Journal article Open Access

Quantitative Characterization of α-Synuclein Aggregation in Living Cells through Automated Microfluidics Feedback Control

Perrino, Giansimone; Wilson, Cathal; Santorelli, Marco; di Bernardo, Diego


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  <identifier identifierType="URL">https://zenodo.org/record/2668451</identifier>
  <creators>
    <creator>
      <creatorName>Perrino, Giansimone</creatorName>
      <givenName>Giansimone</givenName>
      <familyName>Perrino</familyName>
      <nameIdentifier nameIdentifierScheme="ORCID" schemeURI="http://orcid.org/">0000-0001-8095-9139</nameIdentifier>
      <affiliation>Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy</affiliation>
    </creator>
    <creator>
      <creatorName>Wilson, Cathal</creatorName>
      <givenName>Cathal</givenName>
      <familyName>Wilson</familyName>
      <nameIdentifier nameIdentifierScheme="ORCID" schemeURI="http://orcid.org/">0000-0002-0637-8608</nameIdentifier>
      <affiliation>Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy</affiliation>
    </creator>
    <creator>
      <creatorName>Santorelli, Marco</creatorName>
      <givenName>Marco</givenName>
      <familyName>Santorelli</familyName>
      <nameIdentifier nameIdentifierScheme="ORCID" schemeURI="http://orcid.org/">0000-0002-9633-7825</nameIdentifier>
      <affiliation>Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy</affiliation>
    </creator>
    <creator>
      <creatorName>di Bernardo, Diego</creatorName>
      <givenName>Diego</givenName>
      <familyName>di Bernardo</familyName>
      <nameIdentifier nameIdentifierScheme="ORCID" schemeURI="http://orcid.org/">0000-0002-1911-7407</nameIdentifier>
      <affiliation>1. Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy; 2. Department of Chemical, Materials and Industrial Production Engineering, University of Naples Federico II, Piazzale Tecchio 80, 80125 Naples, Italy</affiliation>
    </creator>
  </creators>
  <titles>
    <title>Quantitative Characterization of α-Synuclein Aggregation in Living Cells through Automated Microfluidics Feedback Control</title>
  </titles>
  <publisher>Zenodo</publisher>
  <publicationYear>2019</publicationYear>
  <subjects>
    <subject>Bioengineering</subject>
    <subject>Microfluidics</subject>
    <subject>Feedback control</subject>
    <subject>Gene expression</subject>
    <subject>Synuclein</subject>
    <subject>Aggregation</subject>
  </subjects>
  <dates>
    <date dateType="Issued">2019-04-16</date>
  </dates>
  <resourceType resourceTypeGeneral="JournalArticle"/>
  <alternateIdentifiers>
    <alternateIdentifier alternateIdentifierType="url">https://zenodo.org/record/2668451</alternateIdentifier>
  </alternateIdentifiers>
  <relatedIdentifiers>
    <relatedIdentifier relatedIdentifierType="DOI" relationType="IsIdenticalTo">10.1016/j.celrep.2019.03.081</relatedIdentifier>
    <relatedIdentifier relatedIdentifierType="URL" relationType="IsPartOf">https://zenodo.org/communities/cosy-bio</relatedIdentifier>
  </relatedIdentifiers>
  <rightsList>
    <rights rightsURI="https://creativecommons.org/licenses/by/4.0/legalcode">Creative Commons Attribution 4.0 International</rights>
    <rights rightsURI="info:eu-repo/semantics/openAccess">Open Access</rights>
  </rightsList>
  <descriptions>
    <description descriptionType="Abstract">&lt;p&gt;&lt;strong&gt;Highlights&lt;/strong&gt;&lt;/p&gt;

&lt;p&gt;&amp;bull;&amp;nbsp;&lt;em&gt;In silico&lt;/em&gt;&amp;nbsp;feedback control enables regulation of &amp;alpha;-synuclein expression in yeast&lt;/p&gt;

&lt;p&gt;&amp;bull;&amp;nbsp;&amp;alpha;-Synuclein inclusion formation is strictly concentration, but not time, dependent&lt;/p&gt;

&lt;p&gt;&amp;bull;&amp;nbsp;The aggregation threshold of the &amp;alpha;-synuclein A53T mutant is 56% of the wild-type&lt;/p&gt;

&lt;p&gt;&amp;bull;&amp;nbsp;Autophagy induction speeds up inclusion clearance in the A53T &amp;alpha;-synuclein strain&lt;/p&gt;

&lt;p&gt;&lt;strong&gt;Summary&lt;/strong&gt;&lt;/p&gt;

&lt;p&gt;Aggregation of&amp;nbsp;&amp;alpha;-synuclein&amp;nbsp;and formation of inclusions are hallmarks of Parkinson&amp;rsquo;s disease (PD). Aggregate formation is affected by cellular environment, but it has been studied almost exclusively in cell-free systems. We quantitatively analyzed &amp;alpha;-synuclein inclusion formation and clearance in a yeast cell model of PD expressing either&amp;nbsp;wild-type&amp;nbsp;(WT) &amp;alpha;-synuclein or the disease-associated A53T mutant from the&amp;nbsp;galactose&amp;nbsp;(Gal)-inducible promoter. A computer-controlled microfluidics device regulated &amp;alpha;-synuclein in cells by means of closed-loop feedback control. We demonstrated that inclusion formation is strictly concentration dependent and that the aggregation threshold of the A53T mutant is about half of the WT &amp;alpha;-synuclein (56%). We chemically modulated the proteasomal&amp;nbsp;and autophagic pathways and demonstrated that&amp;nbsp;autophagy&amp;nbsp;is the main determinant of A53T &amp;alpha;-synuclein inclusions&amp;rsquo; clearance. In addition to proposing a technology to overcome current limitations in dynamically regulating&amp;nbsp;protein expression&amp;nbsp;levels, our results contribute to the&amp;nbsp;biology&amp;nbsp;of PD and have relevance for therapeutic applications.&lt;/p&gt;</description>
  </descriptions>
  <fundingReferences>
    <fundingReference>
      <funderName>European Commission</funderName>
      <funderIdentifier funderIdentifierType="Crossref Funder ID">10.13039/501100000780</funderIdentifier>
      <awardNumber awardURI="info:eu-repo/grantAgreement/EC/H2020/766840/">766840</awardNumber>
      <awardTitle>Control Engineering of Biological Systems for Reliable Synthetic Biology Applications</awardTitle>
    </fundingReference>
  </fundingReferences>
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