Daniel Ysselstein

Effects of impaired membrane interactions on α-synuclein aggregation and neurotoxicity.

The post-mortem brains of individuals with Parkinsons disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the proteins aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the proteins neurotoxicity in PD and other synucleinopathy disorders.

Endosulfine-alpha inhibits membrane-induced α-synuclein aggregation and protects against α-synuclein neurotoxicity.

Neuropathological and genetic findings suggest that the presynaptic protein α-synuclein (aSyn) is involved in the pathogenesis of synucleinopathy disorders, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. Evidence suggests that the self-assembly of aSyn conformers bound to phospholipid membranes in an aggregation-prone state plays a key role in aSyn neurotoxicity. Accordingly, we hypothesized that protein binding partners of lipid-associated aSyn could inhibit the formation of toxic aSyn oligomers at membrane surfaces. To address this hypothesis, we characterized the protein endosulfine-alpha (ENSA), previously shown to interact selectively with membrane-bound aSyn, in terms of its effects on the membrane-induced aggregation and neurotoxicity of two familial aSyn mutants, A30P and G51D. We found that wild-type ENSA, but not the non-aSyn-binding S109E variant, interfered with membrane-induced aSyn self-assembly, aSyn-mediated vesicle disruption and aSyn neurotoxicity. Immunoblotting analyses revealed that ENSA was down-regulated in the brains of synucleinopathy patients versus non-diseased individuals. Collectively, these results suggest that ENSA can alleviate neurotoxic effects of membrane-bound aSyn via an apparent chaperone-like activity at the membrane surface, and a decrease in ENSA expression may contribute to aSyn neuropathology in synucleinopathy disorders. More generally, our findings suggest that promoting interactions between lipid-bound, amyloidogenic proteins and their binding partners is a viable strategy to alleviate cytotoxicity in a range of protein misfolding disorders.

Printed optics: phantoms for quantitative deep tissue fluorescence imaging

Three-dimensional (3D) printing allows for complex or physiologically realistic phantoms, useful, for example, in developing biomedical imaging methods and for calibrating measured data. However, available 3D printing materials provide a limited range of static optical properties. We overcome this limitation with a new method using stereolithography that allows tuning of the printed phantoms optical properties to match that of target tissues, accomplished by printing a mixture of polystyrene microspheres and clear photopolymer resin. We show that Mie theory can be used to design the optical properties, and demonstrate the method by fabricating a mouse phantom and imaging it using fluorescence optical diffusion tomography.

Effect of acidic pH on the stability of α-synuclein dimers.

Environmental factors, such as acidic pH, facilitate the assembly of α‐synuclein (α‐Syn) in aggregates, but the impact of pH on the very first step of α‐Syn aggregation remains elusive. Recently, we developed a single‐molecule approach that enabled us to measure directly the stability of α‐Syn dimers. Unlabeled α‐Syn monomers were immobilized on a substrate, and fluorophore‐labeled monomers were added to the solution to allow them to form dimers with immobilized α‐Syn monomers. The dimer lifetimes were measured directly from the fluorescence bursts on the time trajectories. Herein, we applied the single‐molecule tethered approach for probing of intermolecular interaction to characterize the effect of acidic pH on the lifetimes of α‐Syn dimers. The experiments were performed at pH 5 and 7 for wild‐type α−Syn and for two mutants containing familial type mutations E46K and A53T. We demonstrate that a decrease of pH resulted in more than threefold increase in the α‐Syn dimers lifetimes with some variability between the α‐Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96–140 of α‐Syn and this electrostatic effect facilitates the association of the two monomers. Given that dimerization is the first step of α‐Syn aggregation, we posit that the electrostatic effect thereby contributes to accelerating α‐Syn aggregation at acidic pH.

Printed optics: phantoms for quantitative deep tissue fluorescence imaging: publisher's note.

This note points out a number of corrections that were omitted from the published version of the article [Opt. Lett.41, 5230 (2016)OPLEDP0146-959210.1364/OL.41.005230].

Role of Aberrant α-Synuclein–Membrane Interactions in Parkinson’s Disease

A neuropathologic hallmark of Parkinson’s disease is the presence in post-mortem brains of Lewy body inclusions enriched with fibrillar forms of the presynaptic protein α-synuclein (α-syn). α-Syn is natively unfolded in aqueous solution but forms amyloid-like fibrils upon prolonged incubation in physiologic buffers, and it adopts an amphipathic α-helical structure when bound to phospholipid membranes. Although membrane association appears to be critical for the protein’s normal function in modulating neurotransmission, it can also stimulate the formation of potentially toxic α-syn oligomers, particularly when the protein binds the membrane via a partially formed α-helix that leaves the central hydrophobic domain exposed and available to interact with neighboring α-syn molecules. Evidence suggests that α-syn aggregation at the membrane surface results in disruption of the phospholipid bilayer. Aberrant α-syn-membrane interactions may contribute to various mechanisms of α-syn neurotoxicity, including impairment of ER-Golgi trafficking, mitochondrial dysfunction, and the spread of α-syn neuropathology via a prion-like mechanism.