Exploration of a liquid droplet state for α-synuclein in vitro and in vivo

A wide range of human disorders, including Parkinson’s disease (PD), are associated with protein misfolding and aggregation. Misfolded a-synuclein is a major constituent of intracellular inclusions called Lewy bodies, which are a key hallmark of PD. A large body of work shows that a-synuclein can self-assemble into amyloid fibrils via a nucleation dependent pathway. However, the relationship between a-synuclein self-assembly and inclusion formation remains unclear. Recently, it has become apparent that many proteins can phase separate into dense droplets with liquid properties. Aberrant phase separation facilitates the formation of amyloid and intracellular inclusions. As such, we proposed that phase separation by a-synuclein into a liquid droplet state is a critical step in the formation of PD relevant inclusions. In this work, we show that a-synuclein undergoes liquid-liquid phase separation by forming a liquid droplet state, both in vitro and in a Caenorhabditis elegans model of PD, which converts into an amyloid-rich hydrogel. Importantly, this maturation process towards the amyloid state is modulated in the presence of model synaptic vesicles in vitro. As the maturation of a-synuclein droplets can be at least in some cases pathological, we investigated whether a-synuclein phase separation could be pharmacologically modulated. By establishing a robust screening platform, we identified modulators for the maturation of a-synuclein droplets into amyloid. To establish the generality of our observations for a-synuclein droplet formation, we explored phase separation as a proteome-wide concept and developed the FuzDrop method to predict droplet-promoting regions. This method is based on the concept that the droplet state is stabilised by the large conformational entropy associated with non-specific side-chain interactions. Our results indicate that the droplet state could be, at least transiently, accessible to most proteins under conditions found in the cellular environment.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Hardenberg, Maarten

Advisor dc:contributor.advisor

• Vendruscolo, Michele

• protein misfolding
• protein aggregation
• Parkinson's disease
• alpha-synuclein
• Phase separation
• C. elegans

Licence

• https://www.repository.cam.ac.uk/bitstreams/daef7f6c-3772-4df6-8069-6eaa5bd7c512/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.66357

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/319237