Rab11a and HSP90 regulate recycling of extracellular alpha-synuclein

Abstract

Growing evidence suggests that extracellular alpha-synuclein (eSNCA) may play an important role in the pathogenesis of Parkinson's disease (PD) and related synucleinopathies by producing neurotoxicity directly or via activation of glia. However, the mechanisms involved in the trafficking of eSNCA in neurons and/or glia remain unclear. Here, we demonstrated that eSNCA could be resecreted out of neurons via a process modulated by a recycling endosome regulator rab11a in addition to being degraded by an endosome-lysosome system. A quantitative proteomic analysis also revealed numerous proteins through which rab11a might execute its function. One of the candidate proteins, heat shock protein 90 (HSP90), was validated to be interacting with rab11a. Furthermore, geldanamycin, an HSP90 inhibitor, not only prevented resecretion of eSNCA but also attenuated neurotoxicity induced by eSNCA.

Figure 1.

Rab11a and recycling of internalized eSNCA. A, Cells were pulsed with human preaggregated eSNCA for 3 h, washed extensively with PBS followed by adding fresh serum-free medium, and then chased for various time points. At the end of the experiment, the medium was collected and eSNCA was detected at basal (CTL), 3 and 6 h after the chase, along with standard (STD) SNCA. B, MES cells, seeded at 0.05 × 10⁶/well, were treated with fluorescent labeled eSNCA (green) at 250 nm for 3 h before chasing. At an appropriate time, cells were stained with anti-rab11a (red) antibody, followed by examination with confocal microscopy. Note: SNCA was colocalized with rab11a after chase for 3 h (yellow color seen in the insert of the merged image). C, The protein complex of interest was isolated from MES cells homogenate after chasing for 3 h. Coimmunoprecipitation analysis using magnetic beads conjugated with either SNCA (top) or rab11a (bottom) with subsequent pull-down revealed a noticeable protein–protein association between SNCA and rab11a. Input represents original materials. D, MES cells were transfected with rab11a siRNA before treatment with eSNCA. Expression levels of rab11a were analyzed by Western blotting with an anti-rab11a antibody 72 h after gene manipulation, demonstrating that rab11a siRNA effectively inhibited rab11a expression [**p < 0.01, compared with control/nonsense (NS) siRNA groups; β-actin used as loading control]. E, MES cells were transfected with rab11a siRNA before treatment with eSNCA. The amount of eSNCA in condition media was measured by Western blotting analysis with or without rab11a siRNA transfection. A significant blocking in TCA-precipitable eSNCA was found in the condition medium of MES cells transfected with rab11a at 3–6 h chasing. Scale bar, 20 μm.

Figure 2.

HSP90, rab11a, and the exocytosis of internalized eSNCA. A, The protein complex of interest was isolated from MES cells homogenate after eSNCA treatment for 3 h, and detected by anti-HSP90 (top) and anti-rab11a (bottom), respectively. Coimmunoprecipitation analysis using magnetic beads conjugated with either HSP90 or rab11a revealed a clear association between HSP90 and rab11a (Input represents original materials). B, The midbrain slides of PD patients were incubated with anti-HSP90 (1:200) or anti-rab11a (1:180), followed by detection with fluorescent labeled anti-mouse (Alexa Fluor 488) and anti-rabbit (Alexa Fluor 568) secondary antibodies, respectively. Colocalization of two proteins is demonstrated as a yellow color shown in the merged confocal image. C, D, MES cells were pretreated with 1 μm GA before the addition of eSNCA for 3 h before pulse. At the end of the experiment, media and cells were collected, respectively. The amount of extracellular (C) and internalized (D) SNCA was measured. Results were obtained from at least three separate experiments. Scale bar, 10 μm. *p < 0.05.

Figure 3.

HSP90 inhibitor rescued the loss of neurites induced by eSNCA. MES cells, seeded in four-well chamber slides, and grown to confluency overnight, were incubated with rotenone at 5 nm, MPP⁺ at 3 mm, or eSNCA at 250 nm or with a combination (i.e., rotenone plus eSNCA or MPP⁺ plus eSNCA), and with or without pretreatment of GA at 1 μm for 3 h. Data are means ± SD of at least three independent determinations. *p < 0.05; **p < 0.01.