamine depletion was assessed by examining rotational behavior response to apomorphine 21 days after the lesion. A mean contralateral rotation response of 2.7 turns/min after a single injection of apomorphine was observed during the 60 min observation period. The response varied between 128 and 413 full contralateral laps during the 60 min observation period, indicating a pronounced lesion of the nigrostriatal dopaminergic pathway. Earlier studies have shown a strong correlation between the numbers of apomorphine-induced rotations in the 6-OHDA model of parkinsonism and the number of remaining tyrosine hydroxylase immunoreactive cells in substantia nigra pars compacta.32 3956


Furthermore, to verify the efficacy of the dopamine denervation postmortem, the striatal binding of [3H]-CFT, a selective radioligand for the dopamine transporter, was examined.23 The dopamine transporter is enriched in dopaminergic nerve terminals, and previous work has shown that the density of [3H]CFT binding correlates well with the degree of dopaminergic innervation.33,34 In this study, the 6-OHDA injections caused a pronounced (>90%) decrease of [3H]-CFT binding in the lesioned hemisphere (Figure 1). An additional characteristic of dopamine depletion is decrease in tyrosine hydroxylase (TH), and Western blotting showed a strong decrease of TH in the lesioned hemisphere compared to the intact hemisphere (Student’s t test, p > 0.001) (Figure 1). Western Blotting of FKBP-12 Protein. The monoclonal antibody utilized in the present study recognized a single band at 12 kDa. Comparative immunoblotting of intact and dopamine-denervated striata showed a significant (60%) increase in FKBP-12 in the dopamine-denervated lesioned hemisphere (p < 0.05) (Figure 1). Two-Dimensional Gel Electrophoresis. The FKBP-12 protein was detected on the 2D gels after silver staining and software image analysis. The spot patterns from the control and the dopamine-denervated side of the brain were compared, and the differences were further examined. FKBP-12 was highly expressed in the 6-OHDA-treated side of the brain relative to the control side (Figure 2). The spot identity was verified as FKBP-12 using MALDI MS peptide mass fingerprinting with 56% sequence coverage (Figure 3). In Situ Hybridization of FKBP-12 mRNA. In situ hybridization studies showed that FKBP-12 mRNA is widely expressed


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Figure 2. Segment of 2D gel in the mass range 14 kDa and pI 7.5-8.7 presenting the difference between untreated control (left panel) and 6-OHDA-treated (right panel) hemisphere of the striatum. FKBP-12 is clearly visible on the right gel slide corresponding to the lesioned hemisphere, in comparison to the left slide corresponding to untreated control hemisphere.

Figure 3. The full-length amino acid sequence of FKBP-12 (MW ) 11.8 kDa) with sequence coverage (56%) indicated in red. The lower panel shows a schematic figure of the recombinant protein (GST-FKBP-12) that was used in the SPR interaction studies. PreScission Protease cleaves between Q and G.

5). The dorsal and middle areas of the striatum showed a significant increase in the levels of FKBP-12 expression (p < 0.05; 14% and 46%, respectively).

Figure 4. (A) Autoradiogram showing that FKBP-12 mRNA levels are elevated in striatum in the 6-OHDA-denervated hemisphere. (B) Histogram showing the quantification of FKBP-12 mRNA levels in the intact and the denervated hemisphere. * p < 0.05, Student’s t test.

in the brain, with enrichment in the striatum. The dopaminedenervated striatum showed elevated levels (28%) of FKBP-12 mRNA compared to the corresponding intact striatum (p < 0.05) (Figure 4). MALDI MS Profiling of FKBP-12 Directly on Brain Tissue Sections. The MALDI MS profiling analysis was performed at the same coronal levels (consecutive sections) as the dopamine transporter binding assays and in situ hybridization. The ion intensity levels of FKBP-12 were semiquantitatively measured in the dorsal, middle, and ventral parts of the striatum (Figure

FKBP-12 Protein Interaction. Cleavage of Fusionprotein FKBP-12-GST. FKBP-12 was available as a GST-fusion protein (Figure 3). To eliminate nonspecific binding to GST, this tag was cleaved off using a predefined method according to the manufacturer’s description. FKBP-12 was separated from GST by gel filtration, and the eluted fraction was verified by Western blotting. No noncleaved FKBP-12-GST product was detected after purification of the enzymatically cleaved products, which indicates that the enzymatic processing step was fully completed. A full recovery of the protein would give a maximum amount of 43 µg of FKBP-12 in 100 µL of Tris-HCl buffer (cmax ) 0.43 µg/µL) (Figure 3). Immobilization of FKBP-12. FKBP-12 was immobilized on a CM5 chip inside the instrument, for recovery of mouse proteins, or in the external surface prep unit, for recovery of rat proteins, respectively. Typically, about 8000 response units (RU)/flow cell was immobilized, corresponding to a total of about 32 ng of FKBP-12 immobilized on the 4 flow cells inside the instrument and 128 ng on the sensor chip immobilized with the external surface prep unit. The activity of the CM5-FKBP12 sensor chips was tested using a monoclonal FKBP-12 antibody. The antibody bound at 200-400 RU/flow cell and could be completely eluted with 0.25% trifluoroacetic acid (TFA). Binding and Analyte Recovery of FKBP-12-Binding Proteins from Rat and Mouse. The binding and recovery experiments with rat and mouse brain extracts were performed using the Biacore 3000 instrument. Typical levels of binding ranged from 100 to 300 RU/flow cell, corresponding to a total of 0.41.2 ng of protein material. Binding and analyte recovery of proteins from rat were performed in an external unit. Journal of Proteome Research • Vol. 6, No. 10, 2007 3957

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Nilsson, A. Table 1. The Proteins Identified as Binders to FKBP-12 in Rat and Mouse Brain Extractsa Swiss-Prot number

protein name

P63017 P63260 Q04447 P52480 O08709 P63101 P04764 Q07936

Heat shock 70 protein Gamma-actin Creatine kinase, brain M2-type pyruvate kinase 1-Cys peroxiredoxin protein 14-3-3 zeta Alpha enolase Annexin A2

no. of identified mouse rat tryptic peptides

X X X X X X

X X X X X X

8 7 5 8 3 2 3 2

a The number of peptides identified from each protein is indicated in the last column. None of these proteins were observed in the control experiments. A number of previous studies have reported several of these proteins to be affected in neurodegenerative diseases, including HSP-70, 143-3 zeta, and pyruvate kinase.

and rat brain extracts were 14-3-3 zeta/delta (P63102), pyruvate kinase isozymes M1/M2(P11980), 1-cys peroxyredoxin (O08709), and heat shock protein 70 (P14659).

Discussion

Figure 5. Relative ion intensity (y-axis) of FKBP-12 (m/z 11 791) in the unilateral dopamine-depleted rats. The immunophilin FKBP-12 showed increased levels in both (A) dorsal (p < 0.04) and (B) middle (p < 0.04) part of the dopamine-denervated striatum, but was not significantly changed in the (C) ventral part (white bars, non-lesioned side of the striatum; black bars, 6-OHDA-treated side of the striatum).

Blank Chip Controls. In the complex protein mixture of the cytosolic brain fraction, there are several proteins that adsorb nonspecifically to surfaces of the microfluidic system of Biacore 3000 and appear as carryover proteins in the recovered protein solution. These proteins were characterized by performing analyte recovery experiments on an unmodified CM5 chip. Proteins recovered from the blank chip, were myelin basic protein, tubulins, hemoglobin subunits, ribosomal proteins, histone proteins, alpha/beta-caseins, and some microtubule associated proteins. These proteins were considered as nonspecific binders and not proteins that interact with FKBP-12. FKBP-12 Binding Proteins. The MS analysis of the recovered solution revealed several proteins as potential interactors to FKBP-12. These proteins were not detected in the experiments performed on the blank chip and were reproducibly found in experiments performed on the FKBP-12 chip. The proteins identified from the MS analysis as binders to FKBP-12 are summarized in Table 1. The proteins identified in both mouse 3958


In the present study, we have utilized a number of analytical approaches, such as 2D-GE, in situ hybridization, Western blotting, and MALDI MS profiling directly on brain tissue sections, to examine the striatal levels of FKBP-12 mRNA and protein levels in an animal model of PD. We detected an increased level of FKBP-12 mRNA and protein in the striatum of 6-OHDA-denervated rats. We have also identified several proteins that interact with FKBP-12. FKBP-12 was first discovered in immune cells and given the name “immunophilin”.15 FKBP-12 is an ubiquitous protein, existing in all tissues, with particular high density in the brain.8 It interacts with two intracellular calcium channels, the inositol 1,4,5-triphosphate (IP3) receptor and the ryanodine receptor (RyR).15 FKBP-12 is responsible, in complex with calcineurin and calmodulin, for stabilizing these calcium channels by a calcineurin-mediated dephosphorylation of a protein kinase C (PKC)-phosphorylated site on the IP3 receptor. When intracellular calcium increases, calmodulin and calcineurin become activated, causing an association of calcineurin with the IP3 receptor-FKBP-12 complex via FKBP-12. This association causes an activation of the phosphatase activity of calcineurin and results in dephoshorylation of the IP3 receptor and a decrease in calcium release.35 A recent report showed increased levels of FKBP-12 in the caudate, putamen, and globus pallidus in clinical samples from patients with PD.36 In clinical PD cases, FKBP-12 was also shown to be colocalized in R-synuclein positive Lewy bodies,36 which are characteristic intracellular proteinaceous inclusions formed in the brains in PD. The Lewy bodies contain high levels of the protein R-synuclein. Thus, it may be speculated that FKBP-12 is involved in the biochemistry of the proteasome complex that is responsible for degradation of misfolded and damaged proteins.5 Further, FKBP-12, together with growth associated protein (GAP-43), a major neuronal phosphoprotein implicated in neuronal regeneration,37 is also upregulated in growth cones of neurons subjected to nerve crush38 and in PC12 cells subjected to 6-OHDA treatment.39 Calcineurin levels were not increased,38 which may indicate that the rate-limiting step in phosphorylation of neuronal proteins associated with axonal extension is more likely to involve dynamics in FKBP-12 levels rather than calcineurin.38 GAP-43 is thought to play a role in


axonal growth during neuronal development and regeneration and has a major role in intracellular signaling pathways. In differentiated neurons, GAP-43 plays a critical role in axon path finding.40 Administration of 6-OHDA has been shown to cause oxidative stress,41 increased calcium levels, and ultimately apoptosis.17,42 Intracellular calcium levels are maintained by storage mainly in the endoplasmatic reticulum through the RyR and IP3 receptor calcium release channels.43 Oxidative stress can also cause the structural integrity of the lipid bilayer membrane of mitochondria and cells to be impaired due to lipid peroxidation and polyunsaturation from reactions with reactive oxygen species.44 This may lead to impaired calcium homeostasis and disabled storage function of cellular organelles, such as the mitochondria and endoplasmatic reticulum.44 Clinical studies of PD patients have shown increased levels of polyunsaturated lipids and lipid peroxidation indicators, which are indicators of oxidative stress.45 FK506 and related neuroimmunophilins may become useful as neuroprotective treatments of dopamine neurons in PD. However, the present data showing elevated levels of FKBP-12 in striatum will also affect the outcome of treatment with neurophilins as striatum plays a critical role for the basal ganglia function. On the basis of the above-mentioned literature, it is predicted that the elevated levels of FKBP-12 in striatum would prevent calcium-mediated signal transduction. As increased levels of calcium may be detrimental for cells, including striatal neurons, the elevated levels of FKBP-12 may have a protective effect on striatum and basal ganglia function. Our present data would add an additional argument for the possibility that FK506, and related compounds, may be useful in the treatment of PD. The SPR technique is a biosensor technology useful for detecting and studying molecular interactions. The technique is based on detection of mass changes on the sensor chip surface by measuring changes of the incident angle of monochromatic, p-polarized light. The molecule of interest is immobilized onto a sensor chip, interaction partners are passed over the sensor chip through a microfluidic system, and binding can be monitored. In the present study, we used the SPR technology in combination with MS for capturing and identifying binding partners to FKBP-12. The proteins 14-3-3 zeta/ delta, pyruvate kinase isozymes M1/M2, 1-cys peroxyredoxin, and heat shock protein 70 were identified as interacting partners to FKBP-12 in both rat and mouse brain extracts. A number of previous studies have reported several of these proteins to be affected in neurodegenerative diseases.46 The 14-3-3-zeta/delta (protein kinase C inhibitor protein 1) belongs to a family of proteins that are known to bind a large number of different signaling proteins, including kinases, phosphatases, and transmembrane receptors, indicating that it has a role as a general biochemical regulator.47 The levels of 14-3-3 were altered in an experimental model of neurodegenerative disease.46 Previous studies have reported an interaction between the 14-3-3-protein and the translational control factor FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/ mTOR).48 14-3-3 zeta is also known to regulate calcium channels together with calmodulin. In conjunction with calmodulin, the 14-3-3 zeta/delta protein regulates the subcellular distribution of Kir/Gem (Ras-related GTPase) between the cytoplasm and the nucleus thereby affecting Kir/Gem-mediated cell shape modeling and calcium channel activity.49

research articles HSP-70 is a stress-induced heat shock protein that might play an active role in the chaperoning of proteins.50 HSP-70 proteins have been found in Lewy bodies in PD.51 Furthermore, like FKBP-12, HSP-70 is involved in regulating the activity of protein kinases and phosphatases.52,53 Similar to FKBP-12, HSP-70 is known to interact with calmodulin54,55 as well as calcineurin.56 This fact may imply that the interaction between HSP-70 and FKBP-12 is made through an indirect interaction, although we did not find calmodulin and calcineurin among the FKBP12 interacting proteins. This also indicates that we identified only a fraction of the proteins involved in the interaction network around FKBP-12. It should be mentioned that we studied soluble proteins in the present study, and not membranebound proteins. To increase the detection of FKBP-12 interacting proteins, alternative sample preparation protocols, for example, prefractionation, could be used. Pyruvate kinase is one of the main enzymes in glycolysis, regulating the breakdown of glucose to pyruvate. The M2 isoform has been proposed, along with 14-3-3 zeta and R-enolase, to be affected in age-related neurodegenerative diseases such as PD and Alzheimer disease.46 It has further been shown that pyruvate kinase is elevated in neurons undergoing amyloid beta peptide-mediated apoptosis.57 FKBP-12 was, in the present study, analyzed using a number of different techniques. Some of these methods are conventional proteomics and neuroscience methodology (2D-GE, in situ hybridization, Western blot), while other techniques used are more recent, such as profiling of the FKBP-12 protein directly on brain tissue sections by MALDI MS and studying interaction partners of FKBP-12 by SPR. Other approaches, for example, 2D-LC-MS/MS for protein analysis and a peptidomics approach, might have added additional information in the 6-OHDA dopamine-denervated animal model of PD. Further, in previous reports by us, we demonstrated the importance of proper postmortem handling of the brain.58-60 Preserving the biochemical, molecular, and structural sample integrity is essential for correct sample comparisons. We showed that both proteins and neuropeptides, including their post-translational modifications, were subjected to massive degradation already minutes postmortem.59-61 In the present study, the postmortem interval before the sample was frozen was 90 s, which considerably limits the postmortem changes. However, it should be mentioned that some changes due to postmortem degradation might still be present. In conclusion, we have detected increased levels of the immunophilin FKBP-12 in a 6-OHDA dopamine-denervated animal model of PD using 2D-GE, including identification using peptide mass fingerprinting and in situ hybridization. We have further identified and semiquantified FKBP-12 using MALDI MS profiling directly on brain tissue sections. Using protein interaction technology, we showed that FKBP-12 interacted with the proteins, 14-3-3 zeta/delta, pyruvate kinase isozymes M1/M2, and heat shock protein 70. We demonstrated that the immunophilin protein FKBP-12 may be involved in the pathogenesis of PD.

Acknowledgment. This study was sponsored by Swedish Research Council (VR) Grants 2002-2453 and 2004-3417, an institutional grant from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), the K&A Wallenberg Foundation, and the Karolinska Institute Centre for Medical Innovations, Research Programme in Medical Bioinformatics, and the NIH-NIGMS funded Grant 5R01 Journal of Proteome Research • Vol. 6, No. 10, 2007 3959

research articles GM58008. P.S. has been supported by VR, Wiberg’s stiftelse, Osterman’s fund, and Svenska La¨karsa¨llskapet.

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PR070189E

Journal of Proteome Research • Vol. 6, No. 10, 2007 3961

Increased Striatal mRNA and Protein Levels of the Immunophilin

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