Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo

Abstract

In Parkinson's disease (PD) and other α-synucleinopathies, prefibrillar α-synuclein (αS) oligomer is implicated in the pathogenesis. However, toxic αS oligomers observed using in vitro systems are not generally seen to be associated with α-synucleinopathy in vivo. Thus, the pathologic significance of αS oligomers to αS neurotoxicity is unknown. Herein, we show that, αS that accumulate within endoplasmic reticulum (ER)/microsome forms toxic oligomers in mouse and human brain with the α-synucleinopathy. In the mouse model of α-synucleinopathy, αS oligomers initially form before the onset of disease and continue to accumulate with the disease progression. Significantly, treatment of αS transgenic mice with Salubrinal, an anti-ER stress compound that delays the onset of disease, reduces ER accumulation of αS oligomers. These results indicate that αS oligomers with toxic conformation accumulate in ER, and αS oligomer-dependent ER stress is pathologically relevant for PD.

Figure 1.

αS monomers and oligomers accumulate in the ER. A, ER/Ms (P100) obtained from PreS and end-stage (sick) A53T αS Tg mice were solubilized with 0.5% TX-100 and centrifuged to obtain the supernatant (TX-S) and the insoluble pellet (TX-I). Arrowheads, SDS-stable high-MW αS. Bottom, Shorter exposure of the αS blot. B, TX-S_tot and TX-P_tot fractions from the spinal cords of nTg and end-stage A53T αS Tg mice immunoblotted for αS. C, Detergent-insoluble fractions (TX-I from ER/M and TX-P_tot) from two mice immunoblotted for αS. D, ER accumulation of toxic αS oligomers. Dot-blot analysis of SpC microsomes from nTg and A53T αS Tg mice (6 months, PreS, Sick). Also analyzed are total TX-P_tot and C (detergent-free) from sick A53T αS Tg mice. Quantitative analysis of dot-blots showing values normalized for the total αS. Values are given as the mean ± SEM (n = 3–4). *p < 0.05, **p < 0.001, one-way ANOVA. E, Left, Dot-blot analysis of microsomes from diseased A53T mice (P100); in vitro assembled αS (αSF) and Tau (Tau-F) fibrils; or total insoluble fraction from Tg mice (TX-P_tot) shows FILA-1 specificity for αS oligomers. Right, Dot-blots of various microsome fractions obtained from different αS Tg mouse lines [age-matched diseased A53T αS(G2-3), A30P αS(O₂), or WT αS(I2-2)]. F, Dot-blots of ER/M from different areas [α-synucleinopathy-affected areas (SpC) or disease-free areas (Ctx). Right, Dot-blot analysis of ER/M from PreS or diseased (Early, End) mice for FILA-1 or αS. G, ER-associated αS monomer but not the high-MW αS is protected from proteolysis by PK. In the presence of 0.5% TX-100, both αS and the ER-resident grp78 are completely proteolyzed by PK. H, ER/M fractions were first exposed to PK, followed by 0.5% TX-100 extraction, and were fractioned into TX-S and TX-I fractions. The αS monomers remain soluble, but proteolyzed αS fragments remain insoluble. I, Dot-blot analysis of ER/M fractions shown in H for FILA-1 or αS. In the PreS mice, the αS monomer and the oligomers are resistant to PK, indicating that they are in the ER lumen. Even in the absence of PK, TX-100 abolished both A11 and FILA-1 reactivity in PreS samples. I also shows dot-blot analysis of TX-100-soluble and -insoluble ER/M fractions without the prior PK treatment. Note that the FILA-1 signal is abolished with TX-100 in PreS mice but not in Sick mice.

Figure 2.

ER/M accumulation of αS oligomers in human PD cases. A, Dot-blot analysis of BrSt microsome fractions from human control and PD cases. Microsomes were probed with antibodies against αS oligomers and pS129 αS. Quantitative data values are normalized for total αS. Values are given as the mean ± SEM (n = 8 for controls, n = 12 for PD). *p < 0.05, **p < 0.01, Student's t test. B, C, Total αS aggregates (TX-P_tot) and cytosolic fractions from PD brains do not show a significant accumulation of αS oligomers. TX-P_tot (B) or cytosolic (C) fractions from human PD cases were dot-blotted for FILA-1 and αS. The ER/M fractions are also shown in B.

Figure 3.

Salubrinal reduces ER/M accumulation of αS pathology. A, ER/M fractions from Salubrinal (Sal)-treated A53T αS mice were dot-blotted for αS oligomers (A11, FILA-1). B, The graphs showing the quantitative analysis of the dot-blots, which show significant decreases in αS oligomers with the Salubrinal treatment. The values were normalized either for total protein (FILA-1, A11) or for total αS (FILA-1/αS, A11/αS) followed by the average of vehicle (Veh) group. Values are given as the mean ± SEM (n = 3). ***p < 0.001, **p < 0.01, *p < 0.05, Student's t test.