Master Projects

Effect of fluorescent labeling on heterologously seeded alpha-synuclein aggregation

Effect of fluorescent labeling on heterologously seeded alpha-synuclein aggregation

MASTER project   (Published Januari 2019)

Introduction

Parkinsons disease (PD), the second most frequently occurring neurodegenerative disease in the world, is mainly characterized by the loss of dopaminergic neurons and the presence of insoluble proteic complexes, so-called Lewy bodies (LB), in the brain [1]. LB are largely composed of fibrillary aggregates (amyloid) of alpha-synuclein (aSyn) protein, which suggests that aSyn plays an important role in the pathogenesis of PD. In addition, aSyn gene mutations and duplication/triplication are associated with hereditary forms of PD [2].

The aSyn protein is small (14 kDa), intrinsically disordered, and in vivo mainly expressed in dopaminergic neurons in the brain [3]. In vitro aggregation experiments often make use of recombinantly produced aSyn.  In test tubes aSyn aggregates into amyloid fibrils under a variety of environmental conditions (pH, salt, temperature, ..). Aggregation reactions can either be performed from only monomers in solution (unseeded) or in the presence of preformed fibril fragments (seeded) [4]. Two types of seeded aggregations reactions are possible: homologously seeded aggregations (proteins of seeds have same sequence and structure as the monomers) and heterologously seeded aggregations (proteins of seeds have a different sequence and/or structure than the monomers) (Figure 2). 


Figure 2: Different types of amyloid aggregation

In heterologously seeded aggregations it is unclear what allows the seeds to transfer the amyloid fold. Considering the co-occurrence of different neurodegenerative protein aggregation diseases it is very interesting to study the ability of one amyloid protein to induce the aggregation of other amyloid proteins (so-called cross-seeding). Examples of cross-seeding include transfer of the amyloid fold between  aSyn mutants involved in hereditary forms of PD and wild-type aSyn [5] and cross-seeding of an aSyn polymorph and the tau protein involved in Alzheimer’s disease [6].

In unraveling the mechanism of heterologous cross-seeding tracing down the localization of the initial seeds inside the formed fibrils is important . To determine the localization of the seeds fluorescently labelled monomers can be used to prepare seeds for cross-seeding another, differently labeled amyloid protein. Using a fluorescence microscope the seeds can be distinguished from the fibril extensions based on the wavelength of the fluorescence[5]. The result of this procedure may however  be affected by the induction of a different seed morphology (= polymorphism) by using labeled monomers in preparation of the seeds.

 

Description of the project:

The aim of this project is to study the effect of labeling on the aSyn aggregation. For this project, two part will be investigated:

  1. The aggregation kinetics (unseeded, homologously seeded and heterologously seeded) and fibril morphology (using Atomic Force Microscopy) will be compared for unlabeled and labeled aSyn (wild-type and mutants). The results will indicate if fluorescently labeled monomers affect aggregation reactions.
  2. An assay will be developed to specifically label the seeds part of heterologously seeded fibrils after finishing the aggregation reaction. In this way, a possible effect of the fluorescent label on the seed morphology is avoided.

 

Techniques involved:

  • In vitro aSyn aggregation reactions
  • Thioflavin T fluorescence measurements
  • Turbidity measurements
  • Protein labeling
  • Atomic force microscopy
  • Fluorescence microscopy

 

Contact:

Department: NanoBioPhysics (NBP) – UTwente - https://www.utwente.nl/en/tnw/nbp/

Daily supervisor: MSc. Jonathan Vaneyck (PhD student) - j.vaneyck@utwente.nl, Dr. G.M.J. Segers-Nolten (Researcher) - g.m.j.segers-nolten@utwente.nl

Project leader: Prof.Dr. M.M.A.E. Claessens (Chair Nanobiophysics) - m.m.a.e.claessens@utwente.nl