Accumulation of α-synuclein into toxic oligomers or fibrils is implicated in dopaminergic neurodegeneration in Parkinson’s disease. Here we performed a high-throughput, proteome-wide peptide screen to identify protein-protein interaction inhibitors that reduce α-synuclein oligomer levels and their associated cytotoxicity. We find that the most potent peptide inhibitor disrupts the direct interaction between the C-terminal region of α-synuclein and CHarged Multivesicular body Protein 2B (CHMP2B), a component of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III). We show that α-synuclein impedes endolysosomal activity via this interaction, thereby inhibiting its own degradation. Conversely, the peptide inhibitor restores endolysosomal function and thereby decreases α-synuclein levels in multiple models, including female and male human cells harboring disease-causing α-synuclein mutations. Furthermore, the peptide inhibitor protects dopaminergic neurons from α-synuclein-mediated degeneration in hermaphroditic C. elegans and preclinical Parkinson’s disease models using female rats. Thus, the α-synuclein-CHMP2B interaction is a potential therapeutic target for neurodegenerative disorders.
Protein-protein interactions (PPIs) govern virtually all molecular pathways involved in cell growth, differentiation, and survival1,2. Inhibition of PPIs with peptides or small molecules to modulate these pathways has proven to be successful for the treatment of cancers3,4. PPI inhibitors could conceivably be a promising new therapeutic venue for neurodegenerative disorders, such as Parkinson’s disease (PD), for which no disease-modifying therapies exist5.
Wild-type (WT) or mutant α-synuclein protein (a-syn) accumulates in PD to form oligomers that disrupt core cellular systems causing neurodegeneration6. Rescuing these toxic effects by targeting PPIs has been an unexplored therapeutic strategy for PD7,8. However, development of PPI inhibitors that reduce a-syn accumulation will be excessively prolonged if we rely on current strategies based on targeting individual interactions revealed solely by serendipitous discoveries9.
To expedite the discovery of potential peptide therapies, we recently developed a screening system to find candidate peptides that perturb endogenous PPIs. We identified putative PPI inhibitors using computational methods based on PPI motifs and constructed lentiviral libraries of these peptide inhibitors. These libraries contain short linear interaction motifs that mediate a large fraction (~30%) of human PPIs10,11,12.
In this work, we use this unbiased and proteome-wide approach to identify PPI inhibitors that reduce a-syn-mediated neurodegeneration. We discover a peptide inhibitor that disrupts the direct interaction between the C-terminal region of a-syn and CHarged Multivesicular body Protein 2B (CHMP2B). This interaction impairs endolysosomal activity resulting in a-syn accumulation. We demonstrate that the peptide inhibitor restores endolysosomal function and thereby reduces a-syn levels, rescuing dopaminergic neurons from degeneration. Based on our findings, we conclude that the a-syn-CHMP2B interaction is a potential therapeutic target for PD and other neurodegenerative conditions.
Proteomic screen identifies peptides that rescue a-syn cytotoxicity and reduce a-syn oligomers
We used a lentiviral library of 50,549 7-mer peptides enriched in PPI motifs13 on a green fluorescent protein (GFP) scaffold (Fig. 1A). We infected cells in a pooled format and the amino acid sequence of each peptide served as its own barcode. By using a low multiplicity of infection followed by puromycin selection, each infected cell expressed one unique GFP-peptide. We screened for peptides that reduced a-syn-mediated cytotoxicity or a-syn oligomers in parallel (Fig. 1A). To cause cytotoxicity relevant to PD, we induced proteostatic stress in HEK293 cells by inhibiting the proteasome with MG132 and overexpressing human WT a-syn associated with idiopathic PD in one screen or the more toxic mutant A53T a-syn associated with familial PD in a second screen. This combination of a-syn overexpression and pharmacological proteasome inhibition led to death of most cells, with only those that expressed a protective peptide surviving. By extracting genomic DNA from the surviving cells and amplifying the peptide coding sequences, we identified the protective peptides (Fig. 1A). To detect a-syn oligomers, we used a protein fragment complementation assay (PCA) of a-syn oligomerization in HEK293 cells14. In this assay, human A53T a-syn was fused to either the C- or N-terminal half of yellow fluorescent protein (YFP). No fluorescence is detectable while a-syn exists in monomers but, when a-syn oligomerizes, the two halves of YFP come in close enough proximity to form a functional, spontaneously fluorescent protein. This fluorescence has previously been shown to approximate levels of toxic oligomeric conformations of a-syn in cell and rodent models15,16,17,18. By using fluorescence-activated cell sorting (FACS), we isolated cells expressing Flag-tagged peptides that directly or indirectly interfered with a-syn oligomer formation and then identified the peptides from genomic DNA (Fig. 1A). Our screens yielded 10 hits (Table S1). The hit with the most reads (i.e., the most abundant) was the same peptide in all screens: IPIQLKA (named PDpep1) (Table S1). We validated the cytoprotective effects of PDpep1 in HEK293 cells overexpressing WT or A53T a-syn (Figs. 1B, S1A), as well as its ability to reduce WT or A53T a-syn oligomers in a luciferase-based PCA18,19,20 compared to a scrambled control peptide (Figs. 1C, S1B)….