Novel One-step Immunoassays to Quantify α-Synuclein
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Auteurs : Michael Bidinosti ; Derya R. Shimshek ; Brit Mollenhauer [Allemagne] ; David Marcellin ; Tatjana Schweizer ; Gregor P. Lotz ; Michael G. Schlossmacher ; Andreas WeissSource :
- The Journal of Biological Chemistry [ 0021-9258 ] ; 2012.
Abstract
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DOI: 10.1074/jbc.M112.379792
PubMed: 22843695
PubMed Central: 3460466
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Novel One-step Immunoassays to Quantify α-Synuclein</title><author><name sortKey="Bidinosti, Michael" sort="Bidinosti, Michael" uniqKey="Bidinosti M" first="Michael" last="Bidinosti">Michael Bidinosti</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Shimshek, Derya R" sort="Shimshek, Derya R" uniqKey="Shimshek D" first="Derya R." last="Shimshek">Derya R. Shimshek</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Mollenhauer, Brit" sort="Mollenhauer, Brit" uniqKey="Mollenhauer B" first="Brit" last="Mollenhauer">Brit Mollenhauer</name><affiliation wicri:level="1"><nlm:aff id="aff2">Paracelsus-Elena-Klinik, Kassel, Klinikstrasse 16, 34128 Kassel, Germany,</nlm:aff><country xml:lang="fr">Allemagne</country><wicri:regionArea>Paracelsus-Elena-Klinik, Kassel, Klinikstrasse 16, 34128 Kassel</wicri:regionArea></affiliation><affiliation><nlm:aff id="aff3"></nlm:aff></affiliation></author><author><name sortKey="Marcellin, David" sort="Marcellin, David" uniqKey="Marcellin D" first="David" last="Marcellin">David Marcellin</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Schweizer, Tatjana" sort="Schweizer, Tatjana" uniqKey="Schweizer T" first="Tatjana" last="Schweizer">Tatjana Schweizer</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lotz, Gregor P" sort="Lotz, Gregor P" uniqKey="Lotz G" first="Gregor P." last="Lotz">Gregor P. Lotz</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Schlossmacher, Michael G" sort="Schlossmacher, Michael G" uniqKey="Schlossmacher M" first="Michael G." last="Schlossmacher">Michael G. Schlossmacher</name><affiliation><nlm:aff id="aff4"></nlm:aff></affiliation></author><author><name sortKey="Weiss, Andreas" sort="Weiss, Andreas" uniqKey="Weiss A" first="Andreas" last="Weiss">Andreas Weiss</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22843695</idno><idno type="pmc">3460466</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460466</idno><idno type="RBID">PMC:3460466</idno><idno type="doi">10.1074/jbc.M112.379792</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000567</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000567</idno><idno type="wicri:Area/Pmc/Curation">000567</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000567</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Novel One-step Immunoassays to Quantify α-Synuclein</title><author><name sortKey="Bidinosti, Michael" sort="Bidinosti, Michael" uniqKey="Bidinosti M" first="Michael" last="Bidinosti">Michael Bidinosti</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Shimshek, Derya R" sort="Shimshek, Derya R" uniqKey="Shimshek D" first="Derya R." last="Shimshek">Derya R. Shimshek</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Mollenhauer, Brit" sort="Mollenhauer, Brit" uniqKey="Mollenhauer B" first="Brit" last="Mollenhauer">Brit Mollenhauer</name><affiliation wicri:level="1"><nlm:aff id="aff2">Paracelsus-Elena-Klinik, Kassel, Klinikstrasse 16, 34128 Kassel, Germany,</nlm:aff><country xml:lang="fr">Allemagne</country><wicri:regionArea>Paracelsus-Elena-Klinik, Kassel, Klinikstrasse 16, 34128 Kassel</wicri:regionArea></affiliation><affiliation><nlm:aff id="aff3"></nlm:aff></affiliation></author><author><name sortKey="Marcellin, David" sort="Marcellin, David" uniqKey="Marcellin D" first="David" last="Marcellin">David Marcellin</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Schweizer, Tatjana" sort="Schweizer, Tatjana" uniqKey="Schweizer T" first="Tatjana" last="Schweizer">Tatjana Schweizer</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lotz, Gregor P" sort="Lotz, Gregor P" uniqKey="Lotz G" first="Gregor P." last="Lotz">Gregor P. Lotz</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Schlossmacher, Michael G" sort="Schlossmacher, Michael G" uniqKey="Schlossmacher M" first="Michael G." last="Schlossmacher">Michael G. Schlossmacher</name><affiliation><nlm:aff id="aff4"></nlm:aff></affiliation></author><author><name sortKey="Weiss, Andreas" sort="Weiss, Andreas" uniqKey="Weiss A" first="Andreas" last="Weiss">Andreas Weiss</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Biological Chemistry</title><idno type="ISSN">0021-9258</idno><idno type="eISSN">1083-351X</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><bold>Background:</bold> Robust assays for α-synuclein quantification are essential for Parkinson disease therapeutic development.</p><p><bold>Results:</bold> TR-FRET immunoassays were validated for total and oligomeric α-synuclein and used to screen small molecules and kinases that regulate α-synuclein expression.</p><p><bold>Conclusion:</bold> TR-FRET immunoassays are suitable for biomarker development and high-throughput screening.</p><p><bold>Significance:</bold> This is the first platform for large-scale drug discovery and for neuronal pathway analysis of α-synuclein expression regulation.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biol Chem</journal-id><journal-id journal-id-type="iso-abbrev">J. Biol. Chem</journal-id><journal-id journal-id-type="hwp">jbc</journal-id><journal-id journal-id-type="pmc">jbc</journal-id><journal-id journal-id-type="publisher-id">JBC</journal-id><journal-title-group><journal-title>The Journal of Biological Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0021-9258</issn><issn pub-type="epub">1083-351X</issn><publisher><publisher-name>American Society for Biochemistry and Molecular Biology</publisher-name><publisher-loc>9650 Rockville Pike, Bethesda, MD 20814, U.S.A.</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22843695</article-id><article-id pub-id-type="pmc">3460466</article-id><article-id pub-id-type="publisher-id">M112.379792</article-id><article-id pub-id-type="doi">10.1074/jbc.M112.379792</article-id><article-categories><subj-group subj-group-type="heading"><subject>Neurobiology</subject></subj-group></article-categories><title-group><article-title>Novel One-step Immunoassays to Quantify α-Synuclein</article-title><subtitle>APPLICATIONS FOR BIOMARKER DEVELOPMENT AND HIGH-THROUGHPUT SCREENING<xref ref-type="fn" rid="FN1">*</xref></subtitle><alt-title alt-title-type="short">Applications for TR-FRET Quantification of α-Synuclein</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Bidinosti</surname><given-names>Michael</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Shimshek</surname><given-names>Derya R.</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Mollenhauer</surname><given-names>Brit</given-names></name><xref ref-type="aff" rid="aff2"><sup>§</sup></xref><xref ref-type="aff" rid="aff3"><sup>¶</sup></xref></contrib><contrib contrib-type="author"><name><surname>Marcellin</surname><given-names>David</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Schweizer</surname><given-names>Tatjana</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lotz</surname><given-names>Gregor P.</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Schlossmacher</surname><given-names>Michael G.</given-names></name><xref ref-type="aff" rid="aff4"><sup>‖</sup></xref></contrib><contrib contrib-type="author"><name><surname>Weiss</surname><given-names>Andreas</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref><xref ref-type="corresp" rid="cor1"><sup>1</sup></xref></contrib><aff id="aff1">From the<label>‡</label>Neuroscience Discovery Group, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland,</aff><aff id="aff2"><label>§</label>Paracelsus-Elena-Klinik, Kassel, Klinikstrasse 16, 34128 Kassel, Germany,</aff><aff id="aff3">the<label>¶</label>Departments of Neurology and Clinical Neurophysiology, Georg-August University Goettingen, Goettingen, Germany, and</aff><aff id="aff4">the<label>‖</label>Division of Neuroscience, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Ontario, Canada</aff></contrib-group><author-notes><corresp id="cor1"><label>1</label> To whom correspondence should be addressed: <addr-line>Novartis Institutes for Biomedical Research, WSJ-355.2.051.11, CH-4002 Basel, Switzerland.</addr-line> Tel.: <phone>41-79-863-48-27</phone>; E-mail: <email>andreas-1.weiss@novartis.com</email>.</corresp></author-notes><pub-date pub-type="ppub"><day>28</day><month>9</month><year>2012</year></pub-date><pub-date pub-type="epub"><day>27</day><month>7</month><year>2012</year></pub-date><volume>287</volume><issue>40</issue><fpage>33691</fpage><lpage>33705</lpage><history><date date-type="received"><day>8</day><month>5</month><year>2012</year></date><date date-type="rev-recd"><day>24</day><month>7</month><year>2012</year></date></history><permissions><copyright-statement>© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.</copyright-statement><copyright-year>2012</copyright-year></permissions><self-uri xlink:title="pdf" xlink:type="simple" xlink:href="zbc04012033691.pdf"></self-uri><abstract abstract-type="teaser"><p><bold>Background:</bold> Robust assays for α-synuclein quantification are essential for Parkinson disease therapeutic development.</p><p><bold>Results:</bold> TR-FRET immunoassays were validated for total and oligomeric α-synuclein and used to screen small molecules and kinases that regulate α-synuclein expression.</p><p><bold>Conclusion:</bold> TR-FRET immunoassays are suitable for biomarker development and high-throughput screening.</p><p><bold>Significance:</bold> This is the first platform for large-scale drug discovery and for neuronal pathway analysis of α-synuclein expression regulation.</p></abstract><abstract><p>Familial Parkinson disease (PD) can result from α-synuclein gene multiplication, implicating the reduction of neuronal α-synuclein as a therapeutic target. Moreover, α-synuclein content in human cerebrospinal fluid (CSF) represents a PD biomarker candidate. However, capture-based assays for α-synuclein quantification in CSF (such as by ELISA) have shown discrepancies and have limited suitability for high-throughput screening. Here, we describe two sensitive, in-solution, time-resolved Förster's resonance energy transfer (TR-FRET)-based immunoassays for total and oligomeric α-synuclein quantification. CSF analysis showed strong concordance for total α-synuclein content between two TR-FRET assays and, in agreement with a previously characterized 36 h protocol-based ELISA, demonstrated lower α-synuclein levels in PD donors. Critically, the assay suitability for high-throughput screening of siRNA constructs and small molecules aimed at reducing endogenous α-synuclein levels was established and validated. In a small-scale proof of concept compound screen using 384 well plates, signals ranged from <30 to >120% of the mean of vehicle-treated cells for molecules known to lower and increase cellular α-synuclein, respectively. Furthermore, a reverse genetic screen of a kinase-directed siRNA library identified seven genes that modulated α-synuclein protein levels (five whose knockdown increased and two that decreased cellular α-synuclein protein). This provides critical new biological insight into cellular pathways regulating α-synuclein steady-state expression that may help guide further drug discovery efforts. Moreover, we describe an inherent limitation in current α-synuclein oligomer detection methodology, a finding that will direct improvement of future assay design. Our one-step TR-FRET-based platform for α-synuclein quantification provides a novel platform with superior performance parameters for the rapid screening of large biomarker cohorts and of compound and genetic libraries, both of which are essential to the development of PD therapies.</p></abstract><kwd-group><kwd>α-Synuclein</kwd><kwd>Fluorescence Resonance Energy Transfer (FRET)</kwd><kwd>Neurodegeneration</kwd><kwd>Parkinson Disease</kwd><kwd>Protein Aggregation</kwd><kwd>Immunoassay</kwd><kwd>TR-FRET</kwd></kwd-group></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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