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α-synuclein

The protein α-synuclein (αSyn) is crucially involved in Parkinson’s Disease (PD). αSyn aggregation has been linked to early-onset familial forms of PD as well as to the sporadic late-onset forms. Normally, αSyn is a soluble, structurally disorganised protein, found in throughout the brain including in neuronal presynapses. In disease, however, αSyn becomes aggregated and forms highly structured insoluble fibrils that make up Lewy Bodies in patient brains. Although αSyn’s importance in PD has been recognised 20 years ago, the aetiology still remains largely unknown. We are therefore investigating both the normal physiological role of αSyn and the mechanisms that lead to aggregation and disease.

asyn_VesicleBindingCalcium-dependent role of α-synuclein at the pre-synapse. αSyn has been proposed to play a role during exocytosis, endocytosis and the homoeostasis of synaptic vesicles in healthy cells (Lautenschläger et al., 2017). We use super-resolution microscopy and biophysical assays to investigate the interaction between αSyn and synaptic vesicles (Fusco et al., 2016) along with Dr. Alfonso De Simone at Imperial College London using nuclear magnetic resonance (NMR) spectroscopy.

asyn_AFMStructure function relationship of α-synuclein. The structural biology of αSyn will be crucial to understanding the initiation of aggregation. We use atomic force microscopy (AFM) and super resolution microscopy, as well as hydrogen-deuterium exchange mass spectrometry (HDX-MS) in collaboration with Prof. Clemens Kaminski and with Dr. Jonathan Phillips at the University of Exeter, to investigate the structure and aggregation behaviour of aSyn and its mutants (A30P, A53T, E46K, H50Q, G51D).

Prion-like propagation of α-synuclein. αSyn aggregates can propagate their misfolding to normal ‘healthy’ αSyn molecules. We have previously shown, using two colour superresolution microscopy, that αSyn forms different polymorphs (Pinotsi et al., 2014) and that recombinant αSyn can seed endogenous murine αSyn and thereby rescues monomeric αSyn-induced toxicity (Pinotsi et al., 2016). We are currently studying the seeding ability of αSyn in our well-established model system using recombinant human αSyn added to murine αSyn. This allows us to test intervention strategies that may be useful approaches to slow the development of PD. 

asyn_SynaptosomesFactors contributing to PD pathology. In addition to biochemical and biophysical techniques, we also study αSyn aggregation in a cellular model system. Mitochondria, the power stations of the cell, are well known to contribute in PD pathology. They supply ATP for cellular processes, but are also involved in cellular calcium buffering and the regulation of oxidative stress. We are therefore examining whether and how this connects to αSyn aggregation using cellular ATP and calcium imaging, as well as our fluorescence lifetime-based αSyn aggregation sensor (Kaminski Schierle et al., 2011).

Deep mechanistic insights will help us to understand what conveys PD pathology and help target new therapeutic approaches. 

Technologies

Protein purification on FPLC and HPLC

Vesicle purification protocols

Plate reader based aggregation assays

Super-resolution microscopy

P1 Dopaminergic neurons

SH-SY5Y cells

Fluorescence Lifetime Imaging (FLIM)

Link to Gabriele Kaminski Schierle Lab Plasmids