Abstract
Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of protein aggregates (namely Lewy bodies) in dopaminergic neurons in the substantia nigra region of the brain. Alpha-synuclein (α-syn) is the major component of Lewy bodies in PD patients, and impairment of the ubiquitin-proteasome system has been linked to its accumulation. In this work, we developed a tetracycline–inducible expression system, with simultaneous induced expression of α-syn-EGFP and a bright red fluorescent protein marker (mCherry) to screen for potential compounds for degrading α-syn. We identified canthin-6-one as an α-syn lowering compound which promoted both wild type and mutants α-syn degradation in an ubiquitin-proteasome-system (UPS) dependent manner. By CRISPR/Cas9 genome-wide screening technology, we identified RPN2/PSMD1, the 26S proteasome non-ATPase regulatory subunit 1, as the targeting gene for pharmacological activity of canthin-6-one. Finally, we showed that canthin-6-one up-regulates PSMD1 and enhances UPS function by activating PKA.
Highlights
Many neurodegenerative diseases share a common pathogenic mechanism: the aggregation of misfolded proteins lead to cellular toxicity and proteostatic collapse
Canthin-6-One Was Identified as an α-syn Lowering Compound by Using Tet-on 3G α-syn-EGFP/mCherry Dual Fluorescence System
To identify compounds which can degrade α-syn levels in human cells, we engineered a plasmid encoding human α-syn fused with monomeric green fluorescent protein (α-synEGFP)
Summary
Many neurodegenerative diseases share a common pathogenic mechanism: the aggregation of misfolded proteins lead to cellular toxicity and proteostatic collapse. We established a tetracycline–inducible expression system, with a bidirectional tet-responsive promoter that binds the TetOn 3G transactivator protein in the presence of doxycycline, allowing simultaneous induced expression of α-syn-EGFP and a bright red fluorescent protein marker (mCherry) as the reference. In this system, any compound that selectively affects α-syn stability would change the ratio of EGFP/mCherry, which can be monitored by microplate reader efficiently. By using ALP and UPS inhibitor, we confirmed that canthin-6-one induced α-syn degradation dependent of UPS function
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