A kinetic assessment of the C. elegans amyloid disaggregation activity enables uncoupling of disassembly and proteolysis

Abstract

Protein aggregation is a common feature of late onset neurodegenerative disorders, including Alzheimer's disease. In Alzheimer's disease, misassembly of the Abeta peptide is genetically linked to proteotoxicity associated with disease etiology. A reduction in Abeta proteotoxicity is accomplished, in part, by the previously reported Abeta disaggregation and proteolysis activities-under partial control of heat shock factor 1, a transcription factor regulating proteostasis in the cytosol and negatively regulated by insulin growth factor signaling. Herein, we report an improved in vitro assay to quantify recombinant fibrillar Abeta disaggregation kinetics accomplished by the exogenous application of C.elegans extracts. With this assay we demonstrate that the Abeta disaggregation and proteolysis activities of C.elegans are separable. The disaggregation activity found in C.elegans preparations is more heat resistant than the proteolytic activity. Abeta disaggregation in the absence of proteolysis was found to be a reversible process. Future discovery of the molecular basis of the disaggregation and proteolysis activities offers the promise of delaying the age-onset proteotoxicity that leads to neurodegeneration in a spectrum of maladies.

Figure 1

Aβ₄₀ disaggregation activity A: Disaggregation activities of C. elegans PDS as a function of total protein concentration (25–500 μg/mL). Lines show mono-exponential decay function fits to the data (open symbols). B: Disaggregation activities of PDS (20 μg/mL) from Aβ₄₂-expressing (CL for CL2006) and wt (N2) C. elegans fed bacteria expressing hsf-1 RNAi or empty vector controls (EV). Neither the disaggregation rate nor its reduction by hsf-1 downregulation depend on Aβ₄₂ expression. C: PDS-mediated Aβ₄₀ disaggregation is not discernibly inhibited when ATP in the PDS is hydrolyzed by pretreatment with apyrase (AP, 0.1 and 0.5 U, 15 min, 30°C); average relative ThT signals of quintuplicate samples ± SD. D: PDS-mediated (PDS, 25 μg/mL) Aβ₄₀ disaggregation kinetics in the presence of 0.025–0.2% SDS. ThT signals of detergents in buffer were subtracted from the Aβ signals. E: Relative ThT fluorescence signals after 10 h incubation with PDS in the presence of 0.025–0.2% SDS, Tween-20, or NP-40. F: Separation of PDS-mediated disaggregation and proteolysis products of Aβ₄₀ by denaturing PAGE visualized by Western blot analysis (mAb 6E10) of fibrillar Aβ₄₀ before and after PDS treatment. Aβ samples were sonicated for 0, 15, 30, or 60 min before disaggregation. Arrows indicate apparent monomer (M), dimer (D), and trimer (T) bands as well as high-MW aggregate (A) bands. Control: fibrillar Aβ₄₀ incubated for 4 d in the absence of PDS.

Figure 2

Disaggregation activity is distinct from proteolysis. A: Atomic force microscopy images of fibrillar Aβ₄₂ and Aβ₄₀ incubated in the presence or absence of C. elegans PDS (25 μg/mL) and Roche complete protease inhibitor cocktail (PIC). Scale bars = 200 nm, height scale = 20 nm. Incubation with PDS leads to a loss of readily detectable fibrillar structures both in the presence and absence of PIC. B: Kinetic measurement of Thioflavin T fluorescence of Aβ₄₂ incubated for 75 h with PDS in the presence or absence of PIC. C: Western blot of (mAb 6E10) of monomeric Aβ₄₀ peptide (non agg.), Aβ₄₀ aggregates (agg. control), and the disaggregation products resulting from PDS treatment (25 μg/mL) in the absence or presence of PIC. Arrows indicate apparent monomer (M), dimer (D) and high-MW aggregate (A) bands. D: Coomassie staining reveals monomeric and tetrameric bands of non-aggregated (non agg.) Aβ₄₂. Western blot of Aβ₄₂ aggregates (agg. control), and the disaggregation products resulting from PDS treatment (25 μg/mL) in the absence or presence of PIC. Aβ₄₂ is degraded and proteolyzed after incubation with C. elegans PDS for 72 h in the absence of PIC; bands labeled as in C, in addition a soluble aggregate band was observed (A_sol). E: Aβ aliquots from disaggregation assays were analyzed by dot blots and filter retardation assays, respectively, after incubation of Aβ₄₀ for 72 h to quantify the amounts of total Aβ and SDS-resistant Aβ aggregates. F: Quantification of filter retardation assay and dot blot data. Error bars indicate standard deviations in three independent disaggregation experiments. SDS-resistant, aggregated Aβ is reduced after incubation with C. elegans PDS. Total amounts of Aβ peptide remain constant after incubation in the presence of PIC or phenylmethylsulfonylfluoride (PMSF), but decrease in the absence of protease inhibitors.

Figure 3

Disaggregation activity is heat resistant A: Heat-treated C. elegans PDS (25 μg/mL) incubated with fibrillar Aβ₄₀ in the absence of protease inhibitor. PDS was heated to 37, 70, 80 or 95°C for 10 min before incubation with Aβ₄₀ fibrils. B: Dot blot quantification of total Aβ₄₀ using mAB 6E10 after 72 h incubation. C: Aβ₄₀ SDS-resistant aggregate quantification after 72 h incubation (filter retardation, mAB 6E10). Bar graphs show normalized averaged dot blot signals of three independent experiments, error bars indicate standard deviations. Proteolytic activities are inactivated at 70°C, whereas 95°C is needed to inactivate the disaggregation activity D: Fibrillar Aβ₄₂ was incubated with heat-treated C. elegans PDS as in A in the presence or absence of PIC. Disaggregation products were analyzed by Western blotting as in Fig. 2(D). SDS-insoluble aggregate bands (A) and 20 kDa tetramer bands (T) were analyzed quantitatively. Bar graphs show intensities of aggregate (white) and soluble (gray) Aβ₄₂ peptide bands relative to the amounts of Aβ before incubation (control) from four independent experiments, * denotes P < 0.05; ** denotes P < 0.01. E: C. elegans PDS was pretreated without (blue) or with proteinase K (100 μg/mL) (red) for 1 h at 37°C before inactivation of the proteinase K activity by heating to 80°C for 10 min (conditions that leave the disaggregase intact) and then added to fibrillar Aβ₄₀ (15 μM; Aβ monomer).

Figure 4

Disaggregation is reversible in the absence of proteolysis. A: C. elegans PDS was incubated with Aβ₄₀ fibrils for 75 h (as in Fig. 1) in the absence (orange) or presence of PIC (blue) or PMSF (red). Inhibition of proteolysis did not prevent disaggregation, but led to reaggregation of Aβ₄₀ upon prolonged incubation. No disaggregation was observed in the presence of heat-inactivated (95°C) PDS in the presence of PIC (green) or in buffer controls (black). B: Atomic force microscopy image of Aβ₄₀ fibrils after 75 h incubation with PDS in the presence of PIC or PMSF, showing re-emergence of fibrils. Scale bars = 200 nm, height scale = 20 nm. C: Aβ₄₀ peptide (15 μM) was separated into soluble and insoluble fractions by centrifugation at 200,000 × g before and after aggregation and after incubation with PDS (25 μg/mL) in the presence or absence of PIC. Both the pellet and the supernatant were treated with HFIP/TFA to denature Aβ₄₀ allowing it to be quantified by HPLC analysis. D: Addition of untreated PDS (5% v/v) to soluble Aβ₄₀ (15 μM) [buffer and incubation as in Fig. 1(A)] prevents aggregation of Aβ into ThT-binding aggregates (triplicate samples, green lines), whereas heat-inactivated PDS (95°C, triplicate samples, red lines), and PDS in the presence of PIC (triplicate samples, blue lines) ultimately allowed Aβ₄₀ aggregation, apparently after the consumption of the disaggregase activity.