E46K-like α-synuclein mutants increase lipid interactions and disrupt membrane selectivity

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.

Cell models of lipid-rich α-synuclein aggregation validate known modifiers of α-synuclein biology and identify stearoyl-CoA desaturase

“…Similar stepwise changes toward pathology-relevant phenotypes have been observed for 1K–3K with regard to solubility (decrease), native multimerization (decrease), and toxicity (increase) (24). These stepwise effects in cell culture, biophysical data (25), and findings from the αS 3K mouse model (18) (summarized in SI Appendix , Table S1) indicated to us that αS 3K models synucleinopathy-relevant phenotypes and might be exploited as a discovery model (followed by validation in fPD and wt αS excess models). To take advantage of the pronounced 3K::YFP inclusion phenotype while avoiding continuous toxicity, we generated doxycycline (dox)-inducible M17D neuroblastoma cells (M17D/αS-3K::YFP).…”

“…Multiple observations support the concept that αS 3K cells model excessive αS membrane binding. E46K alone exhibits stronger binding to vesicle membranes than does wt αS (16), and this is further amplified in 3K (E35K+E46K+E61K) (25). Accordingly, αS 3K inclusions that are membrane/lipid-rich (17) (Fig.…”

<p>Phosphorylation of Tau and α-Synuclein Induced Neurodegeneration in MPTP Mouse Model of Parkinson’s Disease</p>

“…A-Synuclein is mostly located in presynaptic terminals that are involved in maintaining synaptic vesicle trafficking homeostasis. 19,28 The upregulation, posttranslational modification such as sumoylation and phosphorylation, and polymerization of α-Synuclein is thought to the hallmarks of PD. 20,42 The fibril form of α-Synuclein in the Lewy body is the most toxic in central nervous system.…”

The Role of Lipids in the Initiation of α-Synuclein Misfolding

“…This mutation disrupts an N-terminal KTKEGV motif to KTKKGV and drastically destabilizes physiological aggregation-resistant multimers of α-syn, leading to enhanced levels of aggregation-prone monomeric α-syn ( Dettmer et al, 2015 ). In addition, a recent report argues for a loss of α-syn’s membrane curvature-sensing ability due to E46K mutation, thereby misdirecting α-syn to non-physiological lipid interactants ( Rovere et al, 2019 ). By introducing two additional E > K mutations in other KTKEGV motifs (E35K and E61K, termed “3K” with the E46K mutation), this effect was dose-dependently amplified, resulting in increased monomeric conformations of α-syn and induction of neurotoxic inclusions.…”