Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events

Abstract

There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP-43, FUS, and α-synuclein proteotoxicity by mutating single residues in helix 1, 2, or 3 of the middle domain or the small domain of nucleotide-binding domain 1. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.

Figure 1. Hsp104 MD variants rescue diverse proteotoxicity models

(A) Homology model of the MD and a portion of the small domain of NBD1 of Hsp104. Side chains of key residues are shown as sticks. (B) Δhsp104 yeast strains integrated with galactose-inducible α-syn, FUS, or TDP-43 were transformed with the indicated Hsp104 variant or vector control. Strains were serially diluted fivefold and spotted on glucose (off) or galactose (on) media. (C) Hsp104 variants harboring missense mutations to valine ranging from residue D498 to Y507 were expressed with FUS, TDP-43, or α-syn. (D) Close-up of MD helix 3 from (A). Mutation of D498, A503, D504, or Y507 activates Hsp104. See also Fig. S1.

Figure 2. Hsp104_A503X variants suppress TDP-43 toxicity, aggregation, and mislocalization

(A) Δhsp104 yeast transformed with TDP-43 and Hsp104 variants, or YFP and vector, were serially diluted fivefold and spotted onto glucose (off) or galactose (on). (B) Selected strains from (A) were induced in liquid and growth was monitored by A_600nm. (C) Strains from (B) were induced for 5h, lysed, and immunoblotted. Uninduced (untreated) and heat shocked cells (HS) serve as controls. 3-Phosphoglycerate kinase (PGK1) serves as a loading control. (D) WT, Δire1, or Δatg8 yeast were co-transformed with vector control, or TDP-43 plus vector, or the indicated Hsp104 variant and were serially diluted fivefold and spotted onto glucose (off) or galactose (on). (E) Fluorescence microscopy of cells co-expressing fluorescently tagged TDP-43 and Hsp104^WT, Hsp104^A503V, or vector. Cells were stained with DAPI to visualize nuclei (blue). TDP-43 localization was quantified by counting the number of cells containing colocalized nuclear staining. Values represent means±SEM (n=3). (F) Δhsp104 yeast co-transformed with TDP-43 and vector or the indicated Hsp104 variant were induced with galactose for 5h at 30°C, lysed and processed for s edimentation analysis and quantitative immunoblot. The relative amount of insoluble TDP-43 was determined as a % of the vector control. Values represent means±SEM (n=2). See also Fig. S2, S3, and S4.

Figure 3. Hsp104^A503X variants suppress FUS toxicity and aggregation

(A) Δhsp104 yeast transformed with FUS and the Hsp104 variants, or YFP and vector, were serially diluted fivefold and spotted onto glucose (off) or galactose (on). (B) Selected strains from (A) were induced in liquid and growth was monitored by A_600nm. (C) Strains from (B) were induced for 5h, lysed, and immunoblotted. (D) WT, Δire1, or Δatg8 yeast were co-transformed with vector control, or FUS plus vector, or the indicated Hsp104 variant and were serially diluted fivefold and spotted onto glucose (off) or galactose (on). (E) Fluorescence microscopy of cells co-expressing FUS-GFP and Hsp104^WT, Hsp104^A503V, or vector. Cells were stained with Hoechst dye to visualize nuclei (blue). FUS aggregation was quantified by counting the number of cells containing 0, 1, or more than 1 foci. Values represent means±SEM (n=3). (F) Δhsp104 yeast co-transformed with FUS and vector or the indicated Hsp104 variant were induced with galactose for 5h at 30°C, lysed and processed for sedimentation analysis and quantitative immunoblot. The relative amount of insoluble FUS was determined as a % of the vector control. Values represent means±SEM (n=2). See also Fig. S3 and S4.

Figure 4. Hsp104^A503X variants suppress α-syn toxicity, aggregation, and mislocalization

(A) Δhsp104 yeast co-transformed with two copies of α-syn-YFP and the Hsp104 variants, or YFP and vector, were serially diluted fivefold and spotted onto glucose (off) or galactose (on). (B) Selected strains from (A) were induced in liquid and growth was monitored by A_600nm. (C) Strains from (B) were induced for 8h in galactose, lysed, and immunoblotted. (D) WT, Δire1, or Δatg8 yeast were co-transformed with vector controls, or α-syn plus vector or the indicated Hsp104 variant and were serially diluted fivefold and spotted onto glucose (off) or galactose (on). (E) Fluorescence microscopy of cells co-expressing α-syn-YFP and Hsp104^WT, Hsp104^A503V, or vector. α-Syn localization was quantified by counting the number of cells with plasma membrane fluorescence or cytoplasmic aggregates. Values represent means±SEM (n=3). (F) Δhsp104 yeast co-transformed with α-syn and vector or the indicated Hsp104 variant were induced with galactose for 8h at 30°C, lysed a nd processed for sedimentation analysis and quantitative immunoblot. The relative amount of insoluble α-syn was determined as a % of the vector control. Values represent means±SEM (n=2). See also Fig. S3 and S4.

Figure 5. Hsp104^A503S and Hsp104^A503V-DPLF protect against α-syn toxicity and dopaminergic neurodegeneration in C. elegans

(A) Hsp104 variants and α-syn were coexpressed in the dopaminergic (DA) neurons of C. elegans. Hermaphrodite nematodes have six anterior DA neurons, which were scored at day 7 post-hatching. Hsp104^A503S and Hsp104^A503-VDPLF have significantly greater protective activity than both α-syn alone and the null variant. Normal worms have a full complement of DA neurons at this time. (B) At day 10, there is a decline in worms with normal DA neurons. Hsp104^A503S and Hsp104^A503V-DPLF exhibit greater protective activity when compared to Hsp104^WT and the null variant. Values represent means±SEM (of 3 independent experiments, n=30 per replicate with 3–4 replicates per independent experiment. * p<.05 One-Way ANOVA group). Normal worms have a full complement of DA neurons at this time. (C) Photomicrographs of the anterior region of C. elegans co-expressing GFP with α-syn. Worms expressing α-syn alone (left) exhibit an age dependent loss of DA neurons. Worms expressing α-syn plus Hsp104^A503S (right) exhibit greater neuronal integrity. Arrows indicate degenerating or missing neurons. Triangles indicate normal neurons. See also Fig. S5.

Figure 6. Potentiated Hsp104 variants are tuned differently than Hsp104^WT

(A) ATPase activity of Hsp104 variants. Values represent means±SEM (n=3). (B) Luciferase aggregates were incubated with Hsp104 variant plus (checkered bars) or minus (clear bars) Hsc70 (0.167µM) and Hdj2 (0.167µM). Values represent means±SEM (n=3) (C) Luciferase aggregates were incubated with Hsp104 variant plus or minus: Hsc70 (0.167µM) and Hdj2 (0.073µM); Ssa1 and Ydj1; or Ssa1, Ydj1, and Sse1. Values represent means±SEM (n=3). (D) Increasing concentrations of FITC-casein were incubated with ClpP plus HAP^WT or HAP^A503V. Initial degradation rates were plotted against FITC-casein concentration to determine K_m. Values represent means±SEM (n=3). (E) FITC-casein was incubated with increasing concentrations of Hsp104^WT or Hsp104^A503V. Change in fluorescence polarization was plotted against Hsp104 concentration to determine K_d. Values represent means±SEM (n=3). (F) Kinetics of Hsp104^WT (1µM) or Hsp104_A503V (1µM) binding to FITC-casein (0.1µM) assessed by fluorescence polarization. Values represent means±SEM (n=3). (G, H) RepA_1–70-GFP was incubated with Hsp104 variant and GroEL_trap plus ATP or ATP:ATPγS (3:1). GFP unfolding was measured by fluorescence. Representative data are shown. (I) Buffer, Hsp104^A503V-DWA, or Hsp104^A503V-DPLA was mixed in varying ratios with Hsp104^A503V to create heterohexamer ensembles and luciferase disaggregase activity was assessed. Values represent means±SEM (n=3). Black line denotes the theoretical curve of a probabilistic mechanism where only a single WT subunit is required for disaggregation. (J) Experiments were performed as in (I) for Hsp104^A503V-DWB and Hsp104^{A503VDPLA-DWB}. Theoretical curves are shown wherein adjacent pairs of A503V:A503V or A503V:mutant subunits confer hexamer activity, while adjacent mutant subunits have no activity. Each adjacent A503V:A503V pair has an activity of 1/6. Adjacent A503V:mutant pairs have a stimulated activity (s), and the effect of various s values are depicted. Values represent means±SEM (n=3).

Figure 7. Potentiated Hsp104 variants disaggregate preformed α-syn, TDP-43, and FUS fibrils more efficaciously than Hsp104^WT

(A–C) α-syn fibrils were incubated without or with Hsp104^WT, Hsp104^A503V, Hsp104^A503S, or Hsp104^A503V-DPLF for 1h at 30°C. Fiber disassembly was assessed by ThT fluorescence (A), sedimentation analysis (B), or (C) EM (Bar, 0.5µm). (A, B) Values represent means±SEM (n=2). (D, E) TDP-43 aggregates were incubated with buffer, Hsp104^WT, Hsp104^A503V, or Hsp104^A503S plus or minus Ssa1, Ydj1, and Sse1 for 1h at 30°C. (D) Aggregate dissolution assessed by turbidity. Values represent means±SEM (n=3). (E) Aggregate dissolution assessed by EM. Bar, 0.5µm. (F, G) FUS aggregates were incubated with buffer, Hsp104^WT, Hsp104^A503V, or Hsp104^A503S plus or minus Ssa1, Ydj1, and Sse1 for 1h at 30°C. (F) Aggregate dissolution assessed by turbidity (absorbance at 395nm). Values represent means±SEM (n=3). (G) Aggregate dissolution assessed by EM. Bar, 0.5µm.