The Parkinson-Associated Toxin Paraquat Shifts Physiological α-Synuclein Tetramers toward Monomers That Can Be Calpain-Truncated and Form Oligomers

Abstract

Abnormal aggregation of α-synuclein (αS) is thought to initiate neuronal dysfunction and death in Parkinson disease (PD). In addition to higher-molecular-weight, oligomeric, and polymeric forms of αS associated with neurotoxicity and disease, recent findings indicate the occurrence of physiological tetrameric assemblies in healthy neurons in culture and in brain. Herein, the PD-associated neurotoxin paraquat reduced physiological tetramers and led to calpain-truncated monomers and an approximately 70-kDa apparent oligomer different in size from physiological αS multimers. These truncated and oligomeric forms could also be generated by calpain cleavage of pure, recombinant human αS in vitro. Moreover, they were detected in the brains of tetramer-abrogating, E46K-amplified (3K) mice that model PD. These results indicate that paraquat triggers membrane damage and aberrant calpain activity that can induce a pathologic shift of tetramers toward an excess of full-length and truncated monomers, the accumulation of αS oligomers, and insoluble cytoplasmic αS puncta. The findings suggest that an environmental precipitant of PD can alter αS tetramer/monomer equilibrium, as already shown for several genetically caused forms of PD.

Figure 1

Paraquat (PQ) induces lactate dehydrogenase (LDH) lipid membrane toxicity, aberrant calpain activation, and α-synuclein (αS) deposition. A and B: Quantification (as fold control) of LDH of primary neurons treated with increasing doses of PQ (PQ; A) and prolonged time at 30 μmol/L dose of PQ (B). Cp indicates cotreatment with calpain inhibitor calpeptin. C: Confocal imaging showed increased calpain 1 activity in vivo based on its cell-permeable fluorescent substrate CMAC, and in fixed cells by using an antibody specific for calpain 1 (Calp)–cleaved spectrin in fixed cells. Boxed areas are higher magnifications in the small panels below. D: Immunoreactivity (integrated optic density) is quantified. Cp cotreatment reduced the fluorescence in fixed neurons. E: CMAC enzyme-linked immunosorbent assay. F: Confocal imaging showed PQ-induced αS + puncta (red) and the dendritic marker microtubule-associated protein 2 (Map2; blue). Boxed areas are higher magnifications in small panel below. G: Quantification of αS puncta by the ImageJ built -in particle analyzer. H: Bright-field images of primary neurons were recorded at 12, 24, and 48 hours of PQ challenge, and intersections of neurites were quantified by the ImageJ software version 2.1.0/1.53c. Data are means ± SEM (A, B, and D–H). N = 3 independent experiments (E). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (analysis of variance post hoc Tukey test). Scale bar = 20 μm (C and F). AU, arbitrary unit; clvg, cleavage; Ctl, control; DMSO, dimethyl sulfoxide.

Figure 2

Subtoxic paraquat (PQ) exposure shifts tetramers to monomers, and calpain 1–derived α-synuclein truncation (αS-Δ) and toxic exposure elevates oligomeric forms. A: Increasing PQ exposure of primary neurons and cotreatment with calpeptin (Cp). B: Quantification of experiments. Increasing PQ shifts αS tetramers [visualized by intact cell disuccinimidyl glutarate (DSG) cross-linking] to monomers, and truncated αS-Δ species likely arise from calpain 1 cleavage of monomers, as its inhibition by Cp largely prevents this. Increase in α-spectrin cleavage pattern (lower band intensities) is a control for calpain activity. C: Western blot (WB) analysis of primary neurons exposed to 30 μmol/L PQ for 24 hours, and cotreatment with the calpain inhibitor Cp and quantification of experiments. The DSG intact cell cross-linking showed tetramer loss, and increased truncated αS-Δ and oligomeric (approximately 70-kDa) forms likely arise from calpain 1 cleavage of monomers, as Cp reduces it. D: Quantification of fold change versus control condition (Ctl or Ctl-Cp), by two-way analysis of variance, post-Tukey test. E: WB analysis of (non–cross-linked) sequentially extracted Triton X-100 (TX)–soluble (sol) proteins, detected with antibodies against αS, spectrin (including the calpain-specific breakdown product of spectrin, spectrin-Δ), caveolin 1 (cav1; membrane protein of lipid rafts harboring calpain), actin (loading control). F: Detergent-insoluble (insol; 8M urea/5% SDS-solubilized) αS is detected with antibodies against αS and actin (loading control). G and H: Quantification of signals in WBs shown above (E and F) is fold change versus control condition (Ctl or Ctl + Cp), by two-way analysis of variance, post-Tukey test. Dashed lines indicate quantification of fold change versus control condition (Ctl or Ctl-Cp). Data are means ± SEM (B, D, G, and H). N = 3 experiments (B and C). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (two-way analysis of variance post hoc Tukey test; PQ versus Ctl); #P < 0.05 (PQ versus PQ + Cp). HMW, higher molecular weight.

Figure 3

Calpain 1–derived α-synuclein truncations (αS-Δ) and a disuccinimidyl glutarate (DSG)-stabilized 70-kDa apparent oligomer. A: Schema of cleaving (1.5 μg) pure human recombinant (rec) αS with calpain 1 (2 U), or inhibiting cleavage with the calpain-specific inhibitor calpeptin (Cp; 4 mmol/L) with or without incubation with DSG (0.5 mmol/L) and aliquoting after several minutes. B: Co-incubation with the calpain 1 inhibitor Cp (Cp) prevented αS monomer (mon) cleavage. An apparent dimer of αS (dim) is detected only after Cp exposure, even without paraquat exposure. C: An aliquot of the cleaved rec αS (in A) was DSG cross-linked after calpain 1 treatment, yielding mainly truncated forms, and these were prevented by Cp cotreatment. Stochastically cross-linked multimers are shown in presence of Cp, likely due to the oligomers being sensitive to aberrant calpain 1 cleavage. D: DSG cross-linking at the time of calpain 1 cleavage accentuates a calpain 1–dependent apparent oligomeric αS form of approximately 70 kDa (exceeding the size of 60 kDa). Dim, dimers; Hex, hexamers; Mon, monomers; Oct, octamers; Pen, pentamers; Tet, tetramers; Tri, trimers.

Figure 4

Calpain 1–truncated and oligomeric α-synuclein (αS) in tetramer-abrogating 3K mouse brain. A and E: Recombinant soluble (rec sol) or fibrillized αS (rec fibr) cleaved with calpain 1 (for 75 minutes) and wild-type (WT) or 3K αS mouse brain extracts were subjected to SDS gel electrophoresis and Coomassie staining and blotting to identify gel-slicing sites targeting αS oligomers (approximately 70 kDa) and α-synuclein truncation (αS-Δ; 10 to 12 kDa) in mouse brain (indicated by ]; A) and analyzed by mass spectrometry (E). B: Cortical extracts show full-length, truncated, and αS oligomers in 1K and 3K versus non-transgenic (Ntg) and WT mouse brain. The difference in migration of the human αS is due to the additional positive charges in the K-mutant protein. C: Quantification of truncated versus full-length αS signal in similar expressing WT, 1K, and 3K cortices using the 105 αS antibody that detects full-length and calpain-truncated αS. 3 KL (lower-expressing 3K αS mouse line) and 3K display similar increase in αS-Δ versus 1K and WT. D: Hypothetical model based on data herein. A shift of native αS tetramers to more protease-accessible monomers by familial Parkinson disease (PD) mutations and the amplification of the E46K mutant (E35 + E46 + E61). Paraquat, an environmental toxin linked to a higher PD risk, can also trigger the tetramer/monomer shift, and aberrant activation of calpain 1 results in monomer truncation, which, in turn, promotes aggregates distinct from physiological tetramers. E: Tryptic peptides from liquid chromatography coupled with tandem mass spectroscopy spectra were analyzed using Mascot software version 2.6.2 (Matrix Science Inc., Boston, MA). Green letters are sequences of the human α-synuclein protein that are detected with high confidence. Each underline represents an independent data point for peptide identification. Red underline is modification at a given amino acid. n = 3 per genotype (C). ∗P < 0.05 (one-way analysis of variance post hoc Tukey test). N-acet., N-acetylated.