Microtubule depolymerization potentiates alpha-synuclein oligomerization

Abstract

Parkinson's disease (PD) is associated with perturbed mitochondria function and alpha-synuclein fibrillization. We evaluated potential mechanistic links between mitochondrial dysfunction and alpha-synuclein aggregation. We studied a PD cytoplasmic hybrid (cybrid) cell line in which platelet mitochondria from a PD subject were transferred to NT2 neuronal cells previously depleted of endogenous mitochondrial DNA. Compared to a control cybrid cell line, the PD line showed reduced ATP levels, an increased free/polymerized tubulin ratio, and alpha-synuclein oligomer accumulation. Taxol (which stabilizes microtubules) normalized the PD tubulin ratio and reduced alpha-synuclein oligomerization. A nexus exists between mitochondrial function, cytoskeleton homeostasis, and alpha-synuclein oligomerization. In our model, mitochondrial dysfunction triggers an increased free tubulin, which destabilizes the microtubular network and promotes alpha-synuclein oligomerization.

Keywords: ATP; Parkinson disease; alpha-synuclein; cybrids; mitochondria; tubulin.

Figure 1

Protein oligomerization in PD cybrids. (A) A11 staining is greater in the PD cybrids. Bars, 10 μm. (B) A western blot of alpha-synuclein monomer and oligomers in the PD and CT cybrid lines. (C) Densitometry analysis of alpha-synuclein western blots showing the ratio between alpha-synuclein oligomer and monomer levels corrected for GAPDH. The ratio is increased in the PD cybrid line. (D) Ubiquitination densitometry analysis showed the PD cybrid cells had more ubiquitinated protein. **P < 0.01 and ***P < 0.001, significantly different when compared to the CT cybrid lines.

Figure 2

Tubulin alterations in the PD cybrid lines. (A) Tubulin immunocytochemistry reveals disruption of the microtubule network in the PD cybrid lines. (B) SDS-PAGE analysis shows that in the PD cybrids the free/polymerized tubulin ratio is elevated. (C) After correcting for GADPH content the PD cybrids free/polymerized tubulin ratio was elevated as compared to the control cybrid cell line. **P < 0.01, significantly different as compared to the CT cybrid lines. Bars, 10 μm.

Figure 3

Effects of taxol on tubulin and alpha synuclein. (A) SDS-PAGE showing the effect of 5 nM taxol on free/polymerized tubulin ratios. (B) Densitometry analysis indicated after correcting for GADPH content, 5 nM taxol reduced the free/polymerized tubulin ratio in the PD but not the CT cybrid cells. (C) PAGE showing the effect of 5 nM taxol on alpha-synuclein oligomer levels. (D) Densitometry analysis indicated 5 nM taxol reduced alpha synuclein oligomer levels in the PD cybrid cells. **P < 0.01, significantly different when compared to the untreated CT cybrid cells. ^#P < 0.05 and ^##P < 0.01, significantly different as compared to the untreated PD cybrid line.