p25α Relocalizes in Oligodendroglia from Myelin to Cytoplasmic Inclusions in Multiple System Atrophy
2007; Elsevier BV; Volume: 171; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2007.070201
ISSN1525-2191
AutoresYun Ju Christine Song, Ditte Lundvig, Yue Huang, Wei Gai, Peter Blumbergs, Peter Højrup, Daniel E. Otzen, Glenda M. Halliday, Poul Henning Jensen,
Tópico(s)Muscle Physiology and Disorders
Resumop25α is an oligodendroglial protein that can induce aggregation of α-synuclein and accumulates in oligodendroglial cell bodies containing fibrillized α-synuclein in the neurodegenerative disease multiple system atrophy (MSA). We demonstrate biochemically that p25α is a constituent of myelin and a high-affinity ligand for myelin basic protein (MBP), and in situ immunohistochemistry revealed that MBP and p25α colocalize in myelin in normal human brains. Analysis of MSA cases reveals dramatic changes in p25α and MBP throughout the course of the disease. In situ immunohistochemistry revealed a cellular redistribution of p25α immunoreactivity from the myelin to the oligodendroglial cell soma, with no overall change in p25α protein concentration using immunoblotting. Concomitantly, an ∼80% reduction in the concentration of full-length MBP protein was revealed by immunoblotting along with the presence of immunoreactivity for MBP degradation products in oligodendroglia. The oligodendroglial cell bodies in MSA displayed an enlargement along with the relocalization of p25α, and this was enhanced after the deposition of α-synuclein in the glial cytoplasmic inclusions. Overall, the data indicate that changes in the cellular interactions between MBP and p25α occur early in MSA and contribute to abnormalities in myelin and subsequent α-synuclein aggregation and the ensuing neuronal degeneration that characterizes this disease. p25α is an oligodendroglial protein that can induce aggregation of α-synuclein and accumulates in oligodendroglial cell bodies containing fibrillized α-synuclein in the neurodegenerative disease multiple system atrophy (MSA). We demonstrate biochemically that p25α is a constituent of myelin and a high-affinity ligand for myelin basic protein (MBP), and in situ immunohistochemistry revealed that MBP and p25α colocalize in myelin in normal human brains. Analysis of MSA cases reveals dramatic changes in p25α and MBP throughout the course of the disease. In situ immunohistochemistry revealed a cellular redistribution of p25α immunoreactivity from the myelin to the oligodendroglial cell soma, with no overall change in p25α protein concentration using immunoblotting. Concomitantly, an ∼80% reduction in the concentration of full-length MBP protein was revealed by immunoblotting along with the presence of immunoreactivity for MBP degradation products in oligodendroglia. The oligodendroglial cell bodies in MSA displayed an enlargement along with the relocalization of p25α, and this was enhanced after the deposition of α-synuclein in the glial cytoplasmic inclusions. Overall, the data indicate that changes in the cellular interactions between MBP and p25α occur early in MSA and contribute to abnormalities in myelin and subsequent α-synuclein aggregation and the ensuing neuronal degeneration that characterizes this disease. The myelin sheaths surrounding nerve axons represent a specialized structure produced by oligodendroglia in the central nervous system. The myelin sheath not only facilitates the propagation of action potentials along axons but also supplies trophic support for axons and participates in communication between the axons and oligodendroglia.1Wilkins A Majed H Layfield R Compston A Chandran S Oligodendrocytes promote neuronal survival and axonal length by distinct intracellular mechanisms: a novel role for oligodendrocyte-derived glial cell line-derived neurotrophic factor.J Neurosci. 2003; 23: 4967-4974PubMed Google Scholar The significances of these functions are highlighted by the devastating impact of diseases that primarily target oligodendroglia, eg, multiple sclerosis with profound demyelination and multiple system atrophy (MSA) that develop oligodendroglial cytoplasmic inclusions containing aggregates of the protein α-synuclein. Myelin basic protein (MBP) comprises 30% of the total protein in myelin, making it the second major constituent after proteolipid protein.2Boggs JM Myelin basic protein: a multifunctional protein.Cell Mol Life Sci. 2006; 63: 1945-1961Crossref PubMed Scopus (413) Google Scholar Genetic mutations reducing functional MBP cause substantial neurological symptoms, as demonstrated in shiverer mice and shaker rats.3Carré JL Goetz BD O'Connor LT Bremer Q Duncan ID Mutations in the rat myelin basic protein gene are associated with specific alterations in other myelin gene expression.Neurosci Lett. 2002; 330: 17-20Crossref PubMed Scopus (11) Google Scholar, 4Readhead C Popko B Takahashi N Shine HD Saavedra RA Sidman RL Hood L Expression of a myelin basic protein gene in transgenic shiverer mice: correction of the dysmyelinating phenotype.Cell. 1987; 48: 703-712Abstract Full Text PDF PubMed Scopus (294) Google Scholar p25α is an oligodendroglial-specific phosphoprotein5Takahashi M Tomizawa K Fujita SC Sato K Uchida T Imahori K A brain-specific protein p25 is localized and associated with oligodendrocytes, neuropil, and fiber-like structures of the CA3 hippocampal region in the rat brain.J Neurochem. 1993; 60: 228-235Crossref PubMed Scopus (63) Google Scholar, 6Takahashi M Tomizawa K Ishiguro K Sato K Omori A Sato S Shiratsuchi A Uchida T Imahori K A novel brain-specific 25 kDa protein (p25) is phosphorylated by a Ser/Thr-Pro kinase (TPK II) from tau protein kinase fractions.FEBS Lett. 1991; 289: 37-43Crossref PubMed Scopus (64) Google Scholar also designated tubulin polymerization-promoting protein because of its microtubule-binding activity.7Hlavanda E Kovacs J Olah J Orosz F Medzihradszky KF Ovadi J Brain-specific p25 protein binds to tubulin and microtubules and induces aberrant microtubule assemblies at substoichiometric concentrations.Biochemistry. 2002; 41: 8657-8664Crossref PubMed Scopus (102) Google Scholar Its expression initiates at the time of myelination along with MBP, and p25α can be used as a marker of myelinating oligodendroglia.8Skjoerringe T Lundvig DM Jensen PH Moos T P25α/tubulin polymerization promoting protein expression by myelinating oligodendrocytes of the developing rat brain.J Neurochem. 2006; 99: 333-342Crossref PubMed Scopus (41) Google Scholar Furthermore, the expression of p25α and MBP in myelinating oligodendroglia in the rat brain appears at the same time.8Skjoerringe T Lundvig DM Jensen PH Moos T P25α/tubulin polymerization promoting protein expression by myelinating oligodendrocytes of the developing rat brain.J Neurochem. 2006; 99: 333-342Crossref PubMed Scopus (41) Google Scholar, 9Akiyama K Ichinose S Omori A Sakurai Y Asou H Study of expression of myelin basic proteins (MBPs) in developing rat brain using a novel antibody reacting with four major isoforms of MBP.J Neurosci Res. 2002; 68: 19-28Crossref PubMed Scopus (48) Google Scholar Its normal function is unclear, but accumulating evidence points to a role related to the microtubular system as it co-purifies with kinases directed to the microtubule-binding protein tau and promotes the bundling of microtubules.6Takahashi M Tomizawa K Ishiguro K Sato K Omori A Sato S Shiratsuchi A Uchida T Imahori K A novel brain-specific 25 kDa protein (p25) is phosphorylated by a Ser/Thr-Pro kinase (TPK II) from tau protein kinase fractions.FEBS Lett. 1991; 289: 37-43Crossref PubMed Scopus (64) Google Scholar, 7Hlavanda E Kovacs J Olah J Orosz F Medzihradszky KF Ovadi J Brain-specific p25 protein binds to tubulin and microtubules and induces aberrant microtubule assemblies at substoichiometric concentrations.Biochemistry. 2002; 41: 8657-8664Crossref PubMed Scopus (102) Google Scholar p25α binds tubulin according to a 1:2 stoichiometric model,10Otzen DE Lundvig DM Wimmer R Nielsen LH Pedersen JR Jensen PH p25α is flexible but natively folded and binds tubulin with oligomeric stoichiometry.Protein Sci. 2005; 14: 1396-1409Crossref PubMed Scopus (37) Google Scholar and expression of a fusion protein between human p25α and enhanced green fluorescent protein in transfected cells leads to a colocalization of the fusion protein with microtubules during specific cell-cycle stages.11Lehotzky A Tirian L Tokesi N Lenart P Szabo B Kovacs J Ovadi J Dynamic targeting of microtubules by TPPP/p25 affects cell survival.J Cell Sci. 2004; 117: 6249-6259Crossref PubMed Scopus (57) Google Scholar α-Synuclein is normally a neuron-specific protein that is localized in nerve terminals. However, its aggregation and relocalization to cellular inclusions in a group of neurodegenerative diseases forms the basis for designating these disorders α-synucleinopathies.12Goedert M Spillantini MG Davies SW Filamentous nerve cell inclusions in neurodegenerative diseases.Curr Opin Neurobiol. 1998; 8: 619-632Crossref PubMed Scopus (227) Google Scholar In addition, α-synuclein deposition can also occur in a large variety of central nervous system conditions, including Alzheimer's disease, Gaucher's disease, Niemann-Pick type C1 disease, gangliosidoses, and more recently multiple sclerosis.13Attems J Quass M Jellinger KA Tau and alpha-synuclein brainstem pathology in Alzheimer disease: relation with extrapyramidal signs.Acta Neuropathol (Berl). 2007; 113: 53-62Crossref PubMed Scopus (70) Google Scholar, 14Papadopoulos D Ewans L Pham-Dinh D Knott J Reynolds R Upregulation of α-synuclein in neurons and glia in inflammatory demyelinating disease.Mol Cell Neurosci. 2006; 31: 597-612Crossref PubMed Scopus (34) Google Scholar, 15Saito Y Suzuki K Hulette CM Murayama S Aberrant phosphorylation of α-synuclein in human Niemann-Pick type C1 disease.J Neuropathol Exp Neurol. 2004; 63: 323-328Crossref PubMed Scopus (79) Google Scholar, 16Sidransky E Gaucher disease: complexity in a “simple” disorder.Mol Genet Metab. 2004; 83: 6-15Crossref PubMed Scopus (305) Google Scholar, 17Suzuki K Iseki E Katsuse O Yamaguchi A Katsuyama K Aoki I Yamanaka S Kosaka K Neuronal accumulation of α- and β-synucleins in the brain of a GM2 gangliosidosis mouse model.Neuroreport. 2003; 14: 551-554Crossref PubMed Scopus (45) Google Scholar, 18Wirths O Bayer TA α-Synuclein, Aβ and Alzheimer's disease.Prog Neuropsychopharmacol Biol Psychiatry. 2003; 27: 103-108Crossref PubMed Scopus (36) Google Scholar MSA represents a special disease among the α-synucleinopathies, which primarily are dominated by diseases with intraneuronal Lewy body inclusions such as Parkinson's disease and Lewy body dementias. In MSA, the principal depositing of α-synuclein takes place in oligodendroglial cell bodies as glial cytoplasmic inclusions (GCIs),19Dickson DW Liu W Hardy J Farrer M Mehta N Uitti R Mark M Zimmerman T Golbe L Sage J Sima A D'Amato C Albin R Gilman S Yen SH Widespread alterations of α-synuclein in multiple system atrophy.Am J Pathol. 1999; 155: 1241-1251Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar although accumulation of α-synuclein in neurons also occurs.20Baker KG Huang Y McCann H Gai WP Jensen PH Halliday GM P25α immunoreactive but α-synuclein immunonegative neuronal inclusions in multiple system atrophy.Acta Neuropathol (Berl). 2006; 111: 193-195Crossref PubMed Scopus (22) Google Scholar, 21Wakabayashi K Yoshimoto M Tsuji S Takahashi H α-Synuclein immunoreactivity in glial cytoplasmic inclusions in multiple system atrophy.Neurosci Lett. 1998; 249: 180-182Crossref PubMed Scopus (521) Google Scholar MSA is an aggressive parkinsonian syndrome displaying a widespread development of GCIs and degeneration of both oligodendroglia and neurons, accounting for its symptoms that range from autonomic dysfunctions, parkinsonism, and cerebellar signs. The tissue load of aggregated α-synuclein is high in comparison to Parkinson's disease and Lewy body dementia and is best appreciated by immunoblotting of denatured brain extracts.19Dickson DW Liu W Hardy J Farrer M Mehta N Uitti R Mark M Zimmerman T Golbe L Sage J Sima A D'Amato C Albin R Gilman S Yen SH Widespread alterations of α-synuclein in multiple system atrophy.Am J Pathol. 1999; 155: 1241-1251Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar GCI genesis in MSA remains unclear because oligodendroglial α-synuclein mRNA expression has not been demonstrated in vivo, although oligodendroglia are able to express α-synuclein in culture.22Richter-Landsberg C Gorath M Trojanowski JQ Lee VM α-Synuclein is developmentally expressed in cultured rat brain oligodendrocytes.J Neurosci Res. 2000; 62: 9-14Crossref PubMed Scopus (119) Google Scholar Transgenic oligodendroglial-specific expression of human α-synuclein causes a neurodegenerative phenotype in mice that resembles MSA and clearly demonstrates that neuronal degeneration develops subsequent to an α-synuclein-mediated dysfunction of oligodendroglia.23Shults CW Rockenstein E Crews L Adame A Mante M Larrea G Hashimoto M Song D Iwatsubo T Tsuboi K Masliah E Neurological and neurodegenerative alterations in a transgenic mouse model expressing human α-synuclein under oligodendrocyte promoter: implications for multiple system atrophy.J Neurosci. 2005; 25: 10689-10699Crossref PubMed Scopus (190) Google Scholar, 24Yazawa I Giasson BI Sasaki R Zhang B Joyce S Uryu K Trojanowski JQ Lee VM Mouse model of multiple system atrophy α-synuclein expression in oligodendrocytes causes glial and neuronal degeneration.Neuron. 2005; 45: 847-859Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar We have previously shown that p25α is a potent stimulator of α-synuclein aggregation and demonstrated in MSA that p25α colocalizes with oligodendroglial α-synuclein-immunopositive GCIs.25Lindersson E Lundvig D Petersen C Madsen P Nyengaard JR Hojrup P Moos T Otzen D Gai WP Blumbergs PC Jensen PH p25α stimulates α-synuclein aggregation and is co-localized with aggregated α-synuclein in α-synucleinopathies.J Biol Chem. 2005; 280: 5703-5715Crossref PubMed Scopus (151) Google Scholar To investigate the role of p25α in oligodendroglia, and MSA in particular, this study analyzes the normal association of p25α to myelin, its binding to MBP, and the changes that occur with these components in relation to MSA. We find that p25α is a ligand of MBP and colocalizes with MBP in brainstem myelinated fiber tracts. The colocalization with MBP is lost in MSA where p25α appears to relocate within the oligodendroglia to accumulate in the cell body, enlarging this structure before colocalization with α-synuclein. Along with the relocalization of p25α from myelin, there is a reduction in the level of MBP and an increase in degraded MBP that also becomes relocalized to the oligodendroglial cell bodies. The results suggest a sequence of events in which early pathogenic signals impede the normal cellular function of p25α in myelin resulting in a decreased stability of MBP and a build-up of degraded MBP and p25α in the expanding cell bodies that favor subsequent deposition and fibrillization of α-synuclein leading to neurodegeneration. All chemicals were of analytical grade. Bovine MBP was purchased from Sigma, St. Louis, MO. Recombinant human p25α was expressed in Escherichia coli and purified as described previously.25Lindersson E Lundvig D Petersen C Madsen P Nyengaard JR Hojrup P Moos T Otzen D Gai WP Blumbergs PC Jensen PH p25α stimulates α-synuclein aggregation and is co-localized with aggregated α-synuclein in α-synucleinopathies.J Biol Chem. 2005; 280: 5703-5715Crossref PubMed Scopus (151) Google Scholar Complete ethylenediamine tetraacetic acid (EDTA)-free mini protease inhibitors were purchased from Roche (Indianapolis, IN) and PEFA Bloc SC was from Pentapharm AG (Basel, Switzerland). Kinetic analysis was performed on a Biacore 3000 instrument (Biacore, Uppsala, Sweden) equipped with CM5 sensor chips maintained at 25°C. A continuous flow of CaHBS buffer (150 mmol/L NaCl, 1 mmol/L EGTA, 1.5 mmol/L CaCl2, 0.01% P-20, and 10 mmol/L HEPES, pH 7.4) was passed over the sensor surface at a rate of 5 μl/minute. The carboxylated dextran matrix of the sensor chip flow cells was activated by injection of 60 μl of 0.2 mol/L N-ethyl-N-(3-dimethylaminopropyl) carbodiimide and 0.05 mol/L N-hydroxysuccimide. A solution of recombinant human p25α (80 μl, 100 μg/ml in CaHBS buffer) was then injected at a flow rate of 5 μl/minute. Remaining binding sites were blocked by injection of 35 μl of 1 mol/L ethanolamine, pH 8.5, at the same flow rate. The surface plasmon resonance signal from immobilized p25α generated 4489 Biacore response units equivalent to 190 fmol/mm2. MBP samples (40 μl, in CaHBS buffer) at concentrations of 2 to 100 nmol/L were injected through the flow cell at 5 μl/minute. Binding was expressed in relative response units, ie, the difference in response between the immobilized protein flow cell and a corresponding control flow cell (activated and blocked, but without any protein). Regeneration of the sensor chip after each cycle of analysis was performed by injecting 20 μl of 200 mmol/L Na2CO3, pH 11.0. The kinetic parameter was obtained using BIAevaluation 4.1 (Biacore). Eight hundred μl of a 2 μmol/L MBP solution was added to a 1.7-ml quartz cuvette and p25α was added in 10- to 50-μl steps from an 8 μmol/L stock solution and mixed. After the addition of each aliquot, the solution was allowed to equilibrate for 30 seconds before measuring fluorescence intensity Fobs (excitation at 295 nm, emission at 310 nm, excitation and emission slit widths 10 nm on a LS55 Luminescence Spectrometer; Perkin Elmer, Boston, MA). Intensities of the p25α and MBP stock solutions were recorded separately in the same cuvette before the titration experiment. The fluorescence emission intensity Fexp expected from the titrated solution in the absence of interactions between MBP and p25α was calculated as follows:Fexp=I0MBP*VMBPVMBP+Vp25+I0p25*Vp25VMBP+Vp25 I0p25 and I0MBP are the individual fluorescence intensities of the MBP and p25α stock solutions, whereas VMBP and Vp25 are the volumes of the MBP solution (fixed, 800 μl) and added p25α solution (0 to 400 μl). The difference in the expected and the observed (measured) intensities, Fexp − Fobs, was plotted versus the ratio [p25α]:[MBP]. Far-UV CD studies were performed on a Jasco J-715 spectropolarimeter (Jasco Spectroscopic, Målndal, Sweden) with a Jasco PTC-348W temperature control unit, essentially as described previously10Otzen DE Lundvig DM Wimmer R Nielsen LH Pedersen JR Jensen PH p25α is flexible but natively folded and binds tubulin with oligomeric stoichiometry.Protein Sci. 2005; 14: 1396-1409Crossref PubMed Scopus (37) Google Scholar with protein concentrations of 14 μmol/L MBP and 17 μmol/L p25α. Adult porcine brains were obtained from an Aarhus slaughter house. The cerebellum and medulla oblongata were removed, and the brains were frozen in liquid nitrogen and stored at −80°C. Two separate protocols were used to extract different fractions for different experimental procedures. All steps were performed at 4°C. Brain tissue was homogenized in a solution of 0.32 mol/L sucrose, 50 mmol/L NaF, 20 mmol/L Na4P2O7, 1 mmol/L vanadate, 2 mmol/L EDTA, 2 mmol/L PEFA Bloc SC, and 10 mmol/L Tris-HCl, pH 7.4. The homogenate was centrifuged at 1000 × g for 15 minutes, and the cleared supernatant was centrifuged (145,000 × g, 3 hours). The resulting supernatant was filtered through a 0.45-μm filter (soluble fraction). Protein concentrations were determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). Myelin was isolated from porcine brain according to a previously published protocol.26Norton WT Poduslo SE Myelination in rat brain: method of myelin isolation.J Neurochem. 1973; 21: 749-757Crossref PubMed Scopus (1266) Google Scholar All steps were performed at 4°C. In brief, porcine brain tissue was homogenized in 20 vol (w/v) 0.32 mol/L sucrose and layered over 0.85% sucrose, followed by centrifugation (75,000 × g, 30 minutes). The crude myelin fraction was collected at the interphase of the sucrose layers and resuspended in water and centrifuged (75,000 × g, 15 minutes). The supernatant was discarded, and the myelin pellet was osmotically shocked by resuspension in water followed by centrifugation (12,000 × g, 10 minutes). This step was repeated. The resulting myelin pellet was resuspended in 0.32 mol/L sucrose, layered over 0.85 mol/L sucrose, and centrifuged (75,000 × g, 30 minutes). The purified myelin was collected at the interphase of the two sucrose layers. The extraction of myelin involved the resuspension of myelin (200 μg) in phosphate-buffered saline (PBS; pH 7.2), 1% Triton X-100, 0.1 mol/L Na2CO3, pH 11.5, 1% sodium dodecyl sulfate (SDS), 1% Triton X-100, and 1 mol/L NaCl, and incubated for 30 minutes at 37°C. The myelin samples were centrifuged (13,500 × g, 10 minutes), and both the pellets (P) and supernatants (S) were used. Protein concentrations were determined using the BCA method. Human brain tissue was obtained from the Australian Brain Donor Programs and the South Australia Brain Bank. For all cases (16 MSA) and controls,12Goedert M Spillantini MG Davies SW Filamentous nerve cell inclusions in neurodegenerative diseases.Curr Opin Neurobiol. 1998; 8: 619-632Crossref PubMed Scopus (227) Google Scholar consent for brain autopsy was given and brain removal for research studies approved by the Human Ethics Committees of the institutions involved. The causes of death for the MSA cases were pneumonia (n = 8), cardiorespiratory arrest (n = 6), multiorgan failure, and drowning. The causes of death for the controls were cancer (n = 4), cardiorespiratory arrest (n = 5), renal failure (n = 2), and postoperative complications. Two separate protocols were used: a protocol to extract different fractions for analysis of protein amount and a standard protocol for in situ protein localization. One g of white matter under the superior precentral gyrus (region that does not contribute to clinical presentation and is without cell loss but with GCIs27Su M Yoshida Y Hirata Y Watahiki Y Nagata K Primary involvement of the motor area in association with the nigrostriatal pathway in multiple system atrophy: neuropathological and morphometric evaluations.Acta Neuropathol (Berl). 2001; 101: 57-64PubMed Google Scholar) was sampled from frozen brain hemispheres of four MSA cases (three categorized according to Jellinger and colleagues28Jellinger KA Seppi K Wenning GK Grading of neuropathology in multiple system atrophy: proposal for a novel scale.Mov Disord. 2005; 20: S29-S36Crossref PubMed Scopus (148) Google Scholar as MSA-parkinsonian and one as MSA-cerebellar) and four age-matched controls that had been stored for <2 years at −80°C. Brain proteins were extracted using a previously described protocol.29Anderson JP Walker DE Goldstein JM de Laat R Banducci K Caccavello RJ Barbour R Huang J Kling K Lee M Diep L Keim PS Shen X Chataway T Schlossmacher MG Seubert P Schenk D Sinha S Gai WP Chilcote TJ Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.J Biol Chem. 2006; 281: 29739-29752Crossref PubMed Scopus (879) Google Scholar In brief, tissue was homogenized in a Tris-sucrose homogenization buffer (0.32 mol/L sucrose, 20 mmol/L Tris-Cl, and 5 mmol/L EDTA, pH 7.4), in the presence of a protease inhibitor cocktail (Complete, EDTA-free; Roche). This was followed by sonication (2 × 10 seconds) and centrifugation (23,500 × g, 10 minutes, 4°C), and the resulting supernatant was stored as the soluble particulate fraction. Pellets were resuspended and sonicated (2 × 10 seconds) in a SDS-urea solubilization buffer (6 mol/L urea, 1% SDS, and 5 mmol/L EDTA) (1:5 w/v) and centrifuged (23,500 × g, 10 minutes, room temperature) to obtain the SDS-extractable insoluble particulate fraction. Protein concentrations were determined using the BCA method. Fifteen percent buffered formalin-fixed samples of the pons (region that contributes to a MSA-cerebellar presentation28Jellinger KA Seppi K Wenning GK Grading of neuropathology in multiple system atrophy: proposal for a novel scale.Mov Disord. 2005; 20: S29-S36Crossref PubMed Scopus (148) Google Scholar) were taken from transversely cut slices of either whole or midsagittally dissected brainstems of 12 MSA cases (eight categorized as MSA-parkinsonian and four as MSA-cerebellar28Jellinger KA Seppi K Wenning GK Grading of neuropathology in multiple system atrophy: proposal for a novel scale.Mov Disord. 2005; 20: S29-S36Crossref PubMed Scopus (148) Google Scholar) and eight age-matched controls. Tissue samples were paraffin-embedded, cut at 5 μm on a microtome, and mounted on 3-aminopropyltriethoxysilane (TESPA)-coated slides for immunohistochemical staining. Brain protein samples were added with SDS-loading buffer (4% SDS, 40% glycerol, 50 mmol/L Tris-HCl, pH 6.8) supplemented with 20 mmol/L DTE for reducing SDS-polyacrylamide gel electrophoresis (PAGE) and heated for 3 minutes at 95°C. Proteins were separated by 10 to 16% (w/v) gradient SDS-PAGE. For Western blotting, SDS-PAGE gels were electroblotted onto nitrocellulose or polyvinylidene difluoride membranes (Amersham Biosciences, Buckinghamshire, UK; and Bio-Rad, Hercules, CA). Membranes were blocked in 5% skimmed milk (dissolved in 50 mmol/L NaCl, 0.05% Tween 20, and 20 mmol/L Tris-HCl, pH 7.4), followed by incubation with several primary and secondary antibodies depending on the experiments required and tissues used. To detect p25α, polyclonal rabbit anti-p25α1 antibody raised against recombinant human p25α was used as described previously.25Lindersson E Lundvig D Petersen C Madsen P Nyengaard JR Hojrup P Moos T Otzen D Gai WP Blumbergs PC Jensen PH p25α stimulates α-synuclein aggregation and is co-localized with aggregated α-synuclein in α-synucleinopathies.J Biol Chem. 2005; 280: 5703-5715Crossref PubMed Scopus (151) Google Scholar Monoclonal rat anti-MBP IgG (ab7349; Abcam, Cambridge, UK) or rabbit anti-MBP IgG (18-0444, 1:2000; Zymed Laboratories, South San Francisco, CA) was used to detect MBP. For standardization and internal controls, monoclonal mouse anti-BiP/Grp78 IgG (610978, 1:500; BD Biosciences, Franklin Lakes, NJ) and monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) IgG (4300, 1:4000; Ambion, Austin, TX) were used. Detection was done with enhanced chemiluminescence (Amersham Biosciences; and NEL104; Perkin Elmer, Boston, MA) using secondary antibodies conjugated to horseradish peroxidase (1:1000: DAKO, Glostrup, Denmark; or 1:5000: Sigma; 1:4000: Bio-Rad). Three different columns for chromatography were developed using either recombinant human p25α, anti-p25α1 IgG, or nonimmune IgG immobilized to CNBr-activated Sepharose 4B according to the manufacturer's instructions (Amersham Biosciences). Polyclonal rabbit anti-p25α1 antibody was raised against recombinant human p25α as described previously,25Lindersson E Lundvig D Petersen C Madsen P Nyengaard JR Hojrup P Moos T Otzen D Gai WP Blumbergs PC Jensen PH p25α stimulates α-synuclein aggregation and is co-localized with aggregated α-synuclein in α-synucleinopathies.J Biol Chem. 2005; 280: 5703-5715Crossref PubMed Scopus (151) Google Scholar and rabbit nonimmune IgG was prepared by subjecting preimmune serum to protein A chromatography. All following procedures were performed at 4°C. Soluble cytosol fraction from porcine protocol 1 (see above) was applied to each of the columns. The columns were equilibrated with 90 mmol/L NaCl, 50 mmol/L NaF, 20 mmol/L Na4P2O7, 1 mmol/L vanadate, and 10 mmol/L NaH2PO4, pH 7.4. The columns were eluted with 0.1 mol/L glycine, pH 2.5, and 1-ml fractions were collected. The protein elution profiles were determined by gel electrophoresis and silver staining. Protein bands indicated in Figure 1 were excised from silver-stained gels and subjected to tryptic digestion.30Shevchenko A Wilm M Vorm O Jensen ON Podtelejnikov AV Neubauer G Shevchenko A Mortensen P Mann M A strategy for identifying gel-separated proteins in sequence databases by MS alone.Biochem Soc Trans. 1996; 24: 893-896Crossref PubMed Scopus (196) Google Scholar The resulting peptides were analyzed using a Voyager STR MALDI instrument operating in reflector mode (Applied Biosystems, Foster City, CA). The peptide mass list was analyzed using the PeakErazor program31Hjerno K Kamp RM PeakErazor: A Windows-Based Program for Improving Peptide Mass Searches. Springer Verlag, 2004: 359-370Google Scholar before analysis on the Mascot search engine (Matrix Science, London, UK) for a positive identification in the National Center for Biotechnology Information nonredundant protein database. The spectral data correlated with the MBP sequence using the GPMAW software.32Peri S Steen H Pandey A GPMAW—a software tool for analyzing proteins and peptides.Trends Biochem Sci. 2001; 26: 687-689Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar The soluble cytosol fraction from porcine protocol 1 was subjected to immunoprecipitation by adding 10 μg of anti-p25α1 IgG (50 μl) or 50 μl of anti-MBP IgG, followed by incubation for 16 hours at 4°C. Polyclonal rabbit anti-p25α1 antibody was raised against recombinant human p25α as described previously,25Lindersson E Lundvig D Petersen C Madsen P Nyengaard JR Hojrup P Moos T Otzen D Gai WP Blumbergs PC Jensen PH p25α stimulates α-synuclein aggregation and is co-localized with aggregated α-synuclein in α-synucleinopathies.J Biol Chem. 2005; 280: 5703-5715Crossref PubMed Scopus (151) Google Scholar and monoclonal rat anti-MBP IgG (ab7349; Abcam) was used. Hereafter, 25 μl of protein A-Sepharose (Amersham Biosciences) was added, followed by a 1-hour incubation at 4°C to immobilize the antibody. The beads were washed in an ice-cold buffer (0.5% Triton X-100 and PBS, pH 7.2), and SDS-loading buffer was added. The samples were subjected to gel electrophoresis and Western blotting. Double-fluorescence immunohistochemical labeling to assess myelin changes was performed using previously established methods, including antigen retrieval20Baker KG Huang Y McCann H Gai WP Jensen PH Halliday G
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