{alpha}-synuclein and its A30P mutant affect actin cytoskeletal structure and dynamics

Abstract

The function of alpha-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, alpha-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that alpha-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type alpha-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant alpha-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant alpha-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the alpha-synuclein gene, electroporation of wild-type alpha-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P alpha-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, alpha-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration.

Figure 1.

Effects of α-synucleins on actin polymerization/depolymerization kinetics in cell-free assays. (A) Purified actin and wt α-synuclein (5 μg) separated by SDS-PAGE and stained with Coomassie blue. (B) Kinetics of actin polymerization. Experiments were performed in a fluorometer at 30°C. Purified actin (5 μM; 5% pyrenilated) was polymerized in a solution containing 85 mM NaCl, 30 mM KCl, 1 mM MgCl₂, and 0.1 μM CaCl₂, in the absence (CTRL = controls) or presence of 11 μM wt or A30P α-synuclein. (C) Effects of various concentrations of wt or A30P α-synuclein on the actin polymerization rate at 0.1 μM CaCl₂. (D) Cosedimentation of wt or A30P α-synucleins (11 μM) at 0.1 μM CaCl₂ with actin filaments. Samples were centrifuged at 15,000 × g for 10 min (P1), followed by a second spin at 135,000 × g for 20 min (P2). S, supernatant of the P2 centrifugation (half of the supernatant was loaded). (E) Changes in the maximal rate of actin polymerization induced by wt or A30P α-synuclein (11 μM) at 0.1 or 100 μM CaCl₂. (F) Kinetics of actin depolymerization. Actin was polymerized in high-salt conditions as in B and subsequently diluted 10-fold in buffer without salts (see Materials and Methods), in the absence (CTRL) or presence of 6 μM wt or A30P α-synuclein. (G) Effects of 6 μM wt or A30P α-synuclein on the actin maximum depolymerization rate at 0.1 μM CaCl₂. In C, E, and G values are expressed as percentage of the control. (* p < 0.05).

Figure 2.

Effects of α-synucleins on actin filament structure. (A) Fluorescence microscopy analysis of actin filaments generated by a 30-min incubation at 30°C of 5 μM actin in a solution containing 85 mM NaCl, 30 mM KCl, 1 mM MgCl₂, and 0.1 μM CaCl₂, in the absence (CTRL) or presence of 11 μM wt or A30P α-synuclein. Actin filaments were visualized by labeling with FITC-phalloidin. (B) Length distribution of the filaments shown in A. For each condition, 130–170 bundles from five fields of view were analyzed. Horizontal bars indicate mean values (* p <0.05). (C) Negative staining of actin bundles prepared as in A. Bar, (A) 5 μm; (C) 0.1 μm.

Figure 3.

A30P α-synuclein disrupts the actin cytoskeleton of N2A neuroblastoma cells. (A) Western blot of α-synuclein internalization in N2A cells incubated with 12 μM recombinant α-synuclein in control conditions or after permeabilization with SLO. (B) Distribution of actin and α-synucleins in SLO-permeabilized cells either unloaded (CTRL) or loaded with 12 μM wt or A30P α-synucleins. (C) Western blot analysis of α-synucleins expression in nontransfected (CTRL) or stably transfected N2A cells. (D) Distribution of actin and α-synucleins in nontransfected (CTRL) or stably transfected N2A cells. (A) and (C) β-tubulin is shown as an internal standard. (B and D) Actin was visualized by FITC-labeled phalloidin (green) and α-synuclein by indirect immunofluorescence (red). Notice the disappearance of the peripheral actin spikes with retraction of actin to the perinuclear area in the cells loaded (B) or transfected (D) with A30P α-synuclein, but not in those loaded or transfected with wt α-synuclein. Bars, 10 μm.

Figure 4.

During dynamic remodeling, actin accumulates in restricted zones of A30P α-synuclein–expressing N2A cells. (A) Western blot showing changes in the actin state of polymerization induced by a 5-min incubation with LatA. Filamentous and soluble actin in the cell extracts were separated by a 30 min centrifugation at 355,000 × g (P, pellet; S, supernatant). (B) N2A cells either nontransfected (CTRL) or stably expressing wt or A30P α-synucleins were probed for filamentous actin and α-synuclein as in Figure 3 D. Cells were fixed before addition of LatA (NO LatA), after incubation with the drug (LatA) or after 20 min of recovery (LatA rec). Panels at the bottom right are higher magnifications of cells expressing A30P showing actin-rich foci after 20 min of recovery from LatA. (C) Quantification of the average cell area of N2A cells either nontransfected (CTRL) or expressing wt or A30P α-synuclein, before LatA treatment (NO LatA) and after a 20 min recovery from a 5-min exposure to the drug (rec 20). Bars, 10 μm.

Figure 5.

During actin repolymerization A30P α-synuclein induces the formation of transient actin-rich foci in MDCK cells. (A) Expression of wt or A30P α-synucleins in IPTG-induced clones of MDCK cells. β-Tubulin is shown as an internal standard. (B) Actin distribution in the pellet and supernatant fractions of extracts of MDCK cells treated or not with LatA as in Figure 4A. (C) Actin repolymerization assays. MDCK cells expressing wt or A30P α-synucleins were fixed and probed for F-actin (red) and α-synuclein (green). Cells were fixed before addition of LatA (NO LatA), after 3- and 10-min incubation with the drug (LatA), and after 20, 40, and 60 min of recovery from a 10-min incubation (LatA rec). (D) Middle section of a 3D stack of images on the Z axis showing actin-rich foci in A30P α-synuclein–expressing cells after 20 min of recovery. Right and bottom panels, the Y-Z and X-Z projections at positions marked by the vertical and horizontal gray lines, respectively. (E) Quantification of the cell areas exhibiting actin fluorescence above an arbitrary threshold value in the images of MDCK cells either not induced (CTRL) or expressing wt or A30P α-synucleins. Measurements were performed in cells fixed before LatA addition or at 20-min recovery. A total of 400 × 400 pixels were analyzed in 20 fields of view in five different experiments. The graph highlights the presence of areas of actin compaction in A30P cells at the 20-min recovery time. Bars, 10 μm.

Figure 6.

Co-IP of actin and wt or A30P α-synuclein from homogenates of MDCK cells expressing wt or A30P α-synucleins. (A) Stably transfected MDCK cells, CTRL, and those expressing wt or A30P α-synuclein were lysed under control conditions or after a 20-min recovery from a 10-min incubation with LatA. After a 30 min centrifugation at 355,000 × g, proteins from the pellets and the final supernatants (sups) were stained with anti-α-synuclein and anti-actin antibodies. At 20-min recovery (LatA rec 20 min), F-actin is increased in the A30P α-synuclein–expressing cells, whereas soluble actin is more abundant in the wt α-synuclein–expressing cells. (B) PNS from MDCK cells expressing wt or A30P-α-synuclein treated as in A were subjected to IP with the anti-α-synuclein antibody. In the case of wt α-synuclein the coprecipitated actin was slightly more abundant in the untreated sample, whereas in the case of A30P α-synuclein it was more abundant after LatA recovery. The amount of PNS loaded corresponds to 5% of the total cell extracts. (C) Final supernatant obtained as in A from MDCK cells under control conditions or after 10-min incubation with LatA were subjected to IP as in B. No coprecipitation of soluble actin with A30P α-synuclein was observed, whereas wt α-synuclein coimmunoprecipitated soluble actin especially when its amount was increased by LatA-induced depolymerization.

Figure 7.

Time-lapse microscopy of MDCK cells during LatA treatment and washout. A30P α-synuclein expression inhibits actin reorganization, lamellipodia formation and cell movement. (A) The three rows show frames from time-lapse microscopy videos (Videos 1–3) of CTRL, wt α-synuclein, or A30P α-synuclein–expressing MDCK cell transfected with actin-GFP, during LatA treatment and washout. Notice in the top row the appearance of lamellipodia guiding the cell movement during wash out of LatA; in the low bottom row the appearance, already at 2 min, of large subplasmalemma GFP-actin aggregates persisting up to the end of the experiment. (B) Relative changes of the average cells area in MDCK cells during LatA incubation and recovery as in A. The area is normalized to the −10-min time point. (C) Changes of the rate of cell movement in MDCK during LatA incubation and recovery as in A, expressed as the traveled distance of the nucleus. The points in B and C are averages (±SEM) of three different measurements from independent experiments. Bar, 10 μm.

Figure 8.

Wt and A30P α-synucleins have different effects on cell migration into scratch wounds. (A) Monolayers of MDCK cells, CTRL and those expressing wt or A30P α-synucleins, were cultured until confluence and then scratched with a micropipette tip. Phase-contrast images were collected at different time points after scratching as indicated. (B) The graph shows the migration of MDCK cells as such as those illustrated in A. Each point is the average ± SD of 10 different measurements in five microscopic fields. Bar, (A) 500 μm.

Figure 9.

A30P α-synuclein affects exo- and endocytosis. (A) CTRL, wt, or A30P α-synuclein–expressing MDCK cells were incubated with FM4-64-FX for 5 min, followed by an additional 5-min incubation with the dye in the presence or absence of 200 μM ATP. After washing to eliminate the noninternalized dye the cells were fixed and analyzed for FM fluorescence. Bar, 10 μm. (B) Quantification of total fluorescence from five different fields of two independent experiments. Notice the lack of ATP-induced exo- and endocytosis increase in A30P α-synuclein–expressing cells. (C) Experiment similar to that described in A performed on N2A cells loaded with FM 4-64-FX and either nonstimulated or depolarized with 50 mM KCl. Notice the decreased K⁺-induced exo- and endocytic response in A30P α-synuclein–expressing cells. (B and C) Fluorescence values of are expressed as arbitrary units ± SD; * p < 0.05.

Figure 10.

Wt α-synuclein increases actin instability and A30P α-synuclein induces the formation of actin foci in hippocampal neurons. (A) Immunofluorescence analysis of embryonic hippocampal neurons electroporated either with an empty vector (CTRL) or with the constructs coding for wt or A30P α-synuclein. Neurons were fixed and probed for filamentous actin and α-synucleins at 2DIV, either under control conditions or after a 1-h LatA incubation, followed or not by recovery (LatA rec) for the indicated time points. (B) Higher magnification of the area close to the cell bodies of neurons expressing either exogenous wt or A30P α-synuclein, treated with LatA for 1 h, and fixed after a 1-h recovery. Notice the appreciable increase of the area and the formation of lamellipodia-like structures (asterisks) in the wt α-synuclein neuron and the areas of actin enrichment along the neurites (arrowheads) in the A30P α-synuclein neuron. (C) Statistical analysis of the average length (± SD) of minor and major neurites in embryonic hippocampal neurons electroporated either with an empty vector (CTRL) or with the constructs coding for wt or A30P α-synuclein (□, , and ■, respectively). The minor neurites of the wt α-synuclein–expressing neurons are moderately longer, and the major neurites are shorter, and A30P α-synuclein expressing neurons display a significant decrease of the length of the major neurite. (D) Statistical analysis of the mean cell body area in CTRL, wt α-synuclein, and A30P α-synuclein–loaded embryonic hippocampal neurons, either untreated (NO LatA) or treated with LatA followed or not by recovery. After recovery (LatA rec 1 h, LatA rec 2 h), wt α-synuclein–expressing neurons display a significant increase in the cell body area, compared with CTRL and A30P α-synuclein–expressing neurons. (E) Statistical analysis of the average number of actin foci per neurite (± SD) in CTRL, wt, or A30P α-synuclein–expressing embryonic hippocampal neurons, either untreated (NO LatA) or treated with LatA followed or not by recovery. In A30P α-synuclein neurons the number of actin foci is significantly increased after LatA treatment (LatA) and during the actin cytoskeleton recovery (LatA rec 1 h). (F) Anti-actin immunoprecipitate obtained from the final supernatant isolated from a rat brain homogenate cleared with 1% Triton X-100 and centrifuged at 355,000 × g for 40 min. Actin coimmunoprecipitated endogenous α-synuclein. In the lane labeled FS, 2% of the brain final supernatant was loaded. In Ab, 0.2 μg of the anti-actin antibody was loaded as an internal reference. Statistical analysis in C–E was performed on 40 different images from three experiments. In C–E; * p < 0.05 and ** p < 0.01. Bars, (B and C) 20 μm.

Figure 11.

Hypothetical model of α-synuclein mechanisms of action on the actin cytoskeleton. According to the proposed model, both α-synucleins exert an actin bundling activity. However, only wt α-synuclein sequesters actin monomers. In contrast, A30P α-synuclein induces the formation of actin-rich foci around an A30P α-synuclein core. Blue arches, wt α-synuclein; green arches, A30P α-synuclein.