Aggregation of α-synuclein by DOPAL, the monoamine oxidase metabolite of dopamine

Abstract

Parkinson's disease (PD) is a neurodegenerative disease characterized by the selective loss of dopamine (DA) neurons and the presence of alpha-synuclein (AS) aggregates as Lewy bodies (LBs) in the remaining substantia nigra (SN) neurons. A continuing puzzle in studying PD pathogenesis is that although AS is expressed throughout the brain, LBs and selective dopaminergic cell loss lead to characteristic clinical signs of PD, suggesting that there is a link between AS aggregation and DA metabolism. One potential candidate for this link is the monoamine oxidase (MAO) metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), as neither DA nor DA metabolites other than DOPAL are toxic to SN neurons at physiological concentrations. We tested DOPAL-induced AS aggregation in a cell-free system, in vitro in DA neuron cultures and in vivo with stereotactic injections into the SN of Sprague-Dawley rats by Western blots, fluorescent confocal microscopy and immunohistochemistry. We demonstrate that DOPAL in physiologically relevant concentrations, triggers AS aggregation in the cell-free system, and in cell cultures resulting in the formation of potentially toxic AS oligomers and aggregates. Furthermore, DOPAL injection into the SN of Sprague-Dawley rats resulted in DA neuron loss and the accumulation of high molecular weight oligomers of AS detected by Western blot. Our findings support the hypothesis that DA metabolism via DOPAL can cause both DA neuron loss and AS aggregation observed in PD.

Oligomerization and Membrane-binding Properties of Covalent Adducts Formed by the Interaction of α-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL)

“…For production of 15 N-aS or 13 C, 15 N-aS, cells were grown in minimal media containing 15 N-labeled ammonium chloride or 15 Nlabeled ammonium chloride plus 13 C-labeled glucose, respectively, and the protein expression was induced with 0.8 mM isopropyl 1-thio-␤-D-galactopyranoside for 4 h at 37°C.…”

“…Amide resonance assignments were performed according to previously reported chemical shift assignments for intrinsically unfolded aS (20 -22). HNCA experiments of SDS-bound 13 C, 15 N-aS provided ␣-carbon chemical shifts for secondary shift analysis. For this experiment, 40 mM deuterated SDS was added to 200 M doublelabeled aS-unmodified (aS unmod ) or aS-DOPAL(1:10) monomer.…”

“…Fig. 10C shows the intensity ratio of signals of the 1 H- 15 N HSQC spectrum of aS incubated in the presence of DOPAL (DOPAL/protein ratio ϭ 10) in which the SDS micelles were added to the samples before ((aS ϩ SDS) ϩ DOPAL) or after ((aS ϩ DOPAL) ϩ SDS) incubation at 37°C for 24 h, both normalized by the intensity for aS unmod plus SDS. The continuous line (Fig.…”

“…PRE can establish the physical proximity of different sites within a protein through the degree of broadening observed in the NMR signals caused by the presence of a spin-labeled site (23). Here, PRE was evaluated by measuring the intensity of cross-peaks in the 1 H- 15 N HSQC spectra of MTSL spin-labeled aS, in the absence or presence of ascorbic acid, which was added to the MTSL spin-labeled sample to reduce the nitroxide spin label. PRE measurements for spin-labeled aS unmod mutants G31C, A85C, and P120C showed that spin labels near the N terminus, or in the NAC region (G31C and A85C), result in a broadening of residues in the C-terminal tail in the vicinity of residue 128, whereas the spin label in the C-terminal tail leads to broadening in the NAC region as well as near the N terminus of the protein (Fig.…”

“…Furthermore, the interaction of aS with DOPAL is reported to lead to an increase of the levels of potentially toxic aS aggregates. For instance, the injection of DOPAL into the intranigral region of mouse brains results in the formation of aS oligomers accompanied by death of dopaminergic neurons (15). This means that the interaction between aS and DOPAL might play a central role in the selective degeneration of dopaminergic neurons and could explain the synergism between the toxicity of DA metabolism and aS toxicity.…”