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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    <subfield code="a">Feedback control</subfield>
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    <subfield code="u">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</subfield>
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    <subfield code="a">Quantitative Characterization of α-Synuclein Aggregation in Living Cells through Automated Microfluidics Feedback Control</subfield>
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    <subfield code="a">&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;</subfield>
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    <subfield code="a">10.1016/j.celrep.2019.03.081</subfield>
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