A Downsized and Optimised Intracellular Library-Derived Peptide Prevents Alpha-Synuclein Primary Nucleation and Toxicity Without Impacting Upon Lipid Binding

Abstract

Misfolding and aggregation of alpha-synuclein (αS) within dopaminergic neurons is a key factor in the development and progression of a group of age-related neurodegenerative diseases, termed synucleinopathies, that include Parkinson’s disease (PD). We previously derived a peptide inhibitor from a 209,952-member intracellular library screen by employing the preNAC region (45–54) as a design template. At least six single-point mutations firmly linked to early-onset Parkinson’s disease (E46K, H50Q, G51D, A53T/E/V) are located within this region, strongly implicating a pathogenic role within αS that leads to increased cytotoxicity. A library-derived ten residue peptide, 4554W, was consequently shown to block αS aggregation at the point of primary nucleation via lipid induction, inhibiting its conversion into downstream cytotoxic species. Here we couple truncation with a full alanine scan analysis, to establish the effect upon the αS aggregation pathway relative to 4554W. This revealed the precise residues responsible for eliciting inhibitory interaction and function, as well as those potentially amenable to modification or functionalisation. We find that modification N6A combined with N-terminal truncation results in a peptide of significantly increased efficacy. Importantly, our data demonstrate that the peptide does not directly disrupt αS lipid-binding, a desirable trait since antagonists of αS aggregation and toxicity should not impede association with small synaptic neurotransmitter vesicles, and thus not disrupt dopaminergic vesicle fusion and recycling. This work paves the way toward the major aim of deriving a highly potent peptide antagonist of αS pathogenicity without impacting on native αS function.

Introduction

The misfolding and aggregation of alpha-synuclein (αS), a 140-residue membrane associated neuronal protein, is believed to be the leading cause of several neurodegenerative diseases, referred to collectively as synucleinopathies. This includes Parkinson’s disease (PD), multiple system atrophy (MSA), and Dementia with Lewy Bodies (DLB), accounting for approximately 15% of all known dementia cases.¹ Although understanding of the native function of αS is incomplete, it is believed to be involved in regulating the release of synaptic vesicles, via direct interaction with lipid membranes.²

Previous work to target and detoxify aggregation induced cytotoxicity of αS employed an intracellular Protein-fragment Complementation Assay (PCA).³ A 209,952 member library (Figure 1(a) and (b)), based on preNAC residues 45–54, was screened and resulted in the selection and development of 4554W,⁴ a peptide which has been shown to function by inhibiting the lipid induced primary nucleation step of the αS aggregation pathway.⁵ The known mutations at the point of library design, (E46K, H50Q and A53T/E), along with conserved options displaying similar properties to those within the scaffold region, were included in the library design (Figure 1(a) and (b)). Moreover, the peptide was found to be capable of rescuing αS mediated cytotoxicity in PC-12 cells,⁴ as well as SH-SY5Y human neuroblastoma cells.⁵ The 45–54 region of αS is a particularly compelling antagonist design template (Figure 1(a)), and was chosen since it contains most known early onset familial PD mutations (E46K,6, 7, 8 H50Q,9, 10, 11, 12 G51D,9, 13 A53T,¹⁴ A53E,15, 16 A53V),¹⁷ demonstrating its importance in modulating both intra- and inter-molecular protein–protein interactions (PPIs) that subsequently lead to the accelerated development of αS aggregates, and ultimately PD progression (Figure 2).¹⁸

PreNAC region 45–54 is located within a hydrophobic zipper interface found within aggregated conformations of the protein.19, 20 It forms both intra- and inter-molecular interactions, stabilising two ‘Greek key’ motifs and the interactions between them. These motifs display rotational symmetry about the axis of the majority of currently identified mature αS fibril types,18, 21 highlighting 45–54 as critical in populating αS aggregates within the free energy landscape (Figure 2). Furthermore, new research from ssNMR studies suggest that this area of the protein may also facilitate the conversion of αS from an intrinsically disordered protein (IDP) / helical signature towards these mature β-sheet rich conformations, triggered directly by interaction with the surface of lipid membranes.²² During this process exposure of intermediates that are on-pathway to aggregation could therefore potentially be targeted to mitigate against aggregation into toxic species.

The previously described 4554W peptide4, 5 was shown to function by inhibiting primary nucleation of αS into fibril like structures in the presence of small unilamellar vesicles (SUVs) (Figure 3) composed of the anionic lipid 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), which is able to interact at least in part via positively charged Lys residues within αS.5, 18, 24 The role of DMPS is not fully understood, but levels of phosphatidylserine have been found to increase by ∼36%, in patient brains that display Lewy body pathology but that are devoid of motor symptoms.²⁵ Moreover, DMPS has been suggested to play a role in the regulation of αS-facilitated synaptic vesicle docking, and is linked to SNARE complex formation.26, 27 Therefore, the use of lipid vesicles comprised of DMPS has proven to be a useful tool in probing the effectiveness of molecules able to inhibit primary nucleation.28, 29, 30, 31, 32

Here we combine N/C-terminal deletion constructs with a full alanine scan analysis of the 4554W sequence (Figure 1(d) and (e)), followed by an inspection of the principal steps within the aggregation pathway. We elucidate precisely which residues within 4554W interact with αS, and which are subsequently required to inhibit conversion into toxic conformations. For each construct we sought to establish if efficacy is maintained via modulation at the same point of aggregation as the 4554W parental sequence, or if functional activity was fundamentally altered or lost. Understanding the contribution made by each side chain to the aggregation pathway is key in facilitating the design of more potent peptides, with increased αS affinity and efficacy, as well as the possibility for more drug-like peptidomimetic sequences via appropriate substitutions, or the potential to functionalise groups that are tolerant to change without impacting upon activity with αS….