Aspirin-Mediated Acetylation Protects Against Multiple Neurodegenerative Pathologies by Impeding Protein Aggregation

Abstract

Aims: Many progressive neurological disorders, including Alzheimer's disease (AD), Huntington's disease, and Parkinson's disease (PD), are characterized by accumulation of insoluble protein aggregates. In prospective trials, the cyclooxygenase inhibitor aspirin (acetylsalicylic acid) reduced the risk of AD and PD, as well as cardiovascular events and many late-onset cancers. Considering the role played by protein hyperphosphorylation in aggregation and neurodegenerative diseases, and aspirin's known ability to donate acetyl groups, we asked whether aspirin might reduce both phosphorylation and aggregation by acetylating protein targets.

Results: Aspirin was substantially more effective than salicylate in reducing or delaying aggregation in human neuroblastoma cells grown in vitro, and in Caenorhabditis elegans models of human neurodegenerative diseases in vivo. Aspirin acetylates many proteins, while reducing phosphorylation, suggesting that acetylation may oppose phosphorylation. Surprisingly, acetylated proteins were largely excluded from compact aggregates. Molecular-dynamic simulations indicate that acetylation of amyloid peptide energetically disfavors its association into dimers and octamers, and oligomers that do form are less compact and stable than those comprising unacetylated peptides.

Innovation: Hyperphosphorylation predisposes certain proteins to aggregate (e.g., tau, α-synuclein, and transactive response DNA-binding protein 43 [TDP-43]), and it is a critical pathogenic marker in both cardiovascular and neurodegenerative diseases. We present novel evidence that acetylated proteins are underrepresented in protein aggregates, and that aggregation varies inversely with acetylation propensity after diverse genetic and pharmacologic interventions.

Conclusions: These results are consistent with the hypothesis that aspirin inhibits protein aggregation and the ensuing toxicity of aggregates through its acetyl-donating activity. This mechanism may contribute to the neuro-protective, cardio-protective, and life-prolonging effects of aspirin. Antioxid. Redox Signal. 27, 1383-1396.

Keywords: (protein) acetylation; (protein) aggregation; (protein) phosphorylation; aspirin (acetylsalicylic acid); inflammation; neurodegeneration.

FIG. 1.

Aspirin treatment significantly protects nematodes from loss of chemotaxis after neuronal expression of human tau, TDP-43, or Aβ_1–42 transgenes. Worms of each strain were exposed to aspirin (ASA, 0.5 mM) or vehicle control (V), continuously from the time of hatching. Chemotaxis toward n-butanol was assessed in worms on day 5 posthatch. Significance of control-ASA differences, determined by two-tailed heteroscedastic t-tests: **p ≤ 0.001; ***p ≤ 0.0001. Aβ_1–42, amyloid beta peptide (amino acids 1–42).

FIG. 2.

Aspirin significantly impedes paralysis in Caenorhabditis elegans adults expressing Aβ_1–42 in muscle, by reducing Aβ_1–42 aggregation but not its expression. (A) CL4176 worms were maintained from hatch on 0.5-mM aspirin or vehicle (control). Paralysis was assessed 48 h after an upshift to 25°C (at the L3/L4 transition) to induce Aβ_1–42 synthesis. (B) Without Aβ_1–42 induction, CL4176 worms undergo age-dependent paralysis, presumably due to leaky expression of Aβ_1–42 (4). This decline (dashed trace) was delayed by 0.5-mM aspirin (solid trace). Significance was tested by the two-tailed heteroscedastic t-test (A), or the Gehan-Wilcoxon log-rank test (B). (C) Aβ_1–42 (monomer and oligomers) was quantified by ImageJ on Western blots by using a primary antibody to Aβ_1–42 (AB11132; Abcam). Total signal was not altered in young-adult worms with aspirin treatment (ASA, 0.5 mM) relative to vehicle controls (V) at varying times after induction: 27 h (0% paralysis), 36 h (70%), or 42 h (100%). (D) Amyloid aggregation, measured as spectrally shifted thioflavin-T fluorescence 36 h postinduction, was reduced by 60% in aspirin-treated worms (p < 0.0001).

FIG. 3.

Insoluble protein aggregates in C. elegans muscle expressing Q40::YFP, resolved on 2D gels, increase with age but are attenuated by aspirin treatment. Worms were maintained from hatch on 0.5-mM aspirin (+ASA, panels B, D, F) or vehicle (untreated controls, panels A, C, E), and sarcosyl-insoluble aggregates were isolated at days 1, 3, and 5 after the L4/adult molt (∼3.5, 5.5, and 7.5 days posthatch). Panels display the central regions of 2D gels separating aggregate fractions by isoelectric focusing (horizontal) over the pI ranges indicated, followed by polyacrylamide SDS-gel electrophoresis (vertical). 2D, two-dimensional; pI, isoelectric point; SDS, sodium dodecyl sulfate.

FIG. 4.

Q40::YFP expression does not change with age or aspirin treatment. AM141 worms (expressing Q40::YFP in muscle) were maintained continuously on 0.5-mM aspirin (ASA) or vehicle (V), from hatch. Worms were lysed at 1, 3, or 7 days of adult age, and total protein was extracted. Proteins separated by SDS-PAGE electrophoresis were blotted and probed with an antibody to GFP, which also recognizes YFP. No significant changes in Q40::YFP expression were observed with aspirin treatment or age.

FIG. 5.

Aspirin reduces amyloid accumulation in human neuroblastoma cells expressing an aggregation-prone mutation of amyloid precursor protein (APP_Sw). Amyloid foci were stained with thioflavin T in SH-SY5Y-APP_Sw cells that were treated for 2 days with (A) vehicle [V], or (B) 0.5-mM aspirin [ASA]. (C) Summary of data, quantified by ImageJ, compiled from three independent experiments. Error bars indicate standard error of the mean, and significance was based on a two-tailed heteroscedastic t-test, in each case treating each experiment as a single data point per group. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Aspirin reduces the abundance of low-mobility proteins in insoluble aggregates from neuroblastoma cells, but it does not prevent exclusion of acetylated proteins. Sarcosyl-insoluble aggregates were isolated from SH-SY5Y-APP_Sw cells after growth in medium containing 0.5-mM aspirin (ASA) or vehicle alone (V). (A) SyproRuby staining of total protein from SH-SY5Y-APP_Sw aggregates. (B) Ratios of ASA/control lane intensity in successive gel slices, ±SD (standard deviation) for three independent experiments. Poorly dissolved and low-mobility proteins were 20%–30% less abundant after ASA. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

Aspirin is more effective than salicylate in reducing protein aggregation in C. elegans and human-neuroblastoma models of neurodegeneration. (A) C. elegans strain AM141, expressing Q40::YFP in muscle, was exposed from hatch to 0.5-mM aspirin (ASA), 0.5-mM salicylate (SA), or vehicle control (V). YFP-fluorescence images of worms were captured, and aggregates per worm were counted with ImageJ software at day 1 of adulthood. (B) SH-SY5Y-APP_Sw neuroblastoma cells, expressing an aggregation-prone mutation of amyloid precursor protein (APP_Sw), were assessed for amyloid deposition after treatments (as in A). Cells were detached by brief digestion in trypsin-EDTA, removing pre-existing extracellular amyloid; they were replated at ∼20% confluence, allowed to attach, and finally grown for 48 h (to 70%–80% confluence) in the presence of drug or solvent. Formalin-fixed cells were stained for amyloid with thioflavin T; nuclei were counterstained with DAPI. Amyloid per cell is shown as the ratio of thioflavin-T to DAPI fluorescence (proportional to cell number). Aspirin reduced both aggregate count per worm (A) and amyloid per cell (B), relative to vehicle controls or salicylic acid. Significance (2-tail heteroscedastic t-tests): *p ≈0.02; **p ≈0.006; ****p < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; EDTA, ethylene diamine tetraacetic acid; NS, not significant.

FIG. 8.

Aspirin reduces phosphorylation but increases acetylation of total protein in wild-type C. elegans adults. Total protein was isolated from worms that were maintained on 0.5-mM aspirin (ASA) or vehicle (V), and it was electrophoresed in one dimension. Phosphoprotein was visualized by staining with ProQ Diamond (A), whereas a subset of acetylated proteins was detected by immunostaining with an antibody to acetyl-lysine (B, C). Aggregate proteins for (C) were isolated as described in the “Materials and Methods” section.

FIG. 9.

Aspirin reduces abundance of most aggregate proteins and phosphoproteins, while increasing acetylation of a few proteins, in a nematode model of amyloidopathy. Aggregates from C. elegans strain CL4176, expressing Aβ_1–42 in muscle, were isolated and partitioned by solubility in 1% sarcosyl. (A) Total and cytosolic (nonaggregated) protein profiles are unaffected by aspirin treatment. (B) Most aggregate proteins are reduced in quantity by aspirin treatment, especially in sarcosyl-insoluble fractions (examples are marked with “>,” and exceptions are marked with a white “<”). (C) Most phosphoproteins are reduced in aggregates after aspirin. (D) Very few proteins are acetylated in Aβ_1–42 aggregates, ±aspirin.

FIG. 10.

Acetylated proteins are underrepresented in both soluble and insoluble aggregates of human neuroblastoma cells. Total proteins, cytosolic proteins, and sarcosyl-soluble and -insoluble aggregates were isolated from human neuroblastoma cells (SH-SY5Y_Sw) after growth in medium containing 0.5-mM aspirin (ASA) or vehicle only (V). (A) Western blot of proteins from SH-SY5Y-APP_Sw cells, probed with an antibody to acetyl-lysine. (B) Bars indicate mean ± SD for lane integrals for gels (as in A) from three independent experiments, each normalized to the “total protein” control mean. Aggregate proteins show 35%–80% less acetylation than corresponding total-protein lanes, despite uniform protein loads. Significances, based on two-tailed heteroscedastic t-tests: **p = 0.001, ***p ≤ 0.0006. Asterisks above brackets refer to differences between the bars (groups) connected by each bracket. Asterisks below brackets (just above bars) compare each marked bar with its corresponding (V or ASA) “Total Protein” sample.

FIG. 11.

Acetylation reduces compactness and stability of Aβ_1–42 oligomers. Molecular docking models and molecular-dynamic simulations indicate that lysine acetylation in Aβ_1–42 (“K_ac”) destabilizes its assembly into tetramers and octamers. Optimized docking models are shown for: (A) native Aβ_1–42 tetramer, (B) acetylated Aβ_1–42 tetramer, (C) native Aβ_1–42 octamer, and (D) acetylated Aβ_1–42 octamer. Molecular-dynamic simulations in GROMACS indicate increased dispersion (radius of gyration) for acetylated tetramer (E) and octamer (F), and decreased hydrogen bonding for acetylated octamer (G), relative to their unmodified forms. (E–G) Properties of lysine-acetylated oligomers (K_ac) are shown in red, and those of unmodified Aβ oligomers are indicated in black.

FIG. 12.

Interventions that elevate protein acetylation reduce aggregation, and vice versa. (A) In a C. elegans model of Huntington-like Q40 aggregation, the number of foci was reduced by RNAi knockdown of genes encoding protein deacetylases (sin-3, nceh-1) or by an enhancer of acetylation activity (CTPB); each of these interventions is expected to increase protein acetylation. Conversely, aggregate count was increased by RNAi targeting n-acetyltransferase genes (nat-6, nat-25), and knockdowns were expected to reduce acetylation. Significance was determined by two-tailed heteroscedastic t-tests: ^#p ≈0.07, *p ≤ 0.01, **p < 0.003, and ***p < 0.0005. (B) Fraction paralyzed after inhibition of acetyl transferase or deacetylase genes by RNAi or by chemical activators of acetyltransferases or inhibitors of deacetylases. Significance was determined by two-tailed heteroscedastic t-tests: *p < 0.01, **p < 0.002, and ***p < 0.0005. RNAi, RNA interference.