Selective Insolubility of α-Synuclein in Human Lewy Body Diseases Is Recapitulated in a Transgenic Mouse Model
2001; Elsevier BV; Volume: 159; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63072-6
ISSN1525-2191
AutoresPhilipp J. Kahle, Manuela Neumann, Laurence Ozmen, Veronika I. Müller, Sabine Odoy, Noriko Okamoto, Helmut Jacobsen, Takeshi Iwatsubo, John Q. Trojanowski, Hitoshi Takahashi, Koichi Wakabayashi, Nenad Bogdanović, Peter Riederer, Hans A. Kretzschmar, Christian Haass,
Tópico(s)Nuclear Receptors and Signaling
Resumoα-Synuclein (α-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinson's disease (PD) and LB dementias. α-SYN forms LB-like fibrils in vitro, in contrast to its homologue β-SYN. Here we have investigated the solubility of SYNs in human LB diseases and in transgenic mice expressing human wild-type and PD-associated mutant [A30P]α-SYN driven by the brain neuron-specific promoter, Thy1. Distinct α-SYN species were detected in the detergent-insoluble fractions from brains of patients with PD, dementia with LBs, and neurodegeneration with brain iron accumulation type 1 (formerly known as Hallervorden-Spatz disease). Using the same extraction method, detergent-insolubility of human α-SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse α-SYN nor β-SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic β-SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of α-SYN are absent in β-SYN. In conclusion, the specific accumulation of detergent-insoluble α-SYN in transgenic mice recapitulates a pivotal feature of human LB diseases. α-Synuclein (α-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinson's disease (PD) and LB dementias. α-SYN forms LB-like fibrils in vitro, in contrast to its homologue β-SYN. Here we have investigated the solubility of SYNs in human LB diseases and in transgenic mice expressing human wild-type and PD-associated mutant [A30P]α-SYN driven by the brain neuron-specific promoter, Thy1. Distinct α-SYN species were detected in the detergent-insoluble fractions from brains of patients with PD, dementia with LBs, and neurodegeneration with brain iron accumulation type 1 (formerly known as Hallervorden-Spatz disease). Using the same extraction method, detergent-insolubility of human α-SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse α-SYN nor β-SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic β-SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of α-SYN are absent in β-SYN. In conclusion, the specific accumulation of detergent-insoluble α-SYN in transgenic mice recapitulates a pivotal feature of human LB diseases. α-Synuclein (α-SYN) has been identified as the precursor protein of a nonamyloid β-protein component (NAC) isolated from Alzheimer's disease plaques.1Uéda K Fukushima H Masliah E Xia Y Iwai A Yoshimoto M Otero DA Kondo J Ihara Y Saitoh T Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Scopus (1250) Google Scholar α-SYN was detected immunohistochemically in Lewy bodies (LBs) and Lewy neurites that characterize Parkinson's disease (PD), LB dementia (DLB), LB variant Alzheimer's disease,2Spillantini MG Schmidt ML Lee VM-Y Trojanowski JQ Jakes R Goedert M α-Synuclein in Lewy bodies.Nature. 1997; 388: 839-840Crossref PubMed Scopus (6342) Google Scholar, 3Wakabayashi K Matsumoto K Takayama K Yoshimoto M Takahashi H NACP, a presynaptic protein, immunoreactivity in Lewy bodies in Parkinson's disease.Neurosci Lett. 1997; 239: 45-48Crossref PubMed Scopus (288) Google Scholar, 4Takeda A Mallory M Sundsmo M Honer W Hansen L Masliah E Abnormal accumulation of NACP/α-synuclein in neurodegenerative disorders.Am J Pathol. 1998; 152: 367-372PubMed Google Scholar, 5Irizarry MC Growdon W Gomez-Isla T Newell K George JM Clayton DF Hyman BT Nigral and cortical Lewy bodies and dystrophic nigral neurites in Parkinson's disease and cortical Lewy body disease contain α-synuclein immunoreactivity.J Neuropathol Exp Neurol. 1998; 57: 334-337Crossref PubMed Scopus (364) Google Scholar, 6Baba M Nakajo S Tu P-H Tomita T Nakaya K Lee VM-Y Trojanowski JQ Iwatsubo T Aggregation of α-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies.Am J Pathol. 1998; 152: 879-884PubMed Google Scholar, 7Bayer TA Jäkälä P Hartmann T Havas L McLean C Culvenor JG Li QX Masters CL Falkai P Beyreuther K α-Synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (87) Google Scholar and neurodegeneration with brain iron accumulation type 1 (NBIA1).8Arawaka S Saito Y Murayama S Mori H Lewy body in neurodegeneration with brain iron accumulation type 1 is immunoreactive for α-synuclein.Neurology. 1998; 51: 887-889Crossref PubMed Scopus (159) Google Scholar, 9Wakabayashi K Yoshimoto M Fukushima T Koide R Horikawa Y Morita T Takahashi H Widespread occurrence of α-synuclein/NACP-immunoreactive neuronal inclusions in juvenile and adult-onset Hallervorden-Spatz disease with Lewy bodies.Neuropathol Appl Neurobiol. 1999; 25: 363-368Crossref PubMed Scopus (67) Google Scholar, 10Galvin JE Giasson B Hurtig HI Lee VM-Y Trojanowski JQ Neurodegeneration with brain iron accumulation, type 1 is characterized by α-, β-, and γ-synuclein neuropathology.Am J Pathol. 2000; 157: 361-368Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Neumann M Adler S Schlüter O Kremmer E Benecke R Kretzschmar HA α-Synuclein accumulation in a case of neurodegeneration with brain iron accumulation type 1 (NBIA-1, formerly Hallervorden-Spatz syndrome) with widespread cortical and brainstem-type Lewy bodies.Acta Neuropathol. 2000; 100: 568-574Crossref PubMed Scopus (76) Google Scholar Antibodies directed against both N-terminal and C-terminal epitopes recognized LB filaments,12Spillantini MG Crowther RA Jakes R Hasegawa M Goedert M α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies.Proc Natl Acad Sci USA. 1998; 95: 6469-6473Crossref PubMed Scopus (2462) Google Scholar, 13Arima K Uéda K Sunohara N Hirai S Izumiyama Y Tonozuka-Uehara H Kawai M Immunoelectron-microscopic demonstration of NACP/α-synuclein-epitopes on the filamentous component of Lewy bodies in Parkinson's disease and in dementia with Lewy bodies.Brain Res. 1998; 808: 93-100Crossref PubMed Scopus (219) Google Scholar and the presence of full-length α-SYN was biochemically proven on Western blots of isolated LBs.6Baba M Nakajo S Tu P-H Tomita T Nakaya K Lee VM-Y Trojanowski JQ Iwatsubo T Aggregation of α-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies.Am J Pathol. 1998; 152: 879-884PubMed Google Scholar Moreover, full-length α-SYN is the major fibrillar component of glial cytoplasmic inclusions in multiple system atrophy.14Dickson DW Lin W Liu WK Yen SH Multiple system atrophy: a sporadic synucleinopathy.Brain Pathol. 1999; 9: 721-732Crossref PubMed Scopus (174) Google Scholar The formation of LB-like fibrils is an intrinsic property of α-SYN. 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The causal relationship between α-SYN fibrillization and PD are therefore subject to intense research.24Goldberg MS Lansbury Jr, PT Is there a cause-and-effect relationship between α-synuclein fibrillization and Parkinson's disease?.Nat Cell Biol. 2000; 2: E115-E119Crossref PubMed Scopus (123) Google Scholar Transgenic animals expressing human wild-type [wt]- as well as PD-associated mutant [A53T]α-SYN25Polymeropoulos MH Lavedan C Leroy E Ide SE Dehejia A Dutra A Pike B Root H Rubenstein J Boyer R Stenroos ES Chandrasekharappa S Athanassiadou A Papapetropoulos T Johnson WG Lazzarini AM Duvoisin RC Di Iorio G Golbe LI Nussbaum RL Mutation in the α-synuclein gene identified in families with Parkinson's disease.Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6785) Google Scholar and [A30P]α-SYN26Krüger R Kuhn W Müller T Woitalla D Graeber M Kösel S Przuntek H Epplen JT Schöls L Riess O Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease.Nat Genet. 1998; 18: 106-108Crossref PubMed Scopus (3360) Google Scholar were recently presented. Wild-type and mutant α-SYN assembled into LB-like fibrils in transgenicDrosophila, and a locomotor deficit became apparent with increasing age.27Feany MB Bender WW A Drosophila model of Parkinson's disease.Nature. 2000; 404: 394-398Crossref PubMed Scopus (1740) Google Scholar Somal and neuritic accumulations of wt and mutant α-SYN were observed in transgenic mouse brain.28Masliah E Rockenstein E Veinbergs I Mallory M Hashimoto M Takeda A Sagara Y Sisk A Mucke L Dopaminergic loss and inclusion body formation in α-synuclein mice: implications for neurodegenerative disorders.Science. 2000; 287: 1265-1269Crossref PubMed Scopus (1577) Google Scholar, 29van der Putten H Wiederhold K-H Probst A Barbieri S Mistl C Danner S Kauffmann S Hofele K Spooren WPJM Ruegg MA Lin S Caroni P Sommer B Tolnay M Bilbe G Neuropathology in mice expressing human α-synuclein.J Neurosci. 2000; 20: 6021-6029Crossref PubMed Google Scholar, 30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar Ubiquitination was occasionally detected, but the α-SYN accumulations did not meet ultrastructural criteria of LBs.28Masliah E Rockenstein E Veinbergs I Mallory M Hashimoto M Takeda A Sagara Y Sisk A Mucke L Dopaminergic loss and inclusion body formation in α-synuclein mice: implications for neurodegenerative disorders.Science. 2000; 287: 1265-1269Crossref PubMed Scopus (1577) Google Scholar, 29van der Putten H Wiederhold K-H Probst A Barbieri S Mistl C Danner S Kauffmann S Hofele K Spooren WPJM Ruegg MA Lin S Caroni P Sommer B Tolnay M Bilbe G Neuropathology in mice expressing human α-synuclein.J Neurosci. 2000; 20: 6021-6029Crossref PubMed Google Scholar Masliah and colleagues28Masliah E Rockenstein E Veinbergs I Mallory M Hashimoto M Takeda A Sagara Y Sisk A Mucke L Dopaminergic loss and inclusion body formation in α-synuclein mice: implications for neurodegenerative disorders.Science. 2000; 287: 1265-1269Crossref PubMed Scopus (1577) Google Scholar reported a modest reduction of locomotor performance and van der Putten and colleagues29van der Putten H Wiederhold K-H Probst A Barbieri S Mistl C Danner S Kauffmann S Hofele K Spooren WPJM Ruegg MA Lin S Caroni P Sommer B Tolnay M Bilbe G Neuropathology in mice expressing human α-synuclein.J Neurosci. 2000; 20: 6021-6029Crossref PubMed Google Scholar found that age-dependent degeneration of neuromuscular junctions caused a severe locomotor deficit and premature death in their mice. α-SYN and β-SYN have both been found in the synaptosomal fractions of rodent and human brain.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar, 31Maroteaux L Scheller RH The rat brain synucleins; family of proteins transiently associated with neuronal membrane.Mol Brain Res. 1991; 11: 335-343Crossref PubMed Scopus (298) Google Scholar, 32Shibayama-Imazu T Okahashi I Omata K Nakajo S Ochiai H Nakai Y Hama T Nakamura Y Nakaya K Cell and tissue distribution and developmental change of neuron specific 14 kDa protein (phosphoneuroprotein 14).Brain Res. 1993; 622: 17-25Crossref PubMed Scopus (109) Google Scholar, 33George JM Jin H Woods WS Clayton DF Characterization of a novel protein regulated during the critical period for song learning in the zebra finch.Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (736) Google Scholar, 34Irizarry MC Kim T-W McNamara M Tanzi RE George JM Clayton DF Hyman BT Characterization of the precursor protein of the non-Aβ component of senile plaques (NACP) in the human central nervous system.J Neuropathol Exp Neurol. 1996; 55: 889-895Crossref PubMed Scopus (185) Google Scholar Synaptosomal α-SYN was released into the soluble fraction of human brain biopsies.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar We now report that synaptosomal α-SYN was recovered from the particulate fraction in the case of frozen post mortem brain samples whereas β-SYN was released into the soluble synaptosomal fraction even from archived brain samples. To directly measure SYN solubility, differential detergent extractions were performed. Most of the α-SYN was highly soluble in aqueous buffer and the remainder easily extractable with sodium dodecyl sulfate (SDS). However, detergent-insoluble α-SYN monomers and aggregates were detected in urea extracts from LB disease patient brains, but not in controls. Likewise, some of the human α-SYN was detergent-insoluble in transgenic mouse brains, in sharp contrast to the endogenous mouse α-SYN and β-SYN. The nonamyloidogenic β-SYN failed to form aggregates in vitro because of the lack of amino acids 73 to 83 in the NAC domain. In conclusion, transgenic expression of human α-SYN in mouse brain neurons recapitulates an important aspect of human LB diseases, namely the accumulation of insoluble α-SYN. Rat monoclonal anti-α-SYN 15G711Neumann M Adler S Schlüter O Kremmer E Benecke R Kretzschmar HA α-Synuclein accumulation in a case of neurodegeneration with brain iron accumulation type 1 (NBIA-1, formerly Hallervorden-Spatz syndrome) with widespread cortical and brainstem-type Lewy bodies.Acta Neuropathol. 2000; 100: 568-574Crossref PubMed Scopus (76) Google Scholar, 30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar and mouse monoclonal anti-synaptophysin SY38 hybridoma supernatants were used as described previously.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar Mouse monoclonal anti-α-SYN LB509 and Syn102 were described before.6Baba M Nakajo S Tu P-H Tomita T Nakaya K Lee VM-Y Trojanowski JQ Iwatsubo T Aggregation of α-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies.Am J Pathol. 1998; 152: 879-884PubMed Google Scholar The mouse monoclonal anti-α-SYN MC42 (working dilution, 1:1000) was purchased from Transduction Laboratories (Lexington, KY), and the rabbit polyclonal anti-α-SYN antiserum 3400 (working dilution, 1:20,000) from Affiniti (Mamhead, UK). Mouse-specific anti-α-SYN antiserum 7544 and anti-β-SYN antiserum 6485 have been described previously.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar The rabbit polyclonal anti-NAC antiserum7Bayer TA Jäkälä P Hartmann T Havas L McLean C Culvenor JG Li QX Masters CL Falkai P Beyreuther K α-Synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (87) Google Scholar was used at a working dilution of 1:1000. Mouse monoclonal anti-ubiquitin Ubi-1 (working dilution, 1 μg/ml) was purchased from Zymed (South San Francisco, CA). Goat anti-rat IgG peroxidase conjugate (working dilution, 1:1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG (working dilution, 1:5000) from Sigma (St. Louis, MO). Subcellular fractionation of archived human cerebral cortex samples was performed as previously described for fresh tissue.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar The detergent extraction method of Culvenor and colleagues35Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jäkälä P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar was applied to human and mouse brain with slight modifications. Approximately 0.5 g of brain tissue was homogenized in 10 volumes of TBS+ (Tris-buffered saline plus Complete protease inhibitor cocktail; Roche Diagnostics, Mannheim, Germany) and sonicated. After 5 minutes of centrifugation at 1000 ×g, the supernatants were ultracentrifuged for 1 hour at 130,000 × g. The resulting supernatants represented the buffer-soluble fractions. The pellets were rinsed twice with TBS+ and extracted with 500 μl of 5% SDS in TBS+. All subsequent steps were performed at 24°C. After ultracentrifugation for 30 minutes at 130,000 × g the pellets were re-extracted twice with 5% SDS, and the detergent-soluble supernatants were collected. The bicucullinic acid (BCA) protein assay (Pierce, Rockford, IL) revealed concentrations >1 mg/ml in the first two SDS supernatants that were pooled. The extensively washed detergent-insoluble pellets were squashed in 100 μl of 8 mol/L urea/5% SDS in TBS+ and incubated for at least 10 minutes at room temperature. Then, 80 μl of the resulting suspension were mixed with 20 μl of trichloroacetic acid (TCA) (100%) and allowed to precipitate overnight at 4°C. Protein precipitates were collected by centrifugation, washed with acetone, and resuspended in protein gel-loading buffer containing 6 mol/L urea. Denaturing polyacrylamide gel electrophoresis (PAGE), Western blotting, and probing were done as described previously.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar Equal loading was verified by Coomassie blue staining of the gels after transfer. Enhanced chemiluminescence was generated with SuperSignal (Pierce) or ECLplus (Amersham Pharmacia, Little Chalfont, UK). Human-specific 15G7 band intensities in 25-μg mouse brain cytosol samples were determined relative to 5 ng, 10 ng, 20 ng, and 40 ng recombinant human α-SYN (see below) on the same blot. Mouse-specific 7544 band intensities in 100-μg mouse brain cytosol samples were determined relative to four standards of 75- to 500-ng recombinant mouse α-SYN (see below) on the same blot. Band intensities from densitometric scans were quantified using NIH Image v1.62 freeware (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). Linear regression of the α-SYN standard band intensities revealed correlation coefficients between 0.9 and 1.0. The amplified human [wt]α-SYN-coding sequence was subcloned into the XhoI site of the Thy1 cassette of pTSC21k, and theNotI-linearized DNA was used to generate transgenic C57BL/6 mice as described for [A30P] α-SYN.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar Five founders stably transmitted the transgene, as determined by tail biopsy polymerase chain reaction. Transgene copy number was determined by Southern blotting using as references known amounts of transgene fragment mixed to genomic DNA isolated from nontransgenic littermates. Ten μg of genomicXbaI-KpnI restriction fragments were fractionated by gel electrophoresis and blotted onto Nylon membranes (Roche Molecular Biosciences). A 1.6-kb DNA probe (HindIII-EcoRV fragment of the transgene) was labeled with [33P]dCTP by the random primer method using the Ready-to-Go DNA labeling kit (Pharmacia Biotech). Hybridization was performed overnight at 65°C in 6× standard saline citrate, 10% dextran sulfate, 0.5% SDS. Blots were washed in 2× standard saline citrate (+0.1% SDS) at 65°C for 20 minutes followed by a second wash for 20 minutes at 65°C in 0.2× standard saline citrate (+0.1% SDS). The intensity of the bands was quantified using a phosphorimager scanner. Northern blotting using oligonucleotide probes specific for mRNA of the human α-SYN transgene and mouse β-actin was performed as described previously.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar Fresh mouse brains were fixed in phosphate-buffered 4% paraformaldehyde and embedded in paraffin. Immunocytochemical detection of SYNs was performed as described previously.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar The β-SYN expression vector has been described by Jakes and colleagues.36Jakes R Spillantini MG Goedert M Identification of two distinct synucleins from human brain.FEBS Lett. 1994; 345: 27-32Abstract Full Text PDF PubMed Scopus (913) Google Scholar The mouse α-SYN37Hong L Ko HW Gwag BJ Joe E Lee S Kim Y-T Suh Y-H The cDNA cloning and ontogeny of mouse α-synuclein.NeuroReport. 1998; 9: 1239-1243Crossref PubMed Scopus (34) Google Scholar coding region was amplified from whole brain RNA (High Pure RNA Isolation Kit; Boehringer Mannheim, Mannheim, Germany) by reverse transcriptase-polymerase chain reaction using outer mouse primers (5′-GGAATTCCATATGGATGTGTTCATGAAAGG-3′ and 5′-GGAAT-TCCATATGTTAGGCTTCAGGCTCAT-3′). The coding region of human α-SYN38Okochi M Walter J Koyama A Nakajo S Baba M Iwatsubo T Meijer L Kahle PJ Haass C Constitutive phosphorylation of the Parkinson's disease associated α-synuclein.J Biol Chem. 2000; 275: 390-397Crossref PubMed Scopus (432) Google Scholar was amplified by polymerase chain reaction with outer human primers (5′-TTCATTACATATGGATGTATTCATGAAAGG-3′ and 5′-GGAATTCCATATGTTAGGCTTCAGGTTCGTAG-3′). Codons 73 to 83 of α-SYN were deleted by 4-primer polymerase chain reaction using outer human primers and inner mutagenesis primers 5′-GGAGGAGCAGTGGTGACGGGAGCAGGGAGC-3′ and 5′-GCTCCCTGCTCCCGTCACCACTGCTCCTCC-3′. Amplimers were subcloned into theNdeI site of pET-5a (Promega, Madison, WI), and constructs used to transform Escherichia coli BL21(DE3) pLys. All constructs were sequenced (Medigenomix, Martinsried, Germany). Bacterial cultures were induced with isopropyl-β-d-thiogalactoside for 4 hours, and lysed by freeze/thaw and sonication. After 15 minutes of boiling, the heat-stable 17,000 × g supernatant was loaded onto Q-Sepharose (Pharmacia, Uppsala, Sweden) and eluted with a 25-mmol/L to 500-mmol/L salt gradient. The pooled SYN peak fractions were desalted by Sephacryl S-200 (Pharmacia) gel filtration. Characteristic electron-dense fibrils (data not shown) were formed after 7 days of incubation of 2 mg/ml of purified recombinant SYN proteins in 50 mmol/L HEPES or phosphate (pH 6.9) at 37°C under constant agitation. Aggregates were collected by 100,000 ×g centrifugation, and subjected to the detergent extraction protocol described above. Previous subcellular fractionation experiments with human brain have demonstrated the presence of α-SYN in synaptosomes.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar, 34Irizarry MC Kim T-W McNamara M Tanzi RE George JM Clayton DF Hyman BT Characterization of the precursor protein of the non-Aβ component of senile plaques (NACP) in the human central nervous system.J Neuropathol Exp Neurol. 1996; 55: 889-895Crossref PubMed Scopus (185) Google Scholar In accord with results from rapidly processed rodent brain,30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar, 33George JM Jin H Woods WS Clayton DF Characterization of a novel protein regulated during the critical period for song learning in the zebra finch.Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (736) Google Scholar α-SYN was released into the soluble fraction on hypotonic lysis of the synaptosomes prepared from human biopsy brain.30Kahle PJ Neumann M Ozmen L Müller V Jacobsen H Schindzielorz A Okochi M Leimer U van der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar Using archived cortical tissue, Irizarry and colleagues34Irizarry MC Kim T-W McNamara M Tanzi RE George JM Clayton DF Hyman BT Characterization of the precursor protein of the non-Aβ component of senile plaques (NACP) in the human central nervous system.J Neuropathol Exp Neurol. 1996; 55: 889-895Crossref PubMed Scopus (185) Google Scholar have found a significant portion of α-SYN in the particulate fraction of lysed synaptosomes. Indeed, when subcellular fractionations (Figure 1) were performed with frozen tissue, recovery of α-SYN from the soluble synaptosomal fraction (LS2) was decreased and instead a significant portion of α-SYN was detected in the particulate synaptosomal fraction (LP2). Interestingly, the subcellular fractionation profiles of β-SYN as well as of synaptophysin were the same as previously reported for rap
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