Dataset Information

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

ABSTRACT: Aggregation of ?-synuclein and formation of inclusions are hallmarks of Parkinson's disease (PD). Aggregate formation is affected by cellular environment, but it has been studied almost exclusively in cell-free systems. We quantitatively analyzed ?-synuclein inclusion formation and clearance in a yeast cell model of PD expressing either wild-type (WT) ?-synuclein or the disease-associated A53T mutant from the galactose (Gal)-inducible promoter. A computer-controlled microfluidics device regulated ?-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 ?-synuclein (56%). We chemically modulated the proteasomal and autophagic pathways and demonstrated that autophagy is the main determinant of A53T ?-synuclein inclusions' clearance. In addition to proposing a technology to overcome current limitations in dynamically regulating protein expression levels, our results contribute to the biology of PD and have relevance for therapeutic applications.

SUBMITTER: Perrino G

PROVIDER: S-EPMC6484782 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Perrino Giansimone G Wilson Cathal C Santorelli Marco M di Bernardo Diego D

Cell reports 20190401 3

Aggregation of α-synuclein and formation of inclusions are hallmarks of Parkinson's disease (PD). Aggregate formation is affected by cellular environment, but it has been studied almost exclusively in cell-free systems. We quantitatively analyzed α-synuclein inclusion formation and clearance in a yeast cell model of PD expressing either wild-type (WT) α-synuclein or the disease-associated A53T mutant from the galactose (Gal)-inducible promoter. A computer-controlled microfluidics device regulate ...[more]

PMID: 30995486

Similar Datasets

Project description:Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). Previous studies have demonstrated that METH causes alpha-synuclein (?-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of ?-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of ?-syn is involved in high-dose METH-induced ?-syn aggregation. We measured the levels of ?-syn SUMOylation and these enzymes involved in the SUMOylation cycle in SH-SY5Y human neuroblastoma cells (SH-SY5Y cells), in cultures of C57 BL/6 primary mouse neurons and in brain tissues of mice exposure to METH. We also demonstrated the effect of ?-syn SUMOylation on ?-syn aggregation after METH exposure by overexpressing the key enzyme of the SUMOylation cycle or silencing SUMO-1 expression in vitro. Then, we make introduced mutations in the major SUMOylation acceptor sites of ?-syn by transfecting a lentivirus containing the sequence of WT ?-syn or K96/102R ?-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT ?-syn or K96/102R ?-syn into the mouse striatum. Levels of the ubiquitin-proteasome system (UPS)-related makers ubiquitin (Ub) and UbE1, as well as the autophagy-lysosome pathway (ALP)-related markers LC3, P62 and lysosomal associated membrane protein 2A (LAMP2A), were also measured in SH-SY5Y cells transfected with lentivirus and mice injected with adenovirus. The results showed that METH exposure decreases the SUMOylation level of ?-syn, although the expression of ?-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in ?-syn SUMOylation by UBC9 overexpression relieves METH-induced ?-syn overexpression and aggregation, whereas the decrease in ?-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of ?-syn also aggravate ?-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of ?-syn plays a fundamental part in ?-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure