Yeast reveal a "druggable" Rsp5/Nedd4 network that ameliorates α-synuclein toxicity in neurons

Abstract

α-Synuclein (α-syn) is a small lipid-binding protein implicated in several neurodegenerative diseases, including Parkinson's disease, whose pathobiology is conserved from yeast to man. There are no therapies targeting these underlying cellular pathologies, or indeed those of any major neurodegenerative disease. Using unbiased phenotypic screens as an alternative to target-based approaches, we discovered an N-aryl benzimidazole (NAB) that strongly and selectively protected diverse cell types from α-syn toxicity. Three chemical genetic screens in wild-type yeast cells established that NAB promoted endosomal transport events dependent on the E3 ubiquitin ligase Rsp5/Nedd4. These same steps were perturbed by α-syn itself. Thus, NAB identifies a druggable node in the biology of α-syn that can correct multiple aspects of its underlying pathology, including dysfunctional endosomal and endoplasmic reticulum-to-Golgi vesicle trafficking.

Fig. 1. NAB protects yeast and neurons from α-syn toxicity

(A) NAB structure. (B) Dose-response curves for NAB in yeast proteinopathy models. (C) α–syn-GFP localization in NoTox, HiTox, and HiTox/10 μM NAB. (D) Percent of ROS-positive cells under same conditions as (C). Values are mean +/− SD, N = 3. (E) Immunoblot of Cpy showing ER-Golgi trafficking defect and α-syn protein levels in WT and HiTox cells treated with NAB. (F) Fluorescence microscopy of the six anterior DA neurons in representative C. elegans expressing either GFP or human α–syn and GFP after treatment with DMSO or NAB. Arrows indicate intact DA neuron cell bodies and arrowheads indicate regions where these cells have degenerated. Inlaid values reflect mean +/− SD, N = 3. (G) Representative images of DA-enriched cultures established from embryonic rat midbrains. ‘Control’, untransduced. ‘A53T’, transduced with A53T α-syn virus. Red, MAP2 (neuronal tubulin). Green, TH-positive neurons. Inlaid values reflect mean with control set to 100% and +/− SD, N = 3 for % TH-positive neurons and mean +/− SEM, N = ~75 neurons for neurite length. For all data: *, P<0.05. **, P<0.01. ***, P<0.001 using one-way ANOVA and a Tukey’s test.

Fig. 2. Chemical genetic screens of NAB2 reveal a network centered on the E3 ligase, Rsp5

(A) Efficacy (EC₄₀) in α-syn cells versus growth inhibition (IC₄₀) in WT cells for active analogs. NAB1 is the screen hit and NAB2 the most potent analog. (B) Viable cells recovered after prolonged NAB2 treatment. (C) NAB2 interaction network. Node color reflects screen of origin indicated below. Edges are interactions (see legend) according to String database and literature. VPS23 was deleted after identification of other hits. (D) Heat map of RSP5 variant cell growth in response to increasing NAB2 compared to untreated cells. Mutants include rsp5_G747E and the hypomorphic allele, Δp.rsp5. (E) (Top) Methionine- and Rsp5-dependent Mup1-GFP endocytosis. (Bottom) Mup1-GFP localization in WT and rsp5_G747E strains under indicated conditions. (F) (Left) Schematic of Sna3-GFP endosomal trafficking to the vacuole, where GFP is cleaved. (Right) Immunoblot analysis of Sna3-GFP in WT and rsp5_G747E cells treated with NAB2.

Fig. 3. NAB/Rsp5 network directly impacts rescue of α–syn toxicity

(A) Heat map of NAB2 dose-response in WT and modified α–syn strains. Rescue is relative to EC₁₀₀ for NAB2 in WT α–syn cells. (B) α-syn-GFP localization in WT or rsp5_G747E α-syn cells under indicated conditions. Inlaid values indicate “% of cells with large α–syn foci” (mean +/− SD, N = 3). (C) Immunoblot of Cpy trafficking defect α–syn cells with DMSO or NAB2. *,P<0.05 using one-way ANOVA. (D) Interaction network of α-syn and NAB2 genetic modifiers. α-syn nodes, purple; NAB2 nodes are color coded according to screen of origin (Fig. 2C). RSP5, UBP7, and UBP11 are larger because they both suppress NAB2 growth inhibition and enhance α–syn toxicity (4). Edges between nodes depict physical or genetic interactions. Thicker lines indicate both genetic and physical interactions. Red edges link node interactions between α–syn and NAB2 networks. Remaining α-syn edges are blue.

Fig. 4. NAB2 directly antagonizes α–syn-induced endosomal defects

(A) Methionine-stimulated Mup1-GFP endocytosis in WT or untagged α–syn strains with DMSO or NAB2. Pulse-labeling cells with FM4-64 during the first hour of α–syn expression marked the vacuole. (B) Effects of α-syn on Sna3-GFP localization. Immunoblot shows Sna3-GFP cleavage in response to α–syn and NAB. FM4-64 labeling as in (B). (C) Pulse-labeling of FM4-64 of α-syn cells after 4 hours of expression in the presence or absence of NAB2. (D) Schematic of NAB2 mechanism in antagonizing core and secondary α–syn pathologies.