Selective Insolubility of -Synuclein in Human Lewy Body Diseases Is Recapitulated in a Transgenic Mouse Model
Identifieur interne : 001970 ( Main/Corpus ); précédent : 001969; suivant : 001971Selective Insolubility of -Synuclein in Human Lewy Body Diseases Is Recapitulated in a Transgenic Mouse Model
Auteurs : Philipp J. Kahle ; Manuela Neumann ; Laurence Ozmen ; Veronika Mller ; Sabine Odoy ; Noriko Okamoto ; Helmut Jacobsen ; Takeshi Iwatsubo ; John Q. Trojanowski ; Hitoshi Takahashi ; Koichi Wakabayashi ; Nenad Bogdanovic ; Peter Riederer ; Hans A. Kretzschmar ; Christian HaassSource :
- The American Journal of Pathology [ 0002-9440 ] ; 2001.
Abstract
-Synuclein (-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinsons disease (PD) and LB dementias. -SYN forms LB-like fibrils in vitro, in contrast to its homologue -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]-SYN driven by the brain neuron-specific promoter, Thy1. Distinct -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 -SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse -SYN nor -SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic -SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of -SYN are absent in -SYN. In conclusion, the specific accumulation of detergent-insoluble -SYN in transgenic mice recapitulates a pivotal feature of human LB diseases.
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DOI: 10.1016/S0002-9440(10)63072-6
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Kahle</name><affiliation><mods:affiliation>E-mail: pkahle@pbm.med.uni-muenchen.de</mods:affiliation></affiliation></author><author><name sortKey="Neumann, Manuela" sort="Neumann, Manuela" uniqKey="Neumann M" first="Manuela" last="Neumann">Manuela Neumann</name></author><author><name sortKey="Ozmen, Laurence" sort="Ozmen, Laurence" uniqKey="Ozmen L" first="Laurence" last="Ozmen">Laurence Ozmen</name></author><author><name sortKey="Mller, Veronika" sort="Mller, Veronika" uniqKey="Mller V" first="Veronika" last="Mller">Veronika Mller</name></author><author><name sortKey="Odoy, Sabine" sort="Odoy, Sabine" uniqKey="Odoy S" first="Sabine" last="Odoy">Sabine Odoy</name></author><author><name sortKey="Okamoto, Noriko" sort="Okamoto, Noriko" uniqKey="Okamoto N" first="Noriko" last="Okamoto">Noriko Okamoto</name></author><author><name sortKey="Jacobsen, Helmut" sort="Jacobsen, Helmut" uniqKey="Jacobsen H" first="Helmut" last="Jacobsen">Helmut Jacobsen</name></author><author><name sortKey="Iwatsubo, Takeshi" sort="Iwatsubo, Takeshi" uniqKey="Iwatsubo T" first="Takeshi" last="Iwatsubo">Takeshi Iwatsubo</name></author><author><name sortKey="Trojanowski, John Q" sort="Trojanowski, John Q" uniqKey="Trojanowski J" first="John Q." last="Trojanowski">John Q. Trojanowski</name></author><author><name sortKey="Takahashi, Hitoshi" sort="Takahashi, Hitoshi" uniqKey="Takahashi H" first="Hitoshi" last="Takahashi">Hitoshi Takahashi</name></author><author><name sortKey="Wakabayashi, Koichi" sort="Wakabayashi, Koichi" uniqKey="Wakabayashi K" first="Koichi" last="Wakabayashi">Koichi Wakabayashi</name></author><author><name sortKey="Bogdanovic, Nenad" sort="Bogdanovic, Nenad" uniqKey="Bogdanovic N" first="Nenad" last="Bogdanovic">Nenad Bogdanovic</name></author><author><name sortKey="Riederer, Peter" sort="Riederer, Peter" uniqKey="Riederer P" first="Peter" last="Riederer">Peter Riederer</name></author><author><name sortKey="Kretzschmar, Hans A" sort="Kretzschmar, Hans A" uniqKey="Kretzschmar H" first="Hans A." last="Kretzschmar">Hans A. 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Kahle</name><affiliation><mods:affiliation>E-mail: pkahle@pbm.med.uni-muenchen.de</mods:affiliation></affiliation></author><author><name sortKey="Neumann, Manuela" sort="Neumann, Manuela" uniqKey="Neumann M" first="Manuela" last="Neumann">Manuela Neumann</name></author><author><name sortKey="Ozmen, Laurence" sort="Ozmen, Laurence" uniqKey="Ozmen L" first="Laurence" last="Ozmen">Laurence Ozmen</name></author><author><name sortKey="Mller, Veronika" sort="Mller, Veronika" uniqKey="Mller V" first="Veronika" last="Mller">Veronika Mller</name></author><author><name sortKey="Odoy, Sabine" sort="Odoy, Sabine" uniqKey="Odoy S" first="Sabine" last="Odoy">Sabine Odoy</name></author><author><name sortKey="Okamoto, Noriko" sort="Okamoto, Noriko" uniqKey="Okamoto N" first="Noriko" last="Okamoto">Noriko Okamoto</name></author><author><name sortKey="Jacobsen, Helmut" sort="Jacobsen, Helmut" uniqKey="Jacobsen H" first="Helmut" last="Jacobsen">Helmut Jacobsen</name></author><author><name sortKey="Iwatsubo, Takeshi" sort="Iwatsubo, Takeshi" uniqKey="Iwatsubo T" first="Takeshi" last="Iwatsubo">Takeshi Iwatsubo</name></author><author><name sortKey="Trojanowski, John Q" sort="Trojanowski, John Q" uniqKey="Trojanowski J" first="John Q." last="Trojanowski">John Q. Trojanowski</name></author><author><name sortKey="Takahashi, Hitoshi" sort="Takahashi, Hitoshi" uniqKey="Takahashi H" first="Hitoshi" last="Takahashi">Hitoshi Takahashi</name></author><author><name sortKey="Wakabayashi, Koichi" sort="Wakabayashi, Koichi" uniqKey="Wakabayashi K" first="Koichi" last="Wakabayashi">Koichi Wakabayashi</name></author><author><name sortKey="Bogdanovic, Nenad" sort="Bogdanovic, Nenad" uniqKey="Bogdanovic N" first="Nenad" last="Bogdanovic">Nenad Bogdanovic</name></author><author><name sortKey="Riederer, Peter" sort="Riederer, Peter" uniqKey="Riederer P" first="Peter" last="Riederer">Peter Riederer</name></author><author><name sortKey="Kretzschmar, Hans A" sort="Kretzschmar, Hans A" uniqKey="Kretzschmar H" first="Hans A." last="Kretzschmar">Hans A. Kretzschmar</name></author><author><name sortKey="Haass, Christian" sort="Haass, Christian" uniqKey="Haass C" first="Christian" last="Haass">Christian Haass</name></author></analytic><monogr></monogr><series><title level="j">The American Journal of Pathology</title><title level="j" type="abbrev">AJPA</title><idno type="ISSN">0002-9440</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2001">2001</date><biblScope unit="volume">159</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="2215">2215</biblScope><biblScope unit="page" to="2225">2225</biblScope></imprint><idno type="ISSN">0002-9440</idno></series><idno type="istex">FA10372DB830345DF09BC358DD8C01F9D4FBAF88</idno><idno type="DOI">10.1016/S0002-9440(10)63072-6</idno><idno type="PII">S0002-9440(10)63072-6</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0002-9440</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract">-Synuclein (-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinsons disease (PD) and LB dementias. -SYN forms LB-like fibrils in vitro, in contrast to its homologue -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]-SYN driven by the brain neuron-specific promoter, Thy1. Distinct -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 -SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse -SYN nor -SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic -SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of -SYN are absent in -SYN. In conclusion, the specific accumulation of detergent-insoluble -SYN in transgenic mice recapitulates a pivotal feature of human LB diseases.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>Philipp J. Kahle</name><affiliations><json:string>E-mail: pkahle@pbm.med.uni-muenchen.de</json:string></affiliations></json:item><json:item><name>Manuela Neumann</name></json:item><json:item><name>Laurence Ozmen</name></json:item><json:item><name>Veronika Mller</name></json:item><json:item><name>Sabine Odoy</name></json:item><json:item><name>Noriko Okamoto</name></json:item><json:item><name>Helmut Jacobsen</name></json:item><json:item><name>Takeshi Iwatsubo</name></json:item><json:item><name>John Q. Trojanowski</name></json:item><json:item><name>Hitoshi Takahashi</name></json:item><json:item><name>Koichi Wakabayashi</name></json:item><json:item><name>Nenad Bogdanovic</name></json:item><json:item><name>Peter Riederer</name></json:item><json:item><name>Hans A. Kretzschmar</name></json:item><json:item><name>Christian Haass</name></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Regular Articles</value></json:item></subject><language><json:string>eng</json:string></language><abstract>-Synuclein (-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinsons disease (PD) and LB dementias. -SYN forms LB-like fibrils in vitro, in contrast to its homologue -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]-SYN driven by the brain neuron-specific promoter, Thy1. Distinct -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 -SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse -SYN nor -SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic -SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of -SYN are absent in -SYN. In conclusion, the specific accumulation of detergent-insoluble -SYN in transgenic mice recapitulates a pivotal feature of human LB diseases.</abstract><qualityIndicators><score>9.028</score><pdfVersion>1.7</pdfVersion><pdfPageSize>587 x 786 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>1</keywordCount><abstractCharCount>1194</abstractCharCount><pdfWordCount>6389</pdfWordCount><pdfCharCount>41898</pdfCharCount><pdfPageCount>11</pdfPageCount><abstractWordCount>169</abstractWordCount></qualityIndicators><title>Selective Insolubility of -Synuclein in Human Lewy Body Diseases Is Recapitulated in a Transgenic Mouse Model</title><pii><json:string>S0002-9440(10)63072-6</json:string></pii><genre><json:string>research-article</json:string></genre><host><volume>159</volume><pii><json:string>S0002-9440(10)X6083-7</json:string></pii><pages><last>2225</last><first>2215</first></pages><issn><json:string>0002-9440</json:string></issn><issue>6</issue><genre><json:string>Journal</json:string></genre><language><json:string>unknown</json:string></language><title>The American Journal of Pathology</title><publicationDate>2001</publicationDate></host><categories><wos><json:string>PATHOLOGY</json:string><json:string>CELL BIOLOGY</json:string></wos></categories><publicationDate>2001</publicationDate><copyrightDate>2001</copyrightDate><doi><json:string>10.1016/S0002-9440(10)63072-6</json:string></doi><id>FA10372DB830345DF09BC358DD8C01F9D4FBAF88</id><fulltext><json:item><original>true</original><mimetype>application/pdf</mimetype><extension>pdf</extension><uri>https://api.istex.fr/document/FA10372DB830345DF09BC358DD8C01F9D4FBAF88/fulltext/pdf</uri></json:item><json:item><original>true</original><mimetype>text/plain</mimetype><extension>txt</extension><uri>https://api.istex.fr/document/FA10372DB830345DF09BC358DD8C01F9D4FBAF88/fulltext/txt</uri></json:item><json:item><original>false</original><mimetype>application/zip</mimetype><extension>zip</extension><uri>https://api.istex.fr/document/FA10372DB830345DF09BC358DD8C01F9D4FBAF88/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/FA10372DB830345DF09BC358DD8C01F9D4FBAF88/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a">Selective Insolubility of -Synuclein in Human Lewy Body Diseases Is Recapitulated in a Transgenic Mouse Model</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>ELSEVIER</p></availability><date>2001</date></publicationStmt><notesStmt><note>Supported by grants from the Deutsche Forschungsgemeinschaft (HA 1737/4-1) and the Bavaria California Technology Center (to C. H.).</note><note type="content">Section title: Regular Article</note><note type="content">Figure 1: Subcellular fractionation of frozen human brain tissue. A: Schematic representation of the subcellular fractionation steps, annotations in B correspond to the fractions outlined inA. B: Parietal cortex (0.4 g) from a human control individual was homogenized and subjected to subcellular fractionation. Twenty g of each fraction was TCA precipitated (except postnuclear fraction S1 that was loaded directly) and subjected to denaturing 12.5 PAGE. Sequential Western probing was done with 15G7 anti--SYN, SY38 anti-synaptophysin (SPH), and 6584 anti--SYN, as indicated. Data are representative for three different control cortex fractionations.</note><note type="content">Figure 2: SDS-insoluble -SYN species in LB diseases. A: Schematic representation of the differential extraction steps. See Materials and Methods for details. B: Structure of -SYN and epitopes recognized by anti--SYN antibodies. The imperfect KTKEGV repeats are numbered, the sixth repeat missing in -SYN (see below) isstippled. C: Extracts from temporal cortex of control and DLB brain were prepared, and 10 g of TBS-soluble material, 10 g of SDS-soluble material, and 10 l of urea extracts or TCA precipitates from 50-l urea extracts (as indicated at thebottom) were subjected to denaturing 12.5 PAGE. Western blots were sequentially probed with three different antibodies against -SYN (15G7, 3400, MC42) and anti-ubiquitin (Ubi-1), as indicated on the top. Immunoreactivity was visualized with SuperSignal (for 15G7) or ECLplus.D: TCA precipitates from 50-l urea extracts from temporal cortex of control and DLB brain (left) and from parietal cortex of control and PD brain (right) were loaded on 10 to 20 Tris-tricine gels. The corresponding Western blots were probed with anti-NAC and developed with ECLplus. E: Parietal cortex samples from two controls and two NBIA1 patients were extracted in parallel. TBS-soluble material (10 g, left), SDS-soluble material (25 g,middle), and urea extracts (80 l,right) were separated by SDS-PAGE (TBS-soluble, 15; SDS and urea extracts, 4 to 20 gradient). MC42 and ECLplus were used for Western detection of -SYN. Note that sample NBIA1 no. 2 with higher LB density than NBIA1 no. 1 had also much stronger -SYN immunoreactivity in the urea extract. Nevertheless, the -SYN immunoreactive band pattern of NBIA1 no. 1 was qualitatively the same as of NBIA1 no. 2, as evidenced by a longer exposure of the blot to thefar right. Each experiment is representative for two to three independent extractions. Positions of prestained molecular weight standards are indicated to the left. See text for description of -SYN species denoted to the right.</note><note type="content">Figure 3: Developmental expression of -SYN. Mouse brains were collected from mice at the indicated age, and Western blots prepared from 25-g cytosolic extracts. Wild-type mouse blots were probed with MC42 (top), representative [(Thy1)-h[A30P]-SYN line 18] transgenic mouse blots probed with 3400 (bottom), and developed with ECLplus.</note><note type="content">Figure 4: Immunostainings of brain slices (motor cortex) show specific accumulation of -SYN but not of -SYN in transgenic mice. Animals expressing either [wt]-SYN (AC) or [A30P]-SYN (DF) showed a strong cytosolic labeling of neuronal cells with the human-specific -SYN antibody 15G7 (A andD). A section from a 1-month-old [wt]-SYN-expressing mouse is shown inA to demonstrate the early onset of accumulation of transgenic protein. In contrast, immunostainings with the -SYN-specific antiserum (6485) and the murine-specific -SYN antiserum (7544) only revealed a synaptic staining pattern. Neither accumulation of endogenous murine -SYN (C andF) nor -SYN (B andE) was detectable in neuronal cell bodies. Scale bar in A, 100 m.</note><note type="content">Figure 5: Detergent-insoluble -SYN in transgenic mouse brains. Whole brains of transgenic mice (A: 3- to 4-month-old [wt]-SYN lines 14 and 23, and [A30P]-SYN lines 18 and 31;B: 1-month-old and 1-year-old [A30P]-SYN lines 18 and 31; as indicated at the bottom) and age-matched nontransgenic littermates (lm) were differentially extracted. Buffer- and detergent-soluble proteins (10 g for transgenic human -SYN, 50 g for endogenous mouse SYNs), and TCA precipitate of urea extracts were subjected to denaturing 15 PAGE. Western blots were probed with human (transgene)-specific anti-h-SYN 15G7, endogenous mouse-specific anti-m-SYN 7544, and anti--SYN 6485, as indicated to the left. 15G7-immunoreactive bands were developed with SuperSignal, polyclonal antibody immunoreactivity with ECLplus. A control lane on the urea extract blots contained 10 g of cytosol from a transgenic [A30P]-SYN mouse. The positions of prestained molecular weight markers are indicated to the right. Individual variance of transgenic -SYN expression levels may account for the apparently higher amount of urea extractable mutant h[A30P]-SYN compared to h[wt]-SYN in one experiment (A, exp. 2), but not in two additional experiments (one of them shown as A, exp. 1), and for the apparent increase with age of SDS-soluble -SYN (B) that was not seen in an additional experiment.</note><note type="content">Figure 6: -SYN, but not -SYN aggregates in vitro because of a critical determinant in the NAC domain. A: Recombinant human -SYN aggregates were collected by 100,000 g centrifugation and redissolved in 15 volumes of TBS. The buffer-insoluble material was extracted like the brain samples above. All fractions were TCA precipitated and separated by denaturing 15 PAGE. Western blots were probed with 3400 anti--SYN and developed with SuperSignal. This experiment was repeated three times with the same result. B: Solutions (2 mg/ml) of [wt]-SYN (left), [7383]-SYN (middle), and [wt]-SYN (right) were aggregated for 7 days. After ultracentrifugation, the 100,000 g pellets (pel) were subjected to denaturing 12.5 PAGE. -SYN immunoblots were probed with MC42 and -SYN immunoblots with 6485, and developed with SuperSignal until the band intensities of 1-g freshly dissolved, nonaggregated protein (sol) were comparable. Note the typical retarded electrophoretic motility of recombinant -SYN.36,38 Positions of prestained molecular weight markers are indicated to the right.</note><note type="content">Table 1: The Amount of Insoluble -SYN Correlates with Severity of LB Diseases</note><note type="content">Table 2: -SYN Expression Levels in Transgenic Mice</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">Selective Insolubility of -Synuclein in Human Lewy Body Diseases Is Recapitulated in a Transgenic Mouse Model</title><author><persName><forename type="first">Philipp J.</forename><surname>Kahle</surname></persName><email>pkahle@pbm.med.uni-muenchen.de</email></author><author><persName><forename type="first">Manuela</forename><surname>Neumann</surname></persName></author><author><persName><forename type="first">Laurence</forename><surname>Ozmen</surname></persName></author><author><persName><forename type="first">Veronika</forename><surname>Mller</surname></persName></author><author><persName><forename type="first">Sabine</forename><surname>Odoy</surname></persName></author><author><persName><forename type="first">Noriko</forename><surname>Okamoto</surname></persName></author><author><persName><forename type="first">Helmut</forename><surname>Jacobsen</surname></persName></author><author><persName><forename type="first">Takeshi</forename><surname>Iwatsubo</surname></persName></author><author><persName><forename type="first">John Q.</forename><surname>Trojanowski</surname></persName></author><author><persName><forename type="first">Hitoshi</forename><surname>Takahashi</surname></persName></author><author><persName><forename type="first">Koichi</forename><surname>Wakabayashi</surname></persName></author><author><persName><forename type="first">Nenad</forename><surname>Bogdanovic</surname></persName></author><author><persName><forename type="first">Peter</forename><surname>Riederer</surname></persName></author><author><persName><forename type="first">Hans A.</forename><surname>Kretzschmar</surname></persName></author><author><persName><forename type="first">Christian</forename><surname>Haass</surname></persName></author></analytic><monogr><title level="j">The American Journal of Pathology</title><title level="j" type="abbrev">AJPA</title><idno type="pISSN">0002-9440</idno><idno type="PII">S0002-9440(10)X6083-7</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2001"></date><biblScope unit="volume">159</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="2215">2215</biblScope><biblScope unit="page" to="2225">2225</biblScope></imprint></monogr><idno type="istex">FA10372DB830345DF09BC358DD8C01F9D4FBAF88</idno><idno type="DOI">10.1016/S0002-9440(10)63072-6</idno><idno type="PII">S0002-9440(10)63072-6</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2001</date></creation><langUsage><language ident="en">en</language></langUsage><abstract><p>-Synuclein (-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinsons disease (PD) and LB dementias. -SYN forms LB-like fibrils in vitro, in contrast to its homologue -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]-SYN driven by the brain neuron-specific promoter, Thy1. Distinct -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 -SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse -SYN nor -SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic -SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of -SYN are absent in -SYN. In conclusion, the specific accumulation of detergent-insoluble -SYN in transgenic mice recapitulates a pivotal feature of human LB diseases.</p></abstract><textClass><keywords scheme="keyword"><list><head>Article category</head><item><term>Regular Articles</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2001-09-10">Registration</change><change when="2001">Published</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier doc found" wicri:toSee="Elsevier, no converted or simple article"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 5.1.0//EN//XML" URI="art510.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity></istex:docType><istex:document><article docsubtype="fla"> <item-info> <jid>AJPA</jid> <aid>63072</aid> <ce:pii>S0002-9440(10)63072-6</ce:pii> <ce:doi>10.1016/S0002-9440(10)63072-6</ce:doi> <ce:copyright type="society" year="2001">American Society for Investigative Pathology</ce:copyright> <ce:doctopics> <ce:doctopic> <ce:text>Regular Articles</ce:text> </ce:doctopic> </ce:doctopics> </item-info> <ce:floats> <ce:figure id="fig1"> <ce:label>Figure 1</ce:label> <ce:caption> <ce:simple-para id="spara10">Subcellular fractionation of frozen human brain tissue. <ce:bold>A:</ce:bold> Schematic representation of the subcellular fractionation steps, annotations in <ce:bold>B</ce:bold> correspond to the fractions outlined in<ce:bold>A</ce:bold>. <ce:bold>B:</ce:bold> Parietal cortex (0.4 g) from a human control individual was homogenized and subjected to subcellular fractionation. Twenty μg of each fraction was TCA precipitated (except postnuclear fraction S1 that was loaded directly) and subjected to denaturing 12.5% PAGE. Sequential Western probing was done with 15G7 anti-α-SYN, SY38 anti-synaptophysin (SPH), and 6584 anti-β-SYN, as indicated. Data are representative for three different control cortex fractionations.</ce:simple-para> </ce:caption> <ce:link locator="gr1"></ce:link> </ce:figure> <ce:figure id="fig2"> <ce:label>Figure 2</ce:label> <ce:caption> <ce:simple-para id="spara20">SDS-insoluble α-SYN species in LB diseases. <ce:bold>A:</ce:bold> Schematic representation of the differential extraction steps. See Materials and Methods for details. <ce:bold>B:</ce:bold> Structure of α-SYN and epitopes recognized by anti-α-SYN antibodies. The imperfect KTKEGV repeats are numbered, the sixth repeat missing in β-SYN (see below) is<ce:bold>stippled</ce:bold>. <ce:bold>C:</ce:bold> Extracts from temporal cortex of control and DLB brain were prepared, and 10 μg of TBS-soluble material, 10 μg of SDS-soluble material, and 10 μl of urea extracts or TCA precipitates from 50-μl urea extracts (as indicated at the<ce:bold>bottom</ce:bold>) were subjected to denaturing 12.5% PAGE. Western blots were sequentially probed with three different antibodies against α-SYN (15G7, 3400, MC42) and anti-ubiquitin (Ubi-1), as indicated on the <ce:bold>top</ce:bold>. Immunoreactivity was visualized with SuperSignal (for 15G7) or ECLplus.<ce:bold>D:</ce:bold> TCA precipitates from 50-μl urea extracts from temporal cortex of control and DLB brain (<ce:bold>left</ce:bold>) and from parietal cortex of control and PD brain (<ce:bold>right</ce:bold>) were loaded on 10 to 20% Tris-tricine gels. The corresponding Western blots were probed with anti-NAC and developed with ECLplus. <ce:bold>E:</ce:bold> Parietal cortex samples from two controls and two NBIA1 patients were extracted in parallel. TBS-soluble material (10 μg, <ce:bold>left</ce:bold>), SDS-soluble material (25 μg,<ce:bold>middle</ce:bold>), and urea extracts (80 μl,<ce:bold>right</ce:bold>) were separated by SDS-PAGE (TBS-soluble, 15%; SDS and urea extracts, 4 to 20% gradient). MC42 and ECLplus were used for Western detection of α-SYN. Note that sample NBIA1 no. 2 with higher LB density than NBIA1 no. 1 had also much stronger α-SYN immunoreactivity in the urea extract. Nevertheless, the α-SYN immunoreactive band pattern of NBIA1 no. 1 was qualitatively the same as of NBIA1 no. 2, as evidenced by a longer exposure of the blot to the<ce:bold>far right</ce:bold>. Each experiment is representative for two to three independent extractions. Positions of prestained molecular weight standards are indicated to the <ce:bold>left</ce:bold>. See text for description of α-SYN species denoted to the <ce:bold>right</ce:bold>.</ce:simple-para> </ce:caption> <ce:link locator="gr2"></ce:link> </ce:figure> <ce:figure id="fig3"> <ce:label>Figure 3</ce:label> <ce:caption> <ce:simple-para id="spara30">Developmental expression of α-SYN. Mouse brains were collected from mice at the indicated age, and Western blots prepared from 25-μg cytosolic extracts. Wild-type mouse blots were probed with MC42 (<ce:bold>top</ce:bold>), representative [(Thy1)-h[A30P]α-SYN line 18] transgenic mouse blots probed with 3400 (<ce:bold>bottom</ce:bold>), and developed with ECLplus.</ce:simple-para> </ce:caption> <ce:link locator="gr3"></ce:link> </ce:figure> <ce:figure id="fig4"> <ce:label>Figure 4</ce:label> <ce:caption> <ce:simple-para id="spara40">Immunostainings of brain slices (motor cortex) show specific accumulation of α-SYN but not of β-SYN in transgenic mice. Animals expressing either [wt]α-SYN (<ce:bold>A–C</ce:bold>) or [A30P]α-SYN (<ce:bold>D–F</ce:bold>) showed a strong cytosolic labeling of neuronal cells with the human-specific α-SYN antibody 15G7 (<ce:bold>A</ce:bold> and<ce:bold>D</ce:bold>). A section from a 1-month-old [wt]α-SYN-expressing mouse is shown in<ce:bold>A</ce:bold> to demonstrate the early onset of accumulation of transgenic protein. In contrast, immunostainings with the β-SYN-specific antiserum (6485) and the murine-specific α-SYN antiserum (7544) only revealed a synaptic staining pattern. Neither accumulation of endogenous murine α-SYN (<ce:bold>C</ce:bold> and<ce:bold>F</ce:bold>) nor β-SYN (<ce:bold>B</ce:bold> and<ce:bold>E</ce:bold>) was detectable in neuronal cell bodies. Scale bar in <ce:bold>A,</ce:bold> 100 μm.</ce:simple-para> </ce:caption> <ce:link locator="gr4"></ce:link> </ce:figure> <ce:figure id="fig5"> <ce:label>Figure 5</ce:label> <ce:caption> <ce:simple-para id="spara50">Detergent-insoluble α-SYN in transgenic mouse brains. Whole brains of transgenic mice (<ce:bold>A:</ce:bold> 3- to 4-month-old [wt]α-SYN lines 14 and 23, and [A30P]α-SYN lines 18 and 31;<ce:bold>B:</ce:bold> 1-month-old and 1-year-old [A30P]α-SYN lines 18 and 31; as indicated at the <ce:bold>bottom</ce:bold>) and age-matched nontransgenic littermates (lm) were differentially extracted. Buffer- and detergent-soluble proteins (10 μg for transgenic human α-SYN, 50 μg for endogenous mouse SYNs), and TCA precipitate of urea extracts were subjected to denaturing 15% PAGE. Western blots were probed with human (transgene)-specific anti-hα-SYN 15G7, endogenous mouse-specific anti-mα-SYN 7544, and anti-β-SYN 6485, as indicated to the <ce:bold>left</ce:bold>. 15G7-immunoreactive bands were developed with SuperSignal, polyclonal antibody immunoreactivity with ECLplus. A control lane on the urea extract blots contained 10 μg of cytosol from a transgenic [A30P]α-SYN mouse. The positions of prestained molecular weight markers are indicated to the <ce:bold>right</ce:bold>. Individual variance of transgenic α-SYN expression levels may account for the apparently higher amount of urea extractable mutant h[A30P]α-SYN compared to h[wt]α-SYN in one experiment (<ce:bold>A</ce:bold>, exp. 2), but not in two additional experiments (one of them shown as <ce:bold>A</ce:bold>, exp. 1), and for the apparent increase with age of SDS-soluble α-SYN (<ce:bold>B</ce:bold>) that was not seen in an additional experiment.</ce:simple-para> </ce:caption> <ce:link locator="gr5"></ce:link> </ce:figure> <ce:figure id="fig6"> <ce:label>Figure 6</ce:label> <ce:caption> <ce:simple-para id="spara60">α-SYN, but not β-SYN aggregates <ce:italic>in vitro</ce:italic> because of a critical determinant in the NAC domain. <ce:bold>A:</ce:bold> Recombinant human α-SYN aggregates were collected by 100,000 × <ce:italic>g</ce:italic> centrifugation and redissolved in 15 volumes of TBS+. The buffer-insoluble material was extracted like the brain samples above. All fractions were TCA precipitated and separated by denaturing 15% PAGE. Western blots were probed with 3400 anti-α-SYN and developed with SuperSignal. This experiment was repeated three times with the same result. <ce:bold>B:</ce:bold> Solutions (2 mg/ml) of [wt]α-SYN (<ce:bold>left</ce:bold>), [Δ73–83]α-SYN (<ce:bold>middle</ce:bold>), and [wt]β-SYN (<ce:bold>right</ce:bold>) were aggregated for 7 days. After ultracentrifugation, the 100,000 ×<ce:italic>g</ce:italic> pellets (pel) were subjected to denaturing 12.5% PAGE. α-SYN immunoblots were probed with MC42 and β-SYN immunoblots with 6485, and developed with SuperSignal until the band intensities of 1-μg freshly dissolved, nonaggregated protein (sol) were comparable. Note the typical retarded electrophoretic motility of recombinant β-SYN.<ce:cross-refs refid="bib36 bib38"><ce:sup>36,38</ce:sup></ce:cross-refs> Positions of prestained molecular weight markers are indicated to the <ce:bold>right</ce:bold>.</ce:simple-para> </ce:caption> <ce:link locator="gr6"></ce:link> </ce:figure> <ce:table id="tbl1" frame="topbot" rowsep="0" colsep="0"> <ce:label>Table 1</ce:label> <ce:caption> <ce:simple-para id="spara70">The Amount of Insoluble α-SYN Correlates with Severity of LB Diseases</ce:simple-para> </ce:caption> <tgroup cols="3" altimg="si1.gif"> <colspec colnum="1" colname="col1"></colspec> <colspec colnum="2" colname="col2"></colspec> <colspec colnum="3" colname="col3"></colspec> <thead> <row valign="top" rowsep="1"> <entry align="left">Brain sample</entry> <entry align="center">Insoluble α-SYN</entry> <entry align="center">LB count</entry> </row> </thead> <tbody> <row valign="top"> <entry align="left">Temporal cortex</entry> <entry></entry> <entry></entry> </row> <row valign="top"> <entry align="left"> CON no. 1</entry> <entry align="center">−</entry> <entry align="center">−</entry> </row> <row valign="top"> <entry align="left"> CON no. 2</entry> <entry align="center">−</entry> <entry align="center">−</entry> </row> <row valign="top"> <entry align="left"> CON no. 3</entry> <entry align="center">−</entry> <entry align="center">−</entry> </row> <row valign="top"> <entry align="left"> DLB no. 1</entry> <entry align="center">++</entry> <entry align="center">22.8/mm<ce:sup>2</ce:sup></entry> </row> <row valign="top"> <entry align="left"> DLB no. 2</entry> <entry align="center">+</entry> <entry align="center">6.0/mm<ce:sup>2</ce:sup></entry> </row> <row valign="top"> <entry align="left"> DLB no. 3</entry> <entry align="center">+</entry> <entry align="center">6.6/mm<ce:sup>2</ce:sup></entry> </row> <row valign="top"> <entry align="left">Parietal cortex</entry> <entry></entry> <entry></entry> </row> <row valign="top"> <entry align="left"> CON no. 4</entry> <entry align="center">−</entry> <entry align="center">−</entry> </row> <row valign="top"> <entry align="left"> CON no. 5</entry> <entry align="center">−</entry> <entry align="center">−</entry> </row> <row valign="top"> <entry align="left"> NBIA1 no. 1</entry> <entry align="center">+</entry> <entry align="center">5.3/mm<ce:sup>2</ce:sup></entry> </row> <row valign="top"> <entry align="left"> NBIA1 no. 2</entry> <entry align="center">++</entry> <entry align="center">13.5/mm<ce:sup>2</ce:sup></entry> </row> </tbody> </tgroup> <ce:legend> <ce:simple-para id="spara80">The amount of insoluble α-SYN was estimated from the band strength on urea extract blots (see <ce:cross-ref refid="fig2">Figure 2</ce:cross-ref>). Band intensity: ++, strong; +, weak; −, absent. To determine LB density, vibratome sections of 50 μ thickness from the NBIA1 brains were immunostained with Syn102, and 5-μ-thick sections of DLB brains with 15G7. Five different visual fields (each 0.98 mm<ce:sup>2</ce:sup>) were examined.</ce:simple-para> </ce:legend> </ce:table> <ce:table id="tbl2" frame="topbot" rowsep="0" colsep="0"> <ce:label>Table 2</ce:label> <ce:caption> <ce:simple-para id="spara90">α-SYN Expression Levels in Transgenic Mice</ce:simple-para> </ce:caption> <tgroup cols="6" altimg="si2.gif"> <colspec colnum="1" colname="col1"></colspec> <colspec colnum="2" colname="col2"></colspec> <colspec colnum="3" colname="col3"></colspec> <colspec colnum="4" colname="col4"></colspec> <colspec colnum="5" colname="col5"></colspec> <colspec colnum="6" colname="col6"></colspec> <thead> <row valign="top" rowsep="1"> <entry align="left">Transgenic construct</entry> <entry align="center">Line</entry> <entry align="center">α-SYN transgene copy number</entry> <entry align="center">Transgenic α-SYN mRNA [% actin]</entry> <entry align="center">Transgenic α-SYN protein [ng/μg]</entry> <entry align="center">Endogenous α-SYN protein [ng/μg]</entry> </row> </thead> <tbody> <row valign="top"> <entry align="left">(Thy1)-h[wt]α-SYN</entry> <entry align="char" char=".">6</entry> <entry align="char" char=".">7</entry> <entry align="char" char=".">175</entry> <entry align="center">1.93 ± 0.31</entry> <entry align="center">1.35 ± 0.10</entry> </row> <row valign="top"> <entry align="left">(Thy1)-h[wt]α-SYN</entry> <entry align="char" char=".">10</entry> <entry align="char" char=".">85</entry> <entry align="char" char=".">65</entry> <entry align="center">0.80 ± 0.31</entry> <entry align="center">1.61 ± 0.14</entry> </row> <row valign="top"> <entry align="left">(Thy1)-h[wt]α-SYN</entry> <entry align="char" char=".">14</entry> <entry align="char" char=".">13</entry> <entry align="char" char=".">190</entry> <entry align="center">2.02 ± 0.11</entry> <entry align="center">0.65 ± 0.06</entry> </row> <row valign="top"> <entry align="left">(Thy1)-h[wt]α-SYN</entry> <entry align="char" char=".">23</entry> <entry align="char" char=".">2</entry> <entry align="char" char=".">130</entry> <entry align="center">1.32 ± 0.17</entry> <entry align="center">0.43 ± 0.21</entry> </row> <row valign="top"> <entry align="left">(Thy1)-h[wt]α-SYN</entry> <entry align="char" char=".">38</entry> <entry align="char" char=".">5</entry> <entry align="char" char=".">130</entry> <entry align="center">1.58 ± 0.60</entry> <entry align="center">0.81 ± 0.28</entry> </row> <row valign="top"> <entry align="left">Nontransgenic</entry> <entry></entry> <entry align="center">N/A<ce:cross-ref refid="tbl2fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> <entry align="center">N/A</entry> <entry align="center">N/A</entry> <entry align="center">1.77 ± 0.55</entry> </row> <row valign="top"> <entry align="left">(Thy1)-h[A30P]α-SYN</entry> <entry align="char" char=".">8</entry> <entry align="char" char=".">15–25</entry> <entry align="char" char=".">370</entry> <entry align="center">1.20 ± 0.60</entry> <entry align="center">1.42 ± 0.99</entry> </row> <row valign="top"> <entry align="left">(Thy1)-h[A30P]α-SYN</entry> <entry align="char" char=".">9</entry> <entry align="char" char=".">5</entry> <entry align="char" char=".">30</entry> <entry align="center">0.39 ± 0.05</entry> <entry align="center">1.64 ± 0.05</entry> </row> <row valign="top"> <entry align="left">(Thy1)-h[A30P]α-SYN</entry> <entry align="char" char=".">14</entry> <entry align="char" char=".">10</entry> <entry align="char" char=".">105</entry> <entry align="center">1.2<ce:cross-ref refid="tbl2fn1"><ce:sup>*</ce:sup></ce:cross-ref></entry> <entry align="center">n.d.</entry> </row> <row valign="top"> <entry align="left">(Thy1)-h[A30P]α-SYN</entry> <entry align="char" char=".">18</entry> <entry align="char" char=".">10–15</entry> <entry align="char" char=".">170</entry> <entry align="center">1.43 ± 0.18</entry> <entry align="center">1.59 ± 0.00</entry> </row> <row valign="top"> <entry align="left">(Thy1)-h[A30P]α-SYN</entry> <entry align="char" char=".">31</entry> <entry align="char" char=".">15–20</entry> <entry align="char" char=".">155</entry> <entry align="center">1.69 ± 0.31</entry> <entry align="center">1.26 ± 0.01</entry> </row> </tbody> </tgroup> <ce:legend> <ce:simple-para id="spara100">Five lines each of [wt]α-SYN and [A30P]α-SYN-expressing transgenic mice stably transmitted the transgene. The integrated transgene copy number was determined by quantitative Southern blotting, transgenic α-SYN mRNA expression levels relative to β-actin by quantitative Northern blotting, and the amount of transgenic and endogenous α-SYN protein by quantitative Western blotting. α-SYN protein was measured in three different mouse whole-brain cytosol samples on the same blot. Results are expressed as mean ± SD.</ce:simple-para> </ce:legend> <ce:table-footnote id="tbl2fn1"> <ce:label>*</ce:label> <ce:note-para>Determined indirectly; expression levels relative to the other lines.</ce:note-para> </ce:table-footnote> <ce:table-footnote id="tbl2fn2"> <ce:label>†</ce:label> <ce:note-para>N/A: not applicable.</ce:note-para> </ce:table-footnote> </ce:table> </ce:floats> <head> <ce:article-footnote> <ce:note-para>Supported by grants from the <ce:grant-sponsor id="GS1">Deutsche Forschungsgemeinschaft</ce:grant-sponsor> (<ce:grant-number refid="GS1">HA 1737/4-1</ce:grant-number>) and the Bavaria California Technology Center (to C. H.).</ce:note-para> </ce:article-footnote> <ce:dochead> <ce:textfn>Regular Article</ce:textfn> </ce:dochead> <ce:title>Selective Insolubility of α-Synuclein in Human Lewy Body Diseases Is Recapitulated in a Transgenic Mouse Model</ce:title> <ce:author-group> <ce:author> <ce:given-name>Philipp J.</ce:given-name> <ce:surname>Kahle</ce:surname><ce:cross-ref refid="aff1"><ce:sup>*</ce:sup></ce:cross-ref><ce:cross-ref refid="cor1"><ce:sup>*</ce:sup></ce:cross-ref> <ce:e-address type="email">pkahle@pbm.med.uni-muenchen.de</ce:e-address> </ce:author> <ce:author> <ce:given-name>Manuela</ce:given-name> <ce:surname>Neumann</ce:surname><ce:cross-ref refid="aff2"><ce:sup>†</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Laurence</ce:given-name> <ce:surname>Ozmen</ce:surname><ce:cross-ref refid="aff3"><ce:sup>‡</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Veronika</ce:given-name> <ce:surname>Müller</ce:surname><ce:cross-ref refid="aff1"><ce:sup>*</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Sabine</ce:given-name> <ce:surname>Odoy</ce:surname><ce:cross-ref refid="aff1"><ce:sup>*</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Noriko</ce:given-name> <ce:surname>Okamoto</ce:surname><ce:cross-ref refid="aff1"><ce:sup>*</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Helmut</ce:given-name> <ce:surname>Jacobsen</ce:surname><ce:cross-ref refid="aff3"><ce:sup>‡</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Takeshi</ce:given-name> <ce:surname>Iwatsubo</ce:surname><ce:cross-ref refid="aff4"><ce:sup>§</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>John Q.</ce:given-name> <ce:surname>Trojanowski</ce:surname><ce:cross-ref refid="aff5"><ce:sup>¶</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Hitoshi</ce:given-name> <ce:surname>Takahashi</ce:surname><ce:cross-ref refid="aff6"><ce:sup>‖</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Koichi</ce:given-name> <ce:surname>Wakabayashi</ce:surname><ce:cross-ref refid="aff7"><ce:sup>**</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Nenad</ce:given-name> <ce:surname>Bogdanovic</ce:surname><ce:cross-ref refid="aff8"><ce:sup>††</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Peter</ce:given-name> <ce:surname>Riederer</ce:surname><ce:cross-ref refid="aff9"><ce:sup>‡‡</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Hans A.</ce:given-name> <ce:surname>Kretzschmar</ce:surname><ce:cross-ref refid="aff2"><ce:sup>†</ce:sup></ce:cross-ref> </ce:author> <ce:author> <ce:given-name>Christian</ce:given-name> <ce:surname>Haass</ce:surname><ce:cross-ref refid="aff1"><ce:sup>*</ce:sup></ce:cross-ref> </ce:author> <ce:affiliation id="aff1"> <ce:label>*</ce:label> <ce:textfn>Laboratory for Alzheimer’s and Parkinson’s Disease Research, Ludwig Maximilians University, Munich, Germany</ce:textfn> </ce:affiliation> <ce:affiliation id="aff2"> <ce:label>†</ce:label> <ce:textfn>Department of Biochemistry and the Department of Neuropathology, Ludwig Maximilians University, Munich, Germany</ce:textfn> </ce:affiliation> <ce:affiliation id="aff3"> <ce:label>‡</ce:label> <ce:textfn>Pharma Research Genomics, F. Hoffmann–La Roche Ltd., Basel, Switzerland</ce:textfn> </ce:affiliation> <ce:affiliation id="aff4"> <ce:label>§</ce:label> <ce:textfn>Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan</ce:textfn> </ce:affiliation> <ce:affiliation id="aff5"> <ce:label>¶</ce:label> <ce:textfn>Department of Pathology and Laboratory Medicine, Center for Neurodegenerative Disease Research, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania</ce:textfn> </ce:affiliation> <ce:affiliation id="aff6"> <ce:label>‖</ce:label> <ce:textfn>Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan</ce:textfn> </ce:affiliation> <ce:affiliation id="aff7"> <ce:label>**</ce:label> <ce:textfn>Department of Neuropathology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki, Japan</ce:textfn> </ce:affiliation> <ce:affiliation id="aff8"> <ce:label>††</ce:label> <ce:textfn>Department of Clinical Neuroscience, Geriatric Section, Huddinge Brain Bank, Huddinge, Sweden</ce:textfn> </ce:affiliation> <ce:affiliation id="aff9"> <ce:label>‡‡</ce:label> <ce:textfn>Department of Psychiatry, Clinical Neurochemistry and The National Parkinson Foundation Center of Excellence Research Laboratories, Julius Maximilians University, Würzburg, Germany</ce:textfn> </ce:affiliation> <ce:correspondence id="cor1"> <ce:label>*</ce:label> <ce:text>Address reprint requests to Philipp Kahle or Christian Haass, Laboratory for Alzheimer’s and Parkinson’s Disease Research, Department of Biochemistry, Ludwig Maximilians University of Munich, Schillerstrasse 44, D-80336 Munich, Germany</ce:text> </ce:correspondence> </ce:author-group> <ce:date-accepted day="10" month="9" year="2001"></ce:date-accepted> <ce:abstract> <ce:abstract-sec> <ce:simple-para id="spara110">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 <ce:italic>in vitro</ce:italic>, 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.</ce:simple-para> </ce:abstract-sec> </ce:abstract> </head> <body> <ce:sections> <ce:para id="para10">α-Synuclein (α-SYN) has been identified as the precursor protein of a nonamyloid β-protein component (NAC) isolated from Alzheimer’s disease plaques.<ce:cross-ref refid="bib1"><ce:sup>1</ce:sup></ce:cross-ref> 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,<ce:cross-refs refid="bib2 bib3 bib4 bib5 bib6 bib7"><ce:sup>2-7</ce:sup></ce:cross-refs> and neurodegeneration with brain iron accumulation type 1 (NBIA1).<ce:cross-refs refid="bib8 bib9 bib10 bib11"><ce:sup>8-11</ce:sup></ce:cross-refs> Antibodies directed against both N-terminal and C-terminal epitopes recognized LB filaments,<ce:cross-refs refid="bib12 bib13"><ce:sup>12,13</ce:sup></ce:cross-refs> and the presence of full-length α-SYN was biochemically proven on Western blots of isolated LBs.<ce:cross-ref refid="bib6"><ce:sup>6</ce:sup></ce:cross-ref> Moreover, full-length α-SYN is the major fibrillar component of glial cytoplasmic inclusions in multiple system atrophy.<ce:cross-ref refid="bib14"><ce:sup>14</ce:sup></ce:cross-ref></ce:para> <ce:para id="para20">The formation of LB-like fibrils is an intrinsic property of α-SYN. Purified recombinant α-SYN, but not β-SYN, aggregated <ce:italic>in vitro</ce:italic> to amyloid fibrils resembling those extracted from LBs.<ce:cross-refs refid="bib15 bib16 bib17 bib18 bib19"><ce:sup>15-19</ce:sup></ce:cross-refs> PD risk factors, namely α-SYN mutations<ce:cross-refs refid="bib20 bib21 bib22"><ce:sup>20-22</ce:sup></ce:cross-refs> and oxidative stress,<ce:cross-ref refid="bib23"><ce:sup>23</ce:sup></ce:cross-ref> accelerated α-SYN aggregation. The causal relationship between α-SYN fibrillization and PD are therefore subject to intense research.<ce:cross-ref refid="bib24"><ce:sup>24</ce:sup></ce:cross-ref></ce:para> <ce:para id="para30">Transgenic animals expressing human wild-type [wt]- as well as PD-associated mutant [A53T]α-SYN<ce:cross-ref refid="bib25"><ce:sup>25</ce:sup></ce:cross-ref> and [A30P]α-SYN<ce:cross-ref refid="bib26"><ce:sup>26</ce:sup></ce:cross-ref> were recently presented. Wild-type and mutant α-SYN assembled into LB-like fibrils in transgenic<ce:italic>Drosophila</ce:italic>, and a locomotor deficit became apparent with increasing age.<ce:cross-ref refid="bib27"><ce:sup>27</ce:sup></ce:cross-ref> Somal and neuritic accumulations of wt and mutant α-SYN were observed in transgenic mouse brain.<ce:cross-refs refid="bib28 bib29 bib30"><ce:sup>28-30</ce:sup></ce:cross-refs> Ubiquitination was occasionally detected, but the α-SYN accumulations did not meet ultrastructural criteria of LBs.<ce:cross-refs refid="bib28 bib29"><ce:sup>28,29</ce:sup></ce:cross-refs> Masliah and colleagues<ce:cross-ref refid="bib28"><ce:sup>28</ce:sup></ce:cross-ref> reported a modest reduction of locomotor performance and van der Putten and colleagues<ce:cross-ref refid="bib29"><ce:sup>29</ce:sup></ce:cross-ref> found that age-dependent degeneration of neuromuscular junctions caused a severe locomotor deficit and premature death in their mice.</ce:para> <ce:para id="para40">α-SYN and β-SYN have both been found in the synaptosomal fractions of rodent and human brain.<ce:cross-refs refid="bib30 bib31 bib32 bib33 bib34"><ce:sup>30-34</ce:sup></ce:cross-refs> Synaptosomal α-SYN was released into the soluble fraction of human brain biopsies.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> 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 <ce:italic>in vitro</ce:italic> 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.</ce:para> <ce:section id="cesec10"> <ce:section-title>Materials and Methods</ce:section-title> <ce:section id="cesec20"> <ce:section-title>Antibodies</ce:section-title> <ce:para id="para50">Rat monoclonal anti-α-SYN 15G7<ce:cross-refs refid="bib11 bib30"><ce:sup>11,30</ce:sup></ce:cross-refs> and mouse monoclonal anti-synaptophysin SY38 hybridoma supernatants were used as described previously.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> Mouse monoclonal anti-α-SYN LB509 and Syn102 were described before.<ce:cross-ref refid="bib6"><ce:sup>6</ce:sup></ce:cross-ref> 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.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> The rabbit polyclonal anti-NAC antiserum<ce:cross-ref refid="bib7"><ce:sup>7</ce:sup></ce:cross-ref> 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).</ce:para> </ce:section> <ce:section id="cesec30"> <ce:section-title>Brain Fractionation and Western Blotting</ce:section-title> <ce:para id="para60">Subcellular fractionation of archived human cerebral cortex samples was performed as previously described for fresh tissue.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> The detergent extraction method of Culvenor and colleagues<ce:cross-ref refid="bib35"><ce:sup>35</ce:sup></ce:cross-ref> 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 ×<ce:italic>g</ce:italic>, the supernatants were ultracentrifuged for 1 hour at 130,000 × <ce:italic>g</ce:italic>. 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 × <ce:italic>g</ce:italic> 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.</ce:para> <ce:para id="para70">Denaturing polyacrylamide gel electrophoresis (PAGE), Western blotting, and probing were done as described previously.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> 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 <ce:inter-ref xlink:href="http://rsb.info.nih.gov/nih-image">http://rsb.info.nih.gov/nih-image</ce:inter-ref>). Linear regression of the α-SYN standard band intensities revealed correlation coefficients between 0.9 and 1.0.</ce:para> </ce:section> <ce:section id="cesec40"> <ce:section-title>Generation and Characterization of Transgenic Mice</ce:section-title> <ce:para id="para80">The amplified human [wt]α-SYN-coding sequence was subcloned into the <ce:italic>Xho</ce:italic>I site of the Thy1 cassette of pTSC21k, and the<ce:italic>Not</ce:italic>I-linearized DNA was used to generate transgenic C57BL/6 mice as described for [A30P] α-SYN.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> Five founders stably transmitted the transgene, as determined by tail biopsy polymerase chain reaction.</ce:para> <ce:para id="para90">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 genomic<ce:italic>Xba</ce:italic>I-<ce:italic>Kpn</ce:italic>I restriction fragments were fractionated by gel electrophoresis and blotted onto Nylon membranes (Roche Molecular Biosciences). A 1.6-kb DNA probe (<ce:italic>Hin</ce:italic>dIII-<ce:italic>Eco</ce:italic>RV fragment of the transgene) was labeled with [<ce:sup>33</ce:sup>P]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.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref></ce:para> <ce:para id="para100">Fresh mouse brains were fixed in phosphate-buffered 4% paraformaldehyde and embedded in paraffin. Immunocytochemical detection of SYNs was performed as described previously.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref></ce:para> </ce:section> <ce:section id="cesec50"> <ce:section-title>Expression and <ce:italic>in Vitro</ce:italic> Aggregation of Recombinant SYNs</ce:section-title> <ce:para id="para110">The β-SYN expression vector has been described by Jakes and colleagues.<ce:cross-ref refid="bib36"><ce:sup>36</ce:sup></ce:cross-ref> The mouse α-SYN<ce:cross-ref refid="bib37"><ce:sup>37</ce:sup></ce:cross-ref> 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-SYN<ce:cross-ref refid="bib38"><ce:sup>38</ce:sup></ce:cross-ref> 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 the<ce:italic>Nde</ce:italic>I site of pET-5a (Promega, Madison, WI), and constructs used to transform <ce:italic>Escherichia coli</ce:italic> BL21(DE3) pLys. All constructs were sequenced (Medigenomix, Martinsried, Germany).</ce:para> <ce:para id="para120">Bacterial cultures were induced with isopropyl-β-<ce:small-caps>d</ce:small-caps>-thiogalactoside for 4 hours, and lysed by freeze/thaw and sonication. After 15 minutes of boiling, the heat-stable 17,000 × <ce:italic>g</ce:italic> 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.</ce:para> <ce:para id="para130">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 ×<ce:italic>g</ce:italic> centrifugation, and subjected to the detergent extraction protocol described above.</ce:para> </ce:section> </ce:section> <ce:section id="cesec60"> <ce:section-title>Results</ce:section-title> <ce:section id="cesec70"> <ce:section-title>Recovery of α-SYN from the Particulate Synaptosomal Lysate Fraction Post Mortem</ce:section-title> <ce:para id="para140">Previous subcellular fractionation experiments with human brain have demonstrated the presence of α-SYN in synaptosomes.<ce:cross-refs refid="bib30 bib34"><ce:sup>30,34</ce:sup></ce:cross-refs> In accord with results from rapidly processed rodent brain,<ce:cross-refs refid="bib30 bib33"><ce:sup>30,33</ce:sup></ce:cross-refs> α-SYN was released into the soluble fraction on hypotonic lysis of the synaptosomes prepared from human biopsy brain.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> Using archived cortical tissue, Irizarry and colleagues<ce:cross-ref refid="bib34"><ce:sup>34</ce:sup></ce:cross-ref> have found a significant portion of α-SYN in the particulate fraction of lysed synaptosomes. Indeed, when subcellular fractionations (<ce:cross-ref refid="fig1">Figure 1</ce:cross-ref><ce:float-anchor refid="fig1"></ce:float-anchor>) 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 rapidly processed human biopsy samples.<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> Thus, there seems to be a shift of α-SYN but not β-SYN into the pelletable synaptosomal fraction post mortem. This effect could be either because of altered membrane affinity and/or decreased solubility of α-SYN.</ce:para> </ce:section> <ce:section id="cesec80"> <ce:section-title>Detergent-Insoluble α-SYN in LB Disease Brain</ce:section-title> <ce:para id="para150">Sequential detergent extraction methods have been successfully used to detect α-SYN in brains of patients with α-synucleinopathies.<ce:cross-refs refid="bib6 bib10 bib35 bib39"><ce:sup>6,10,35,39</ce:sup></ce:cross-refs> We have adapted the method of Culvenor and colleagues<ce:cross-ref refid="bib35"><ce:sup>35</ce:sup></ce:cross-ref> (<ce:cross-ref refid="fig2">Figure 2A</ce:cross-ref><ce:float-anchor refid="fig2"></ce:float-anchor>) to detect insoluble α-SYN molecules in human LB disease brain and in transgenic mice expressing human α-SYN.</ce:para> <ce:para id="para160">Buffer- and detergent-soluble monomeric α-SYN was detected in brains from human controls as well as from LB disease patients (<ce:cross-ref refid="fig2">Figure 2</ce:cross-ref>). In contrast to control brain urea extracts that were virtually devoid of α-SYN, strong immunoreactivity was found in urea extracts from PD and DLB patients (<ce:cross-ref refid="fig2">Figure 2, C and D</ce:cross-ref>). The detergent-insoluble α-SYN was characterized using four different antibodies raised against distinct epitopes (<ce:cross-ref refid="fig2">Figure 2B</ce:cross-ref>). Monomeric α-SYN migrated as a 16- to 19-kd band [(α-SYN)<ce:inf>1</ce:inf>]. Anti-NAC, but not C-terminal antibodies detected a previously unrecognized α-SYN species with slightly retarded electrophoretic motility (α-SYN)<ce:inf>p17</ce:inf>) (<ce:cross-ref refid="fig2">Figure 2D</ce:cross-ref>). This band was unlikely to be cross-reactive β-SYN, because specific anti-β-SYN did not reveal any signal in urea extracts (data not shown). In addition, all four anti-α-SYN antibodies recognized ∼40- to 45-kd double bands [(α-SYN)<ce:inf>2</ce:inf>; consistent with α-SYN dimers and/or a recently described membrane-bound form of α-SYN<ce:cross-ref refid="bib40"><ce:sup>40</ce:sup></ce:cross-ref>], multiple bands in the 60- to 80-kd range [(α-SYN)<ce:inf>n</ce:inf>; putative α-SYN oligomers), and higher molecular weight aggregates (<ce:cross-ref refid="fig2">Figure 2</ce:cross-ref>). Interestingly, an ∼25-kd band [(α-SYN)<ce:inf>p25</ce:inf>] was consistently observed in urea extracts from LB disease patients (<ce:cross-ref refid="fig2">Figure 2</ce:cross-ref>). This band was reminiscent of an O-glycosylated form of α-SYN recently described by Shimura and colleagues.<ce:cross-ref refid="bib41"><ce:sup>41</ce:sup></ce:cross-ref> Consistent with this notion was the absence of ubiquitin immunoreactivity of (α-SYN)<ce:inf>p25</ce:inf> (<ce:cross-ref refid="fig2">Figure 2C</ce:cross-ref>), indicating that this species did not correspond to mono-ubiquitinated α-SYN.</ce:para> <ce:para id="para170">A similar differential extraction pattern for α-SYN was found for another LB disease. NBIA1 is a neurodegenerative synucleinopathy formerly known as Hallervorden-Spatz disease that is characterized by α-SYN inclusions similar to LBs as well as by axonal spheroids that contain immunoreactive α-, β-, and γ-SYN.<ce:cross-refs refid="bib8 bib9 bib10 bib11"><ce:sup>8-11</ce:sup></ce:cross-refs> We have analyzed brain samples from two NBIA1 patients described elsewhere (case no. 1,<ce:cross-ref refid="bib42"><ce:sup>42</ce:sup></ce:cross-ref> case no. 2<ce:cross-ref refid="bib10"><ce:sup>10</ce:sup></ce:cross-ref>). Monomeric α-SYN was detected in the buffer-soluble and detergent-soluble fractions. The decrease in the relative amount of buffer-soluble α-SYN in both NBIA1 samples (<ce:cross-ref refid="fig2">Figure 2E</ce:cross-ref>) may be indicative of defective synaptic integrity in these patients.<ce:cross-ref refid="bib10"><ce:sup>10</ce:sup></ce:cross-ref> Alternatively, the soluble pool of α-SYN might be depleted as α-SYN monomers and aggregates accumulated in the detergent-insoluble fraction (<ce:cross-ref refid="fig2">Figure 2E</ce:cross-ref>). Limited N-terminal degradation to a 14-kd band ([ΔN]α-SYN) was occasionally noted, but the bulk of insoluble α-SYN was full-length protein. Higher molecular weight α-SYN bands were observed even in the SDS fractions from brain of NBIA1 case no. 2, who also had much more intense α-SYN immunoreactivity in the urea extracts than NBIA1 case no. 1 (<ce:cross-ref refid="fig2">Figure 2E</ce:cross-ref>). In fact, there was a positive correlation between the α-SYN immunoreactivity in urea extracts and LB density in the two NBIA1 patients and the three DLB patients (<ce:cross-ref refid="tbl1">Table 1</ce:cross-ref><ce:float-anchor refid="tbl1"></ce:float-anchor>).</ce:para> </ce:section> <ce:section id="cesec90"> <ce:section-title>Expression Pattern of α-SYN in Transgenic Mice</ce:section-title> <ce:para id="para180">To generate a rodent model of α-synucleinopathy, we have generated transgenic mice expressing human wt and PD-associated mutant [A30P]α-SYN under the control of a brain-specific pan-neuronal promoter, Thy1. The (Thy1)-[A30P]α-SYN mice described previously<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> and newly generated strains of (Thy1)-[wt]α-SYN mice were of identical genetic background.</ce:para> <ce:para id="para190">As expected for the Thy1 cassette,<ce:cross-ref refid="bib43"><ce:sup>43</ce:sup></ce:cross-ref> expression of transgenic α-SYN was undetectable in the first postnatal week, then increased sharply to reach a plateau around 1 month (<ce:cross-ref refid="fig3">Figure 3</ce:cross-ref><ce:float-anchor refid="fig3"></ce:float-anchor>). The developmental onset of transgene expression paralleled that of endogenous α-SYN, except for the low but significant early postnatal expression of endogenous α-SYN (<ce:cross-ref refid="fig3">Figure 3</ce:cross-ref>). The putative embryonic/perinatal function of α-SYN in mouse brain<ce:cross-refs refid="bib37 bib44"><ce:sup>37,44</ce:sup></ce:cross-refs> remains to be elucidated.</ce:para> <ce:para id="para200">In adult animals, expression levels of transgenic mRNA and protein generally correlated with each other (except in the [A30P]α-SYN-expressing mouse line 8, which had very high mRNA levels but intermediate protein amounts) (<ce:cross-ref refid="tbl2">Table 2</ce:cross-ref><ce:float-anchor refid="tbl2"></ce:float-anchor>). In contrast, the transgene copy number was no predictor of expression levels (<ce:cross-ref refid="tbl2">Table 2</ce:cross-ref>). Approximately 0.1% (w/w) of total adult transgenic mouse brain cytosolic protein was α-SYN, as determined by quantitative Western blot analysis (<ce:cross-ref refid="tbl2">Table 2</ce:cross-ref>). Individual quantification of mouse and human α-SYN revealed up to threefold overexpression levels of the transgenic protein.</ce:para> <ce:para id="para210">At these rather moderate overexpression levels, aberrant subcellular localization of human α-SYN was apparent in transgenic mouse brain sections. The human (transgene)-specific antibody showed somal and neuritic accumulations in both [wt]α-SYN and [A30P]α-SYN mice (<ce:cross-ref refid="fig4">Figure 4, A and D</ce:cross-ref><ce:float-anchor refid="fig4"></ce:float-anchor>), in addition to neuropil (presumably synaptic) staining. Compact LBs were not observed in transgenic mice. Nevertheless, swollen α-SYN-positive neurites as observed in [wt]α-SYN and [A30P]α-SYN transgenic mice (<ce:cross-ref refid="fig4">Figure 4, A and D, inserts</ce:cross-ref>) were a prominent feature in brain sections from LB disease patients.<ce:cross-refs refid="bib2 bib9 bib30"><ce:sup>2,9,30</ce:sup></ce:cross-refs> In sharp contrast, endogenous mouse α-SYN showed only the normal neuropil staining pattern (<ce:cross-ref refid="fig4">Figure 4, C and F</ce:cross-ref>). Likewise, our β-SYN antibody detected only a normal synaptic staining pattern in sections from transgenic mouse brain (<ce:cross-ref refid="fig4">Figure 4, B and E</ce:cross-ref>).</ce:para> </ce:section> <ce:section id="cesec100"> <ce:section-title>Transgenic Human α-SYN But Not Endogenous Mouse SYNs in Detergent-Insoluble Fractions</ce:section-title> <ce:para id="para220">As the presence of SDS-insoluble α-SYN seemed to be a diagnostic criterion for LB diseases in human brain, we applied the above method to transgenic mouse brains expressing human α-SYN in brain neurons. Although the overexpression levels of transgenic α-SYN were rather moderate, a portion of human α-SYN was specifically detected in urea extracts of detergent-insoluble fractions from transgenic mouse brains (<ce:cross-ref refid="fig5">Figure 5</ce:cross-ref><ce:float-anchor refid="fig5"></ce:float-anchor>). Both transgenic [A30P] α-SYN and [wt] α-SYN were found in SDS-insoluble fractions (<ce:cross-ref refid="fig5">Figure 5A</ce:cross-ref>). Insoluble transgenic α-SYN became detectable parallel to the onset of transgene expression (<ce:cross-ref refid="fig3">Figure 3</ce:cross-ref>) and persisted for at least 1 year (<ce:cross-ref refid="fig5">Figure 5B</ce:cross-ref>). The onset of expression of insoluble transgenic α-SYN was concomitant with the appearance of cytosolic accumulations (<ce:cross-ref refid="fig4">Figure 4A</ce:cross-ref>).</ce:para> <ce:para id="para230">In sharp contrast, endogenous mouse α-SYN as well as β-SYN were entirely soluble in buffer and detergent. In a semiquantitative manner, Western blots were sequentially probed with human (transgene)-specific anti-α-SYN, mouse (endogenous)-specific anti-α-SYN, and anti-β-SYN, and the signal of a control lane on the same blot (10 μg transgenic mouse brain cytosol) was used for normalization. Exposure times yielding comparable signals from the control lane demonstrated that SDS-insoluble endogenous α-SYN or β-SYN in transgenic α-SYN-positive urea extracts were undetectable (<ce:cross-ref refid="fig5">Figure 5</ce:cross-ref>).</ce:para> <ce:para id="para240">Taken together, cytoplasmic accumulation and detergent-insolubility of transgenic human α-SYN represent specific pathological alterations in transgenic mouse brain that are reminiscent of human LB diseases.</ce:para> </ce:section> <ce:section id="cesec110"> <ce:section-title>Lack of an Aggregation-Promoting Stretch of Amino Acids in the NAC Domain of β-SYN</ce:section-title> <ce:para id="para250">To determine whether the detergent insolubility of α-SYN in LB disease brain and transgenic mice was a consequence of aggregation,<ce:italic>in vitro</ce:italic> formed aggregates of α-SYN were subjected to a similar differential extraction procedure used for brain tissue (see above). α-SYN aggregated at a concentration of 2 mg/ml was partially solubilized by resuspension in 15 volumes of TBS+. However, 5% SDS was required for complete solubilization (<ce:cross-ref refid="fig6">Figure 6A</ce:cross-ref><ce:float-anchor refid="fig6"></ce:float-anchor>). In contrast, β-SYN failed to form insoluble aggregates <ce:italic>in vitro</ce:italic> (<ce:cross-ref refid="fig6">Figure 6B</ce:cross-ref>).</ce:para> <ce:para id="para260">β-SYN lacks amino acids homologous to residues 73 to 83 of α-SYN. Codons 73 to 83 were specifically deleted by site-directed mutagenesis yielding [Δ73–83]α-SYN. In parallel <ce:italic>in vitro</ce:italic> aggregation assays, [Δ73–83]α-SYN behaved like β-SYN in that it practically lost its capability to form 100,000 × <ce:italic>g</ce:italic> pellets after 1 week incubation at 37°C (<ce:cross-ref refid="fig6">Figure 6B</ce:cross-ref>). Thus, amino acids 73 to 83 in the N-terminal half of NAC are critical for α-SYN aggregation, and their absence in β-SYN accounts for the loss of aggregation capacity of the nonamyloidogenic β-SYN.</ce:para> </ce:section> </ce:section> <ce:section id="cesec120"> <ce:section-title>Discussion</ce:section-title> <ce:para id="para270">PD, DLB, and NBIA1 are characterized immunohistochemically by α-SYN-immunoreactive intraneuronal inclusions (LBs) and dystrophic neurites. Biochemically, detergent-insoluble α-SYN was found to be diagnostic for these diseases. We have performed differential detergent extractions to evaluate the potential development of α-synucleinopathy in transgenic mice expressing human α-SYN in brain neurons. Like in human LB diseases, detergent-insoluble human α-SYN was detected in transgenic mouse brain. In striking contrast, endogenous mouse α-SYN and β-SYN were not found in the urea extracts. These results demonstrate that a transgenic mouse model recapitulates some specific features of α-synucleinopathies.</ce:para> <ce:para id="para280">The source of detergent-insoluble α-SYN may not only be solid LBs. It is noteworthy that the NBIA1 case with much urea-extractable α-SYN had abundant dystrophic neurites (not shown). Moreover, diffuse accumulation of α-SYN in neuronal cell bodies was occasionally reported for human α-synucleinopathies.<ce:cross-refs refid="bib45 bib46"><ce:sup>45,46</ce:sup></ce:cross-refs> The accumulations of human α-SYN in transgenic mouse brain neurons did not meet ultrastructural criteria of LBs. Nevertheless, detergent-insoluble transgenic α-SYN was specifically detectable in these mice. Thus, a portion of transgenic α-SYN is converted to a less soluble form that might represent an early form of α-synucleinopathy. It remains to be shown if the decreased solubility of transgenic α-SYN is a mere consequence of overexpression, or if there is some secondary processing that is peculiar to human transgenic α-SYN in mouse brain.</ce:para> <ce:para id="para290">Both PD-associated [A30P]α-SYN and human [wt]α-SYN were detected in detergent-insoluble fractions. This is of note because the overwhelming majority of PD patients have no mutation in the α-SYN gene.<ce:cross-ref refid="bib47"><ce:sup>47</ce:sup></ce:cross-ref> The faster <ce:italic>in vitro</ce:italic> aggregation rate of concentrated solutions of mutant α-SYN<ce:cross-ref refid="bib22"><ce:sup>22</ce:sup></ce:cross-ref> was apparently not reflected by greater pathology of human mutant α-SYN compared to [wt]α-SYN in transgenic mice.<ce:cross-refs refid="bib29 bib30"><ce:sup>29,30</ce:sup></ce:cross-refs> The rather moderate expression levels of transgenic α-SYN (<ce:cross-ref refid="tbl2">Table 2</ce:cross-ref>) make it unlikely that the critical concentration required for recombinant α-SYN aggregation <ce:italic>in vitro</ce:italic> (28 μmol/L)<ce:cross-ref refid="bib48"><ce:sup>48</ce:sup></ce:cross-ref> was reached in neuronal cytosol. Perhaps the differences in aggregation kinetics between wt and mutant α-SYN are not evident at concentrations reached in transgenic mouse neurons. Because α-SYN expression is not elevated enough in PD patients to allow spontaneous aggregation, additional risk factors are likely to exist that favor the aggregation at subcritical α-SYN concentrations. Similar risk factors may act in rodents. For example, the mitochondrial complex I inhibitor, rotenone, was recently shown to elicit PD-like alterations in rat brain, including the formation of α-SYN inclusions and selective loss of striatonigral dopaminergic neurons.<ce:cross-ref refid="bib49"><ce:sup>49</ce:sup></ce:cross-ref></ce:para> <ce:para id="para300">Potentially aggregation-promoting posttranslational modifications of α-SYN include phosphorylation,<ce:cross-ref refid="bib38"><ce:sup>38</ce:sup></ce:cross-ref> nitration,<ce:cross-ref refid="bib50"><ce:sup>50</ce:sup></ce:cross-ref> and glycation.<ce:cross-ref refid="bib51"><ce:sup>51</ce:sup></ce:cross-ref> Moreover, perturbation of proteosomal degradation should be considered. LBs contain ubiquitinated α-SYN. It was reported that in human brain, α-SYN needs to be converted to a slower migrating species (termed “αSp22”) to become a substrate for the ubiquitin ligase, parkin,<ce:cross-ref refid="bib41"><ce:sup>41</ce:sup></ce:cross-ref> We have found an α-SYN species with similar retarded electrophoretic motility running at apparent 25-kd [(α-SYN)<ce:inf>p25</ce:inf>] in the detergent-insoluble fractions from LB disease brain (<ce:cross-ref refid="fig2">Figure 2</ce:cross-ref>), but not in transgenic mice (<ce:cross-ref refid="fig5">Figure 5</ce:cross-ref>). It is possible that posttranslational modifications characteristic for human LB diseases have to occur in transgenic mice to allow true LB formation in an animal model.</ce:para> <ce:para id="para310">In contrast to the amyloidogenic α-SYN, the close homologue β-SYN was absent from amyloid deposits. β-SYN has no intrinsic capacity to form amyloid fibrils. β-SYN lacks the sequence GVTAVAQKTVE corresponding to amino acids 73 to 83 of α-SYN in the NAC domain. Indeed, the deletion mutant protein [Δ73–83]α-SYN was aggregation-deficient. Similar findings were recently reported by Giasson and colleagues<ce:cross-ref refid="bib52"><ce:sup>52</ce:sup></ce:cross-ref> for a slightly shifted deletion mutant, namely [Δ71–82]α-SYN. Interestingly, the region critical for α-SYN aggregation, which is missing in β-SYN, overlaps with one of the imperfectly conserved KTKEGV repeats. Such repeats are characteristic for the lipid-binding N-terminal half of α-SYN that has a predicted amphipathic helix structure comparable with apolipoprotein A.<ce:cross-refs refid="bib53 bib54"><ce:sup>53,54</ce:sup></ce:cross-refs> It will be interesting to determine whether the aggregation-promoting region of α-SYN acquires an amphipathic β-strand conformation upon fibrillization.</ce:para> <ce:para id="para320"><ce:italic>In vitro</ce:italic> aggregates of α-SYN clearly required harsher solubilization methods than β-SYN. Nevertheless, hardly any SDS-insoluble protein was found in urea extracts of <ce:italic>in vitro</ce:italic> aggregated recombinant α-SYN. Thus, the massive amounts of α-SYN in urea extracts from LB disease and transgenic mouse brain represent a pathological form of α-SYN that may not be fully reproduced by simple aggregation <ce:italic>in vitro</ce:italic>. Thus, α-SYN transgenic mice provide an <ce:italic>in vivo</ce:italic> model that exhibits an important aspect of human α-synucleinopathy, namely selective insolubility of α-SYN.</ce:para> </ce:section> </ce:sections> <ce:acknowledgment> <ce:section-title>Acknowledgements</ce:section-title> <ce:para id="para330">We thank R. Jakes and M. Goedert for the gift of β-SYN expression plasmid, K. Beyreuther and T. Hartmann for providing anti-NAC, W. Franke and R. Leube for the donation of anti-synaptophysin, and H. 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type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Laurence</namePart><namePart type="family">Ozmen</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Veronika</namePart><namePart type="family">Mller</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Sabine</namePart><namePart type="family">Odoy</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Noriko</namePart><namePart type="family">Okamoto</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Helmut</namePart><namePart type="family">Jacobsen</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Takeshi</namePart><namePart type="family">Iwatsubo</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">John Q.</namePart><namePart type="family">Trojanowski</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Hitoshi</namePart><namePart type="family">Takahashi</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Koichi</namePart><namePart type="family">Wakabayashi</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Nenad</namePart><namePart type="family">Bogdanovic</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Peter</namePart><namePart type="family">Riederer</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Hans A.</namePart><namePart type="family">Kretzschmar</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Christian</namePart><namePart type="family">Haass</namePart><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article"></genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2001</dateIssued><dateValid encoding="w3cdtf">2001-09-10</dateValid><copyrightDate encoding="w3cdtf">2001</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract>-Synuclein (-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinsons disease (PD) and LB dementias. -SYN forms LB-like fibrils in vitro, in contrast to its homologue -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]-SYN driven by the brain neuron-specific promoter, Thy1. Distinct -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 -SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse -SYN nor -SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic -SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of -SYN are absent in -SYN. In conclusion, the specific accumulation of detergent-insoluble -SYN in transgenic mice recapitulates a pivotal feature of human LB diseases.</abstract><note>Supported by grants from the Deutsche Forschungsgemeinschaft (HA 1737/4-1) and the Bavaria California Technology Center (to C. H.).</note><note type="content">Section title: Regular Article</note><note type="content">Figure 1: Subcellular fractionation of frozen human brain tissue. A: Schematic representation of the subcellular fractionation steps, annotations in B correspond to the fractions outlined inA. B: Parietal cortex (0.4 g) from a human control individual was homogenized and subjected to subcellular fractionation. Twenty g of each fraction was TCA precipitated (except postnuclear fraction S1 that was loaded directly) and subjected to denaturing 12.5 PAGE. Sequential Western probing was done with 15G7 anti--SYN, SY38 anti-synaptophysin (SPH), and 6584 anti--SYN, as indicated. Data are representative for three different control cortex fractionations.</note><note type="content">Figure 2: SDS-insoluble -SYN species in LB diseases. A: Schematic representation of the differential extraction steps. See Materials and Methods for details. B: Structure of -SYN and epitopes recognized by anti--SYN antibodies. The imperfect KTKEGV repeats are numbered, the sixth repeat missing in -SYN (see below) isstippled. C: Extracts from temporal cortex of control and DLB brain were prepared, and 10 g of TBS-soluble material, 10 g of SDS-soluble material, and 10 l of urea extracts or TCA precipitates from 50-l urea extracts (as indicated at thebottom) were subjected to denaturing 12.5 PAGE. Western blots were sequentially probed with three different antibodies against -SYN (15G7, 3400, MC42) and anti-ubiquitin (Ubi-1), as indicated on the top. Immunoreactivity was visualized with SuperSignal (for 15G7) or ECLplus.D: TCA precipitates from 50-l urea extracts from temporal cortex of control and DLB brain (left) and from parietal cortex of control and PD brain (right) were loaded on 10 to 20 Tris-tricine gels. The corresponding Western blots were probed with anti-NAC and developed with ECLplus. E: Parietal cortex samples from two controls and two NBIA1 patients were extracted in parallel. TBS-soluble material (10 g, left), SDS-soluble material (25 g,middle), and urea extracts (80 l,right) were separated by SDS-PAGE (TBS-soluble, 15; SDS and urea extracts, 4 to 20 gradient). MC42 and ECLplus were used for Western detection of -SYN. Note that sample NBIA1 no. 2 with higher LB density than NBIA1 no. 1 had also much stronger -SYN immunoreactivity in the urea extract. Nevertheless, the -SYN immunoreactive band pattern of NBIA1 no. 1 was qualitatively the same as of NBIA1 no. 2, as evidenced by a longer exposure of the blot to thefar right. Each experiment is representative for two to three independent extractions. Positions of prestained molecular weight standards are indicated to the left. See text for description of -SYN species denoted to the right.</note><note type="content">Figure 3: Developmental expression of -SYN. Mouse brains were collected from mice at the indicated age, and Western blots prepared from 25-g cytosolic extracts. Wild-type mouse blots were probed with MC42 (top), representative [(Thy1)-h[A30P]-SYN line 18] transgenic mouse blots probed with 3400 (bottom), and developed with ECLplus.</note><note type="content">Figure 4: Immunostainings of brain slices (motor cortex) show specific accumulation of -SYN but not of -SYN in transgenic mice. Animals expressing either [wt]-SYN (AC) or [A30P]-SYN (DF) showed a strong cytosolic labeling of neuronal cells with the human-specific -SYN antibody 15G7 (A andD). A section from a 1-month-old [wt]-SYN-expressing mouse is shown inA to demonstrate the early onset of accumulation of transgenic protein. In contrast, immunostainings with the -SYN-specific antiserum (6485) and the murine-specific -SYN antiserum (7544) only revealed a synaptic staining pattern. Neither accumulation of endogenous murine -SYN (C andF) nor -SYN (B andE) was detectable in neuronal cell bodies. Scale bar in A, 100 m.</note><note type="content">Figure 5: Detergent-insoluble -SYN in transgenic mouse brains. Whole brains of transgenic mice (A: 3- to 4-month-old [wt]-SYN lines 14 and 23, and [A30P]-SYN lines 18 and 31;B: 1-month-old and 1-year-old [A30P]-SYN lines 18 and 31; as indicated at the bottom) and age-matched nontransgenic littermates (lm) were differentially extracted. Buffer- and detergent-soluble proteins (10 g for transgenic human -SYN, 50 g for endogenous mouse SYNs), and TCA precipitate of urea extracts were subjected to denaturing 15 PAGE. Western blots were probed with human (transgene)-specific anti-h-SYN 15G7, endogenous mouse-specific anti-m-SYN 7544, and anti--SYN 6485, as indicated to the left. 15G7-immunoreactive bands were developed with SuperSignal, polyclonal antibody immunoreactivity with ECLplus. A control lane on the urea extract blots contained 10 g of cytosol from a transgenic [A30P]-SYN mouse. The positions of prestained molecular weight markers are indicated to the right. Individual variance of transgenic -SYN expression levels may account for the apparently higher amount of urea extractable mutant h[A30P]-SYN compared to h[wt]-SYN in one experiment (A, exp. 2), but not in two additional experiments (one of them shown as A, exp. 1), and for the apparent increase with age of SDS-soluble -SYN (B) that was not seen in an additional experiment.</note><note type="content">Figure 6: -SYN, but not -SYN aggregates in vitro because of a critical determinant in the NAC domain. A: Recombinant human -SYN aggregates were collected by 100,000 g centrifugation and redissolved in 15 volumes of TBS. The buffer-insoluble material was extracted like the brain samples above. All fractions were TCA precipitated and separated by denaturing 15 PAGE. Western blots were probed with 3400 anti--SYN and developed with SuperSignal. This experiment was repeated three times with the same result. B: Solutions (2 mg/ml) of [wt]-SYN (left), [7383]-SYN (middle), and [wt]-SYN (right) were aggregated for 7 days. After ultracentrifugation, the 100,000 g pellets (pel) were subjected to denaturing 12.5 PAGE. -SYN immunoblots were probed with MC42 and -SYN immunoblots with 6485, and developed with SuperSignal until the band intensities of 1-g freshly dissolved, nonaggregated protein (sol) were comparable. Note the typical retarded electrophoretic motility of recombinant -SYN.36,38 Positions of prestained molecular weight markers are indicated to the right.</note><note type="content">Table 1: The Amount of Insoluble -SYN Correlates with Severity of LB Diseases</note><note type="content">Table 2: -SYN Expression Levels in Transgenic Mice</note><subject><genre>Article category</genre><topic>Regular Articles</topic></subject><relatedItem type="host"><titleInfo><title>The American Journal of Pathology</title></titleInfo><titleInfo type="abbreviated"><title>AJPA</title></titleInfo><genre type="Journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">200112</dateIssued></originInfo><identifier type="ISSN">0002-9440</identifier><identifier type="PII">S0002-9440(10)X6083-7</identifier><part><detail type="volume"><number>159</number><caption>vol.</caption></detail><detail type="issue"><number>6</number><caption>no.</caption></detail><extent unit="issue pages"><start>1977</start><end>2409</end></extent><extent unit="pages"><start>2215</start><end>2225</end></extent></part></relatedItem><identifier type="istex">FA10372DB830345DF09BC358DD8C01F9D4FBAF88</identifier><identifier type="DOI">10.1016/S0002-9440(10)63072-6</identifier><identifier type="PII">S0002-9440(10)63072-6</identifier><accessCondition type="use and reproduction" contentType="">© 2001American Society for Investigative Pathology</accessCondition><recordInfo><recordContentSource>ELSEVIER</recordContentSource><recordOrigin>American Society for Investigative Pathology, ©2001</recordOrigin></recordInfo></mods></metadata><enrichments><istex:catWosTEI uri="https://api.istex.fr/document/FA10372DB830345DF09BC358DD8C01F9D4FBAF88/enrichments/catWos"><teiHeader><profileDesc><textClass><classCode scheme="WOS">PATHOLOGY</classCode><classCode scheme="WOS">CELL BIOLOGY</classCode></textClass></profileDesc></teiHeader></istex:catWosTEI></enrichments><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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