Modulation of α-Synuclein Fibrillation and Toxicity by 4-Phenylbutyric Acid

Abstract

The protein misfolding and aggregation of α-synuclein (α-Syn) into neurotoxic amyloids underlies the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). Emerging evidence suggests that 4-phenylbutyrate (PBA) may play a role as a potential chemical chaperone for targeting α-Syn aggregation, but its molecular mechanism remains largely unknown. Using in vitro assays, we demonstrate that PBA treatment alters the pattern of α-Syn aggregation, as evidenced by reduced formation of oligomeric species and its increased susceptibility to proteolytic cleavage under the influence of PBA. Proteinase K (PK) assays, surface plasmon resonance (SPR), Nile red assays, and cytotoxicity assays indicate that PBA interacts with the extensive hydrophobic contacts of α-Syn oligomers and significantly reduces α-Syn-amyloid-induced toxicity. Furthermore, using thioflavin T-based assays, we elucidated the kinetics of PBA-mediated modulation of α-Syn aggregation, highlighting its role in accelerating the formation of α-Syn amyloid fibrils. Molecular dynamics (MD) simulations suggest PBA's role in the destabilization of the C-terminus in α-Syn oligomers through multiple residue interactions. Collectively, our findings provide compelling evidence for the neuroprotective potential of PBA in targeting protein misfolding and aggregation in PD and suggest an avenue for disease-modifying interventions in neurodegenerative disorders.

Keywords: 4-phenylbutyric acid; amyloid protein; molecular dynamics simulations; oligomers; α-synuclein fibrillation.

Figure 1

Aggregation kinetics profile of α-synuclein in the absence (control: α-synuclein) and presence of 1 μM, 100 μM, and 1000 μM PBA concentrations, showing the sigmoidal curve of aggregation indicative of nucleation-dependent polymerization. The graph shows means ± SD (n = 3 replicates).

Figure 2

(A) Congo red absorption and (B) Nile red fluorescence in the absence and presence of 1 μM, 100 μM, and 1000 μM PBA concentrations.

Figure 3

Far UV-CD spectra of α-Syn at different time intervals and concentrations of PBA: (A) α-Syn only, (B) α-Syn +1 μM, (C) 100 μM, and (D) 1000 μM PBA.

Figure 4

SPR sensorgram illustrates the binding interaction of PBA with α-Syn.

Figure 5

SDS PAGE analysis of the proteinase K digestion assay of α-Syn fibrils in the presence and absence of PBA. (1) Ladder, (2) α-Syn fibrils with proteinase K, (3) α-Syn fibrils in the presence of 1 μM PBA and proteinase K, (4) α-Syn fibrils in the presence of 100 μM PBA and proteinase K, and (5) α-Syn fibrils with 1000 μM PBA and proteinase K.

Figure 6

Representative AFM images showing the morphology of α-Syn fibrils at different time intervals at the scale of 1 μm. (A) α-Syn at 12 h, (B) α-Syn at 45 h, (C) α-Syn at 85 h, (D) α-Syn + 1000 μM PBA at 12 h, (E) α-Syn + 1000 μM PBA at 45 h, and (F) α-Syn + 1000 μM PBA at 85 h.

Figure 7

Characterization of α-Syn fibrils using fluorescence microscopy at different time points at a scale of 100 μm: representative fluorescence images (A) in the absence of PBA at 45 h, (B) in the presence of 1000 μM PBA at 45 h, (C) in the absence of PBA at 85 h, and (D) in the presence of 1000 μM PBA at 85 h.

Figure 8

Representative DLS profiles of (A) α-Syn in the absence of PBA and in the presence of PBA at (B) 1 μM, (C) 100 μM, and (D) 1000 μM concentrations at 85 h.

Figure 9

MTT assay of α-Syn in the absence (control) and presence of 1 μM, 100 μM, and 1000 μM PBA concentrations. For the MTT assay, ± SD was calculated from the average of 3 wells. Statistical data analysis was performed using one-way ANOVA, and Dunnett’s post-hoc test was employed for multiple comparison; *P < 0.05 and ***P < 0.001 indicate statistically significant differences.

Figure 10

Initial and final conformations of the 25 ns molecular dynamics (MD) simulations. Initial (A) and final (B–D) conformations of unbound trimeric α-Syn and initial (E) and final (F–H) conformations of α-Syn with PBA are shown. Each α-Syn chain is represented as a different-colored cartoon, while PBA molecules are shown as black spheres.

Figure 11

Binding of two PBA molecules (A,B) to trimeric α-Syn following 25 ns MD simulation replicant 1. Each α-Syn chain is represented as a differently colored cartoon, while PBA molecules are shown as salmon spheres. (C) The orientation between the trihelical bundles following 25 ns for unbound (darker shades) and PBA-bound (lighter shades) systems.

Figure 12

Initial and final conformations of the 25 ns molecular dynamics (MD) simulations. Initial (A) and final (B–D) conformations of tetramer α-Syn unbound and initial (E) and final (F–H) conformations of α-Syn with PBA are shown. Each α-Syn chain is represented as a different-colored cartoon, while PBA molecules are shown as black spheres.

Figure 13

Typical binding model between PBA and tetrameric α-Syn. The binding of four PBA molecules (A–D) to tetrameric α-Syn following 25 ns MD simulation of the first replicant. Each α-Syn chain is represented as a differently colored cartoon, while PBA molecules are shown as salmon spheres. (E) The orientation between the tetra-helical bundles following 25 ns for unbound (darker shades) and PBA-bound (lighter shades) systems. Each PBA molecule is marked a–d to indicate which panel it is associated with.