Alpha Synuclein only Forms Fibrils In Vitro when Larger than its Critical Size of 70 Monomers.

Santiago Enrique Sanchez,Daniel R. Whiten,Eric Hidari,Francesco Simone Ruggeri,Georg Meisl,David Klenerman

doi:10.1002/cbic.202100285

Santiago Enrique Sanchez, Daniel R. Whiten + Show 4 more

Open Access

https://doi.org/10.1002/cbic.202100285

Copy DOI

Abstract

The aggregation of α‐synuclein into small soluble aggregates and then fibrils is important in the development and spreading of aggregates through the brain in Parkinson's disease. Fibrillar aggregates can grow by monomer addition and then break into fragments that could spread into neighboring cells. The rate constants for fibril elongation and fragmentation have been measured but it is not known how large an aggregate needs to be before fibril formation is thermodynamically favorable. This critical size is an important parameter controlling at what stage in an aggregation reaction fibrils can form and replicate. We determined this value to be approximately 70 monomers using super‐resolution and atomic force microscopy imaging of individual α‐synuclein aggregates formed in solution over long time periods. This represents the minimum size for a stable α‐synuclein fibril and we hypothesis the formation of aggregates of this size in a cell represents a tipping point at which rapid replication occurs.

Highlights

A body of data has shown how intrinsically disordered proteins such as beta amyloid, tau and α-synuclein aggregate in the test-tube and the rate constants for the key molecular steps have recently been determined.[1,2,3,4,5,6] α-Synuclein monomers have been shown to rapidly form small globular proteinase-Ksensitive aggregates by monomer addition which undergo a slow structural conversion to fibrillar proteinase-K-resistant aggregates, that can rapidly grow into fibrils by further addition of monomers.[1,6] Experiments over longer times have shown that these fibrils can fragment into smaller aggregates and replicate in the test-tube5
Aggregates were imaged with Aptamer-DNA Point Accumulation for Imaging in Nanoscale Topography (AD-PAINT) using a total internal reflection fluorescence (TIRF) microscope, as has been previously published.[12]
These super-resolution images make it possible to distinguish morphological differences between the highly linear α-synuclein fibrils and the comparatively more abundant, smaller, globular species which make up the vast majority of aggregates formed near physiological concentrations of αsynuclein (1 μM) (Figure 1a)

Summary

Introduction

It can be shown that quite generally this replication rate is determined by the product of the elongation and multiplication rates.[8] Through measurements of the aggregation of purified α-synuclein, the rate constants for elongation and fragmentation of fibrillar aggregates of α-synuclein have recently been determined.[4] it is not known how large an aggregate needs to be before conversion from a pre-fibrillar to intermediate and mature cross-β fibrillar forms is favorable. This quantity is important since it defines the minimum size of a replicationcompetent fibrillar aggregate. We use superresolution fluorescence microscopy and atomic force microscopy to directly measure the size distributions of α-synuclein aggregates formed in vitro and determine the critical size at which fibrillar aggregates are formed

Results and Discussion

Conclusion

Conflict of Interest