Abstract
Parkinson's disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn's interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous Glu-to-Lys substitutions in αSyn's N-terminal region (i.e. E35K and E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA–responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other “synucleinopathies.” In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants' avidity for synaptic vesicle–like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn's curvature selectivity. Indiscriminate binding can shift αSyn's subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies.
Highlights
Parkinson’s disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein ␣-synuclein (␣Syn) in its pathogenesis
Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA–responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other “synucleinopathies.” In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like ␣Syn mutants by studying their structure and membrane-binding and remodeling abilities
Parkinson’s disease (PD)3 is a progressive neurodegenerative disorder characterized in its early stages by resting tremor, bradykinesia, and rigidity because of loss of dopaminergic neurons in the substantia nigra [1]
Summary
To characterize whether the Glu-to-Lys mutations induced any change in the structure of the lipid-unbound protein, CD spectra of the recombinant mutants in buffer were measured. CD-monitored lipid-into-protein titrations can provide accurate estimates of the number of lipids per binding site (N) and macroscopic structural information regarding the coil-to-helix transition, their estimates of the apparent microscopic binding constants (kB) are poor For this reason, and to confirm the previous results by means of an independent technique, we measured the binding curves of WT ␣Syn and its Glu-to-Lys mutants to SUVs by isothermal titration calorimetry (ITC). The effects of Glu-to-Lys mutations are generally mild in the SUV titrations (possibly because of the overall high affinities), robust changes are evident in the LUV titrations, especially after curve fitting with our N-independent binding site model (Fig. 6, C and D). Ing in a greater number of bound ␣Syn molecules and more profound bilayer remodeling
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