Membrane binding, internalization, and sorting of alpha-synuclein in the cell

Abstract

Alpha-synuclein (aSyn) plays a crucial role in Parkinson's disease (PD) and other synucleinopathies, since it misfolds and accumulates in typical proteinaceous inclusions. While the function of aSyn is thought to be related to vesicle binding and trafficking, the precise molecular mechanisms linking aSyn with synucleinopathies are still obscure. aSyn can spread in a prion-like manner between interconnected neurons, contributing to the propagation of the pathology and to the progressive nature of synucleinopathies. Here, we investigated the interaction of aSyn with membranes and trafficking machinery pathways using cellular models of PD that are amenable to detailed molecular analyses. We found that different species of aSyn can enter cells and form high molecular weight species, and that membrane binding properties are important for the internalization of aSyn. Once internalized, aSyn accumulates in intracellular inclusions. Interestingly, we found that internalization is blocked in the presence of dynamin inhibitors (blocked membrane scission), suggesting the involvement of the endocytic pathway in the internalization of aSyn. By screening a pool of small Rab-GTPase proteins (Rabs) which regulate membrane trafficking, we found that internalized aSyn partially colocalized with Rab5A and Rab7. Initially, aSyn accumulated in Rab4A-labelled vesicles and, at later stages, it reached the autophagy-lysosomal pathway (ALP) where it gets degraded. In total, our study emphasizes the importance of membrane binding, not only as part of the normal function but also as an important step in the internalization and subsequent accumulation of aSyn. Importantly, we identified a fundamental role for Rab proteins in the modulation of aSyn processing, clearance and spreading, suggesting that targeting Rab proteins may hold important therapeutic value in PD and other synucleinopathies.

Keywords: Alpha-synuclein; Parkinson’s disease; Rab proteins; Spreading; Uptake.

Fig. 1

Recombinant aSyn monomers and fibrils are internalized by H4 cells. a Recombinant aSyn monomers (aSyn Mono) or fibrils (aSyn Fibrils) were added to the cell culture medium and incubated for 24 h. b SDS-PAGE and immunoblot analysis of the recombinant monomeric or fibrillar species of aSyn used in the experiments. The monomers show also the presence of a small fraction of dimers, as displayed by the faint band at 35 kDa. In the fibril preparation, one can observe the presence of higher molecular weight (HMW) species that are stable even on an SDS-PAGE. c Western blot (WB) of H4 cells after treatment with aSyn, confirming the internalization of aSyn monomers or fibrils as seen by the increase in the levels of aSyn in cells that were treated with monomers or fibrils (actin is used as a loading control). d Quantification of the immunoblots. Statistical test was performed using one-way ANOVA followed by Tukey’s post-hoc tests, *p < 0.01. e Epifluorescence microscopy of cells treated as indicated. Scale bar: 30 μm. f TIRF microscopy of cells treated with aSyn monomers or fibrils (green) and stained with Phalloidin (red) confirm intracellular localization of aSyn. Scale bar: 30 μm

Fig. 2

aSyn associates with membranes and forms high molecular weight species during the internalization process in H4 cells. a Immunoblot of the biotinylation assay of cells treated with aSyn monomers or fibrils (tubulin is used as a loading control). b Quantification of the levels of aSyn present in the biotinylated fraction (membrane-associated aSyn). Statistical test was performed using one-way ANOVA followed by Tukey’s post-hoc tests, *p < 0.01 c Dot blot of the size exclusion chromatography fractions of lysates of untreated cells, cells treated with 1 μM of aSyn monomers, and cells treated with 1 μM of aSyn fibrils. The black box highlights monomeric aSyn, while the red boxes highlight the presence of high molecular weight species of aSyn. d Triton X-100 fractionation, with the soluble (left panel) and the insoluble fractions (right panel), treated as described

Fig. 3

aSyn partially colocalizes with Rab5A-GFP and Rab7-GFP in H4 cells. a ICC of cells transfected with Rab5A-GFP (right side of the panel) or with Rab7-GFP (left side) and then treated with 1 μM of aSyn monomers or fibrils. b Pearson correlation coefficient reveals colocalization of aSyn and Rab5A, and of aSyn and Rab7 in cells treated with aSyn monomers, but not with fibrils. Scale bar: 30 μm

Fig. 4

aSyn accumulates in Rab 4A-positive vesicles. ICC on H4 cells transfected with Rab4A-GFP and treated with 1 μM of aSyn monomers or fibrils. Inset: zoom and separated channels of Rab4A-GFP aSyn. Arrows point to the large inclusions aSyn (in red, panel on the right) matching with the GFP-positive Rab4A vesicles (in green, panel on the left). Scale bar: 30 μm

Fig. 5

Membrane binding is essential for the aSyn internalization and inclusion formation. a and b Immunoblot and quantification of the comparison of WT aSyn and two mutants (A30P and A11P/V70P) with different membrane-binding properties using a biotinylation assay. Dotted bars refer to the band corresponding to aSyn dimers (aSyn**), and clear bars refer to aSyn monomers (aSyn*). c ICC and d immunoblotting of non-treated (NT) cells, or cells treated with WT, A30P, or A11P/V70P aSyn for 24 h. Scale bar: 30 μm. e Quantification of the immunoblot in panel D. Dotted bars refer to the band corresponding to aSyn dimers (aSyn**), and clear bars refer to aSyn monomers (aSyn*). Statistical tests were performed using one-way ANOVA with repeated-measures for grouped analysis, followed by Tukey’s post-hoc tests. Data were expressed as mean ± SEM and a 0.5% general significance level was defined, with significance levels as follows: *: p < 0.05; **: p < 0.01; ***: p < 0.001. Statistical significance is indicated with the symbol “#” for the monomers, “+” for the dimers, and “*” for the combination between monomers and dimers

Fig. 6

The A30P and A11P/V70P aSyn mutants are less internalized than WT aSyn. a ICC and b Immunoblotting of cells transfected with Rab4A–GFP and treated as in experiments shown in Fig. 5. d and e ICC and Immunoblotting of cells transfected with Rab5A-GFP and treated as above. g and h ICC and Immunoblotting of cells transfected with Rab5ACA-GFP (constitutively active) and treated as above. c, f and i Quantifications of the immunoblots in panels b, e and h. Dotted bars refer to the band corresponding to aSyn dimers (aSyn**), and clear bars refer to aSyn monomers (aSyn*). Statistical tests were performed using one-way ANOVA with repeated-measures for grouped analysis, followed by Tukey’s post-hoc tests. Data are expressed as mean ± SEM and a 0.5% general significance level was defined, with significance levels as follows: *: p < 0.05; **: p < 0.01; ***: p < 0.001. Statistical significance is indicated with the symbol “#” for the monomers, “+” for the dimers, and “*” for the combination between monomers and dimers. Scale bar: 30 μm

Fig. 7

Rab7 reduces the formation of dimers in cells treated with WT aSyn monomers. a ICC and b Immunoblotting of H4 cells transfected with Rab7-GFP and treated as described above. d and e The overexpression of the Rab7 dominant negative (DN) does not affect the degradation of the internalized aSyn. c and f Quantifications of the immunoblots in panels B and E. Dotted bars refer to the band corresponding to aSyn dimers (aSyn**), and clear bars refer to aSyn monomers (aSyn*). Statistical tests were performed using one-way ANOVA with repeated-measures for grouped analysis, followed by Tukey’s post-hoc tests. Data were expressed as mean ± SEM and a 0.5% general significance level was defined, with significance levels as follows: *: p < 0.05; **: p < 0.01; ***: p < 0.001. The significance is shown with the symbol “#” for the monomers, with the symbol “+” for the dimers and with the symbol “*” for the sum between monomers and dimers. Scale bar: 30 μm

Fig. 8

Blocking endocytosis and autophagy modulates the internalization and degradation of aSyn. a ICC of Rab4A-GFP expressed in H4 cells treated with 1 μM aSyn monomers and with vehicle, PitStop 30 μM or with Dyngo 50 μM. Both PitStop and Dyngo are inhibitors of the endocytic processes. b Quantification of the aSyn mean fluorescence intensity in the three conditions. c ICC of Rab7-GFP-expressing cells treated with 1 μM aSyn monomers and with vehicle, Bafilomycin 100 nM (Baf 100 nM), or with Chloroquine 50 μM (Chlq 50 μM). Bafilomycin and Chloroquine are inhibitors of lysosomal acidification and, therefore, autophagy-lysosome pathway (ALP). (D) Quantification of the aSyn mean fluorescence intensity in the three conditions. Scale bar: 30 μm

Fig. 9

Model for the proposed mechanism of aSyn internalization and interaction with the membrane and trafficking machinery. After interacting with the plasma membrane, aSyn monomers are internalized through the endocytic pathway. Rab4A plays an important role in the sorting of aSyn and on the transport from/to the plasma membrane. aSyn is then found in the early endosome and, it is possible that during the progress from early to late endosome, due to acidification of the environment, aSyn monomers start to aggregate and form high molecular weight species. Afterwards, late endosomes fuse with lysosomes, where aSyn can be degraded. It is possible that, during any of those steps, aSyn is released to the cytoplasm and to the extracellular milieu, leading to its potential accumulation and spreading of pathology