Wild type human α-synuclein (a.a. 1–140) was bacterially expressed in E. coli BL21 (DE3) from a pET28b plasmid (Novagen), as previously described (Figure 1A; Pivato et al., 2012). CC dimer was produced by adding terminal GC residues after a.a. 140 of the α-synuclein sequence, which allowed the molecules to be covalently linked by disulfide bonds at their C-termini (Figure 1B). NC dimer was produced by including two full-length copies of α-synuclein in the same plasmid, thus generating a dimer as a single polypeptide (Figure 1B). After expression and purification, α-synuclein dimers were isolated from monomers using anionic exchange and then lyophilized for long-term storage. Prior to use, the lyophilized dimers were resuspended in lamprey internal solution (180 mM KCl, 10 mM HEPES K⁺, pH 7.4). Any unwanted higher molecular weight α-synuclein oligomers or aggregates that may have formed during the lyophilization process were removed by passing the protein sample through a Microcon^® YM-100 centrifugal filter. Prior to injection, all preparations of monomeric and dimeric α-synuclein were run on a 12% SDS-PAGE gel and Coommassie stained in order to confirm purity and molecular weights (Figure 1C).

Liposomes were generated using 16:0–18:1 phosphatidylcholine (PC), or alternatively 16:0–18:1 1-phosphatidic acid (PA) and PC in a 1:1 ratio (Avanti Polar Lipids, Alabaster, AL, USA). The PC component of all liposomes included 1% fluorescently tagged 18:1–12:0 nitrobenzoxadiazole-PC (NBD-PC). Liposomes were prepared as previously described (Busch et al., 2014). Briefly, 1 mg of total lipids was added to 200 μl of 2:1 chloroform:methanol and dried over a stream of nitrogen. The membranes were swollen in 300 mM sucrose at 37°C for 20 min and then vortexed, thus forming large, heterogeneous liposomes. Liposomes were sonicated in order to generate small, uniform liposomes (~30 nm diameter) and then ultracentrifuged at 80,000× g for 20 min at 25°C to remove any large liposomes by sedimentation. Liposome binding was performed as previously described (Burré et al., 2012; Busch et al., 2014). The small, sonicated liposomes (17 μl) were incubated with 5 μg of α-synuclein in HKE buffer (25 mM HEPES, pH 7.4, 150 mM KCl, 1 mM EDTA) for 2 h at room temperature (RT; 100 μl total volume). Samples were then loaded onto the bottom of an Accudenz gradient (40%, 35%, 30%, 0% from bottom to top; 800 μl total; Accurate Chemical and Scientific Corp., Westbury, NY, USA) and ultracentrifuged at 280,000× g for 3 h at RT (Figure 2A). After ultracentrifugation, samples were separated into eight equal fractions, and fluorescence measurements were made on each fraction using a Nanodrop 3300 in order to determine the distribution of liposomes. In all experiments, it was determined that >90% of the liposomes floated up into fractions 1–3 (Figure 2B). The amount of α-synuclein in each fraction was subsequently determined by Western blotting, as in prior studies (Figure 2C; Burré et al., 2012; Busch et al., 2014). α-Synuclein was detected using a pan-synuclein antibody raised against the N-terminus of human α-synuclein, which recognized both monomeric and dimeric α-synuclein (1:1000; ab53726; Abcam, Cambridge, MA, USA). The secondary antibody used was a goat anti-rabbit HRP conjugated IgG (H + L) (Thermo Scientific, Waltham, MA, USA). Protein bands were detected using Pierce™ ECL Western blotting substrate (Thermo Scientific, Waltham, MA, USA). α-Synuclein band intensity was quantified in each fraction using FIJI 2.0.0 software, and the percentage of liposome-bound α-synuclein in fractions 1–3 was determined in n = 3–4 independent experiments for each experimental condition.

All animal procedures were approved by the Institutional Animal Care and Use Committee at the Marine Biological Laboratory in Woods Hole, MA and in accordance with standards set by the National Institutes of Health. Lampreys (Petromyzon marinus; 11–13 cm) were anesthetized in MS-222 (0.1 g/L; Western Chemicals Inc., Ferndale, WA, USA), after which 2–3 cm pieces of spinal cords were microdissected and pinned in a Sylgard-lined petri dish containing fresh, oxygenated Lamprey Ringer (100 mM NaCl, 2.1 mM KCl, 1.8 mM MgCl₂, 4 mM glucose, 2 mM HEPES, 0.5 mM L-glutamine, 2.6 mM CaCl_2, pH 7.4). CC dimer (50 μM), NC dimer (80 μM), Dynasore (80 μM; Abcam ab120192; Cambridge, MA, USA) and monomeric α-synuclein (160 μM; rPeptide), all diluted in lamprey internal solution to the indicated stock concentrations, were loaded into glass microelectrodes and subsequently injected into lamprey giant RS axons using small pulses of nitrogen delivered via a Toohey Spritzer. All reagents were co-injected with a fluorescent dextran approximating their molecular weights in order to determine how much reagent was injected and how far it spread via axonal diffusion. For experiments with α-synuclein dimers, we co-injected a 40 kDa fluorescein dextran (Thermo Fisher Scientific, D1845; Waltham, MA, USA). For Dynasore experiments, we co-injected a 3 kDa fluorescein dextran (Thermo Fisher Scientific, D3309; Waltham, MA, USA). For experiments with monomeric α-synuclein, we co-injected a 10 kDa Alexa Fluor^® 488 dextran (Thermo Fisher Scientific, D22910; Waltham, MA, USA). The spread of the fluorescent dye, as well as its intensity along the axon relative to the injection site, were monitored during the injection in order to estimate the final intra-axonal concentration of the proteins (see Figure 3B). By the fluorescence measurements, the α-synuclein and other reagents were diluted >10–20 times from the stock concentration in typical injection experiments.

After the injections were complete, the lamprey giant RS axons were stimulated intracellularly with short current pulses (30–70 nA; 1 ms) in order to trigger action potentials (20 Hz, 5 min). Spinal cords were fixed immediately in 3% glutaraldehyde, 2% paraformaldehyde in 0.1 M Na cacodylate, pH 7.4, processed with 2% osmium in 2% potassium ferrocyanide (EM Sciences, Hatfield, PA, USA), stained en bloc with 2% uranyl acetate and embedded in EMbed 812 resin, as previously described (Morgan et al., 2004, 2013b; Busch et al., 2014). In order to evaluate the tissue quality and to identify the injected axon, histological staining was performed on 1 μm sections stained with a 1% toluidine blue, 1% sodium borate solution for 1–2 min (see Figure 3A, for example). For electron microscopy, spinal cords were thin sectioned at 70 nm onto formvar coated copper slot grids, post stained with 2% uranyl acetate and 0.4% lead citrate, and subsequently imaged using a JEOL JEM 200CX transmission electron microscope at 37,000× or 59,000× magnification. For each experimental condition, all synapses containing a clearly defined presynaptic active zone were imaged in series. Images that contained the center of the active zone were selected for further morphometric analyses (n = 10–26 synapses per condition).

As described previously, morphometric analyses were performed on all synaptic membranes within a 1 μm radius of the presynaptic active zone using FIJI 2.0.0 by a researcher blinded to the experimental conditions (Morgan et al., 2004, 2013b; Busch et al., 2014). These analyses included the number of SVs, size of plasma membrane (PM) evaginations, number and size of membranous “cisternae”, and number and stage of CCPs and CCVs per synapse, as determined from single thin sections. SVs were defined as small, clear round vesicles <100 nm in diameter. PM evaginations were measured by drawing a straight, 1 μm line from the edge of the active zone to the nearest point on the axolemma in either direction, measuring the curved distance between these points, then recording the average from each synapse. “Cisternae” were defined as larger, atypical vesicles >100 nm in diameter. CCP/Vs were determined by the presence of an electron dense coat around the vesicle membrane. In addition, a total membrane analysis was performed on each synapse in order to determine how the synaptic membranes were redistributed after acute perturbations. Here, the SV and CCP/V membrane areas were determined by multiplying the surface area of a sphere (4πr²) by the number of SVs or CCP/Vs at each synapse, respectively, where r = d/2 and d = the average diameter of 200 SVs measured from each experimental condition. Cisternae and PM measurements were obtained by multiplying the summed cisternae perimeters and length of PM evaginations by the section thickness (70 nm) in order to obtain the total amount of membrane area. For the analysis on the necks of CCPs (Figure 6), the neck width was measured at its narrowest point, and the neck length was measured from the PM to the beginning of the clathrin coat. All graphed data represent the mean ± SEM per section per synapse. Graphs were generated using Origin Pro 7.0 (OriginLab, Northampton, MA, USA). As previously described (Busch et al., 2014), 3D reconstructions of five serial electron micrographs were generated using Reconstruct software version 1.1.0.0 (Fiala, 2005). PM and cisternae were rendered as trace slabs; SVs and CCP/Vs were rendered as 50 nm and 90 nm spheres, respectively; active zone was rendered as a Boissonnat surface.

Human α-synuclein (140 a.a) comprises an N-terminal domain that mediates membrane binding and folds into an amphipathic helix upon interaction with small, highly curved vesicles; a central core non-amyloid component (NAC) domain that mediates self-association; and a less structured C-terminus containing acidic residues and prolines (Figure 1A). The goal of this study was to determine how α-synuclein dimers affect SV trafficking. We therefore synthesized two stable recombinant α-synuclein dimers, as described in the “Materials and Methods” section and a prior study (Pivato et al., 2012). CC dimer was generated by covalent linkage of two α-synuclein molecules at their C-termini (Figure 1B). NC dimer was generated by expression of two α-synuclein molecules in tandem in a single polypeptide, thereby bringing together the N- terminus of one α-synuclein with the C-terminus of the other (Figure 1B). While the exact conformations of endogenous α-synuclein dimers are unknown, formation of parallel and anti-parallel multimers was implied through the use of bimolecular fluorescence complementation assays (Wang et al., 2014). Thus, the recombinant α-synuclein dimers, which likely adopt parallel and anti-parallel conformations, allowed us to test the efficacy of each form. The α-synuclein dimers were stable in SDS-PAGE under non-reducing conditions (Figure 1C). While monomeric α-synuclein ran at ~14 kDa by SDS-PAGE, CC and NC dimers ran higher around ~28 kDa, as expected for these larger proteins (Figure 1C).

A prior study showed that the α-synuclein CC and NC dimers exhibited many biochemical properties similar to monomeric α-synuclein, including folding, fibrillation and aggregation, albeit with slightly altered dynamics (Pivato et al., 2012). Here, we additionally characterized the lipid binding capabilities of the α-synuclein dimers. Monomeric α-synuclein binds in vitro to anionic phospholipids when presented in small, highly-curved liposomes (30–50 nm diameter; Davidson et al., 1998; Burré et al., 2010, 2012, 2015; Busch et al., 2014). Lipid binding induces the N-terminal domain of α-synuclein to adopt an alpha helical conformation, which likely contributes to its ability to bind to SVs in vivo (Maroteaux et al., 1988; Davidson et al., 1998; Fortin et al., 2005). We previously showed that α-synuclein point mutations that reduced alpha helical content and liposome binding also greatly reduced the synuclein-induced vesicle trafficking defects at lamprey RS synapses, implicating membrane binding as a significant contributor to the observed synaptic defects (Busch et al., 2014). These findings are consistent with studies at mammalian synapses (Nemani et al., 2010; Xu et al., 2016; Eguchi et al., 2017).

To estimate whether α-synuclein dimers might also induce vesicle trafficking defects, we therefore tested the lipid binding capacity of the α-synuclein dimers using a well-established liposome binding assay (Figure 2; Burré et al., 2010, 2012; Busch et al., 2014). Briefly, monomeric or dimeric α-synuclein was incubated with small, sonicated liposomes (~30 nm) and then loaded onto the bottom of an Accudenz gradient. After ultracentrifugation and separation into eight fractions (Figure 2A), fluorometer measurements indicated that 90%–95% of the liposomes floated into the top three fractions (Figure 2B). Figure 2B shows representative fluorometer data obtained from liposomes made with PC or a 1:1 mixture of PC and PA. Consequently, any liposome-bound α-synuclein, as detected by Western blotting, will also be present in fractions 1–3, while unbound α-synuclein will remain in fractions 4–8 (Figure 2A). As previously reported (Burré et al., 2012; Busch et al., 2014), monomeric α-synuclein exhibited very little binding to PC liposomes, but bound robustly to PC/PA liposomes (Figures 2C,D; Monomeric α-synuclein—PC: 12.3 ± 7.4%, n = 4; PC/PA: 96.9 ± 1.6%, n = 3; Student’s t-test, p = 0.0002). Similarly, CC and NC dimers exhibited stronger binding to PC/PA liposomes than to PC only liposomes, as shown by a shift of the protein into the top fractions of the column (Figures 2C,D). Quantification of Western blot band intensities revealed significantly greater binding of the α-synuclein dimers to PC/PA liposomes, compared to liposomes containing only PC (Figure 2D; CC dimer—PC: 21.2 ± 2.5%, n = 3; PC/PA: 64.4 ± 9.7%, n = 3; Student’s t-test, p = 0.01; NC dimer—PC: 30.0 ± 3.5%, n = 3; PC/PA: 51.1 ± 3.9%, n = 3; Student’s t-test, p = 0.02). When directly compared, α-synuclein, CC and NC dimers exhibited no difference in binding to PC liposomes (ANOVA p > 0.05; Figure 2E). However, CC and NC dimers did exhibit a 34% and 47% reduction in binding to PC/PA liposomes, respectively (ANOVA p < 0.05; Figure 2E). Thus, while α-synuclein dimers preferentially bind to liposomes containing an anionic phospholipid such as PA, they also bind with reduced efficacy compared to monomeric α-synuclein, predicting that they may produce distinct effects on SV trafficking.

Next, we wanted to determine whether and how acute introduction of α-synuclein dimers affects SV trafficking. We used as our experimental model lamprey giant RS synapses, which are large (1–2 μm in diameter) glutamatergic, en passant synapses that reside along the perimeter of the giant axons (20–40 μm in diameter) within the ventromedial spinal cord (Figure 3A). α-Synuclein CC and NC dimers were acutely delivered via microinjection into the giant RS axons, providing direct access of the proteins to the presynapses (Figures 3B,C). Co-injection of a fluorescent dye with a similar molecular weight allowed us to approximate the concentrations of the α-synuclein dimers along the axon with respect to the injection site (Figure 3B). After dimer injections, the axons were stimulated intracellularly with action potentials (20 Hz, 5 min). Immediately following the stimulation period, spinal cords were fixed and processed for electron microscopy for visualization of RS synapses. The RS synapses consist of large vesicle clusters (~100–150 SVs/section; 1000–2000 SVs total) positioned along the axolemma and centered around an electron dense active zone (Figures 3C,D). We examined the ultrastructural effects of CC and NC dimers on synapses located within three regions of the axon (Figure 3B): (1) 25–225 μm from the injection site where α-synuclein dimer concentrations were higher (estimated 5–8 μM; see “Materials and Methods” section); (2) 250–390 μm from the injection site where α-synuclein dimer concentrations were lower (estimated 0.5–4 μM); and (3) >400 μm from the injection site where no exogenous α-synuclein was present (0 μM). This last region allowed us to assess the normal morphologies of untreated, stimulated synapses, thereby providing an internal control. The maximal amount of exogenous α-synuclein injected (5–8 μM) was only 2–3 times greater than the current estimate of endogenous α-synuclein at synapses, which is 3–6 μM (Westphal and Chandra, 2013). Thus, the additional α-synuclein introduced to synapses is commensurate with the two- to three-fold overexpression levels observed in PD and animal models (Singleton et al., 2003; Rockenstein et al., 2005; Nemani et al., 2010; Scott et al., 2010).

Under these stimulation conditions, control RS synapses exhibited large SV clusters, shallow PM evaginations and only a few CCPs (Figures 4A,D). Compared to unstimulated synapses (Figure 3D), stimulated synapses have slightly fewer vesicles, less compact SV clusters and more CCPs, indicating that vesicle recycling is occurring. In comparison, at synapses treated with low concentrations of α-synuclein CC dimer (α-Syn-CC-L), the SV clusters were even smaller; PM evaginations were more extended; and, large vesicles (>100 nm diameter) with irregular shapes, which we call “cisternae,” were more abundant (Figures 4B,E). In addition, there were greater numbers of clathrin-coated pits (CCPs), including complex tubules ending in clathrin coats, budding from the PM (Figures 4B,E; see insets). A loss of SVs combined with an increase in PM and endocytic intermediates is consistent with an inhibition of SV endocytosis from the PM. At higher concentrations of CC dimer (α-Syn-CC-H), a similar phenotype was observed with fewer SVs, larger PM evaginations and more cisternae and CCPs, but the effects were even more pronounced (Figures 4C,F). 3D reconstructions of serial sections corroborated the endocytic phenotypes observed in single sections and also further emphasized the build up of CCPs at the PM (Figures 4D–F; yellow spheres and insets). In contrast, the free CCVs did not appear to be dramatically affected (Figures 4E,F, white spheres). A higher magnification view of the synaptic membranes revealed that control synapses typically exhibited only one or several CCPs with constricted necks (Figure 4G). In comparison, synapses treated with CC dimer often exhibited rows of constricted CCPs with normal appearance emanating directly from the PM (Figure 4H, arrowheads), as well as atypical complex buds and tubules ending in multiple clathrin coats (Figures 4I–K, arrowheads), suggesting a defect in vesicle fission during SV endocytosis.

Morphometric analyses further corroborated that α-synuclein CC dimer induced endocytic defects at synapses. The number of SVs was significantly reduced at synapses treated with CC dimer, compared to controls, especially at higher concentrations (Figure 4L; Control: 112.4 ± 11.7 SVs, n = 26 synapses, 3 axons; α-Syn-CC-L: 88.5 ± 7.8 SVs, n = 27 synapses, 2 axons; α-Syn-CC-H: 45 ± 5.3 SVs, n = 25 synapses, 2 axons; ANOVA p = 3.3 × 10⁻⁶; Tukey’s post hoc). The mean diameter of the remaining SVs was not significantly altered (Control: 52.2 ± 0.7 nm, n = 200 SVs; α-Syn-CC-L: 50.9 ± 0.6 nm, n = 200 SVs; α-Syn-CC-H: 53.7 ± 0.9 nm, n = 200 SVs; ANOVA p = 0.03; Tukey’s post hoc). PM evaginations became progressively larger with increased concentrations of CC dimer (Figure 4M; Control: 2.0 ± 0.2 μm, n = 26 synapses, 3 axons; α-Syn-CC-L: 2.6 ± 0.2 μm, n = 27 synapses, 2 axons; α-Syn-CC-H: 3.7 ± 0.3 μm, n = 25 synapses, 2 axons; ANOVA p = 9.7 × 10⁻⁷, Tukey’s post hoc). Both the number and size of cisternae were also significantly increased after treatment with CC dimer (Figures 4N,O) (# Cisternae—Control: 2.9 ± 0.4 cisternae, n = 26 synapses, 3 axons; α-Syn-CC-L: 5.7 ± 1.0 cisternae, n = 27 synapses, 2 axons; α-Syn-CC-H: 7.5 ± 0.9 cisternae, n = 25 synapses, 2 axons; ANOVA p = 6.5 × 10⁻⁴, Tukey’s post hoc; Size Cisternae—Control: 0.37 ± 0.01 μm, n = 76 cisternae, 26 synapses; α-Syn-CC-L: 0.38 ± 0.01 μm, n = 153 cisternae, 27 synapses; α-Syn-CC-H: 0.49 ± 0.03 μm, n = 186 cisternae, 25 synapses; ANOVA p = 2.3 × 10⁻⁴, Tukey’s post hoc). In addition, the total number of CCPs and CCVs combined (i.e., “clathrin coats”) was also significantly increased (Figure 4P; Control: 3.4 ± 0.3 coats, n = 26 synapses, 3 axons; α-Syn-CC-L: 7.1 ± 0.8 coats, n = 27 synapses, 2 axons; α-Syn-CC-H: 7.1 ± 0.8 coats, n = 25 synapses, 2 axons; ANOVA p = 0, Tukey’s post hoc). We also performed a comprehensive analysis of the total membrane distribution at synapses. This analysis revealed that the loss of SV membrane area was compensated by greater membrane area in the PM, cisternae and clathrin coat compartments (Figure 4Q; Control: 1.4 ± 0.1 μm², n = 26 synapses, 3 axons; α-Syn-CC-L: 1.3 ± 0.08 μm², n = 27 synapses, 2 axons; α-Syn-CC-H: 1.3 ± 0.08 μm², n = 25 synapses, 2 axons; ANOVA p = 0.84, Tukey’s post hoc). Taken together, these data indicate that α-synuclein CC dimer induced a dose-dependent inhibition of SV endocytosis from the PM, including effects on the clathrin pathway. The atypical CCPs observed further suggested that CC dimer impairs vesicle fission during clathrin-mediated endocytosis.

Similarly, we also examined the synaptic effects produced by NC dimer. Acute introduction of α-synuclein NC dimer produced nearly identical effects on SV endocytosis, but the effects were slightly more pronounced even at the lower concentrations. Both low and high concentrations of NC dimer impaired synaptic morphology, resulting in progressively smaller SV clusters (Figures 5A–F,L; Control: 133.1 ± 15.8 SVs, n = 10 synapses, 2 axons; α-Syn-NC-L: 63.5 ± 8.3 SVs, n = 24 synapses, 1 axon; α-Syn-NC-H: 33.2 ± 5.2 SVs, n = 23 synapses, 1 axon; ANOVA p = 9.96 × 10⁻⁹; Tukey’s post hoc). At the higher concentrations of NC dimer, the mean SV diameter was slightly larger than at control synapses (Control: 54.0 ± 0.6 nm, n = 200 SVs, 10 synapses; α-Syn-NC-L: 55.7 ± 0.6 nm, n = 200 SVs, 10 synapses; α-Syn-NC-H: 56.5 ± 0.8 nm, n = 200 SVs, 10 synapses; ANOVA p = 0.02; Tukey’s post hoc). PM evaginations were significantly larger after treatment with NC dimer (Figures 5A–F,M; Control: 1.9 ± 0.2 μm, n = 10 synapses, 2 axons; α-Syn-NC-L: 4.0 ± 0.3 μm, n = 24 synapses, 1 axon; α-Syn-NC-H: 4.0 ± 0.4 μm, n = 23 synapses, 1 axon; ANOVA p = 0.00011, Tukey’s post hoc). As with CC dimer, the number and size of cisternae were also significantly increased (Figures 5N,O; # Cisternae—Control: 3.0 ± 0.6 cisternae, n = 10 synapses, 2 axons; α-Syn-NC-L: 9.6 ± 0.8 cisternae, n = 24 synapses, 1 axon; α-Syn-CC-H: 8.3 ± 1.3 cisternae, n = 23 synapses, 1 axon; ANOVA p = 0.0026, Tukey’s post hoc; Size Cisternae—Control: 0.39 ± 0.01 μm, n = 30 cisternae, 10 synapses; α-Syn-NC-L: 0.42 ± 0.01 μm, n = 219 cisternae, 24 synapses; α-Syn-NC-H: 0.49 ± 0.02 μm, n = 188 cisternae, 23 synapses; ANOVA p = 0.0039, Tukey’s post hoc). As with CC dimer, NC dimer also induced aberrant CCP budding from the PM (Figures 5E–K), resulting in greater numbers of CCPs with normal appearance (Figure 5H) as well as atypical, complex budding structures extending from the PM (Figures 5I–K, arrowheads). The total number of clathrin coats was significantly greater than at control synapses (Figure 5P; Control: 1.8 ± 0.3 coats, n = 10 synapses, 2 axons; α-Syn-NC-L: 8.2 ± 0.9 coats, n = 24 synapses, 1 axon; α-Syn-NC-H: 7.2 ± 0.9 coats, n = 23 synapses, 1 axon; ANOVA p = 2.81 × 10⁻⁴, Tukey’s post hoc). Total membrane analysis revealed that the loss of SV membrane area induced by NC dimer was compensated by greater membrane area in the PM, cisternae, and CCPs and CCVs (Figure 5Q; Control: 1.57 ± 0.17 μm², n = 10 synapses, 1 axon; α-Syn-NC-L: 1.44 ± 0.09 μm², n = 23 synapses, 1 axon; α-Syn-NC-H: 1.25 ± 0.12 μm², n = 24 synapses, 1 axon; ANOVA p = 0.21, Tukey’s post hoc). Thus, as with CC dimer, NC dimer also impaired clathrin-mediated SV endocytosis with apparent effects on the vesicle fission process.

Clathrin-mediated SV endocytosis proceeds through several morphologically distinct stages (Figure 6A; Brodin et al., 2000; Morgan et al., 2002, 2013a; Saheki and De Camilli, 2012). As in our prior studies, we have defined them according to the stages of clathrin coat formation (Morgan et al., 2004, 2013b; Bourne et al., 2006; Busch et al., 2014). Initiation of clathrin coat formation begins when the adaptor proteins AP180 and AP2 recruit clathrin triskelions to the PM (Stage 1). Following maturation of the clathrin coat (Stage 2), the GTPase dynamin is recruited to the neck of the CCP, where its GTPase activity leads to constriction and vesicle fission (Stage 3). Once separated from the PM, the free CCV is uncoated by the ATP activity of the chaperone protein Hsc70 (Stage 4). The aberrant vesicle budding observed with CC and NC dimers suggested that they may be affecting the process of vesicle budding at the fission step. To test this, we examined the number of CCPs (Stage 1–3) and CCVs (Stage 4) at synapses after treatment with the α-synuclein dimers. Both CC and NC dimers selectively and significantly increased the number of Stage 3 CCPs with constricted necks, indicating an impairment in vesicle fission (Figures 6B,C; CC dimer: Stage 1-Control: 0.1 ± 0.1 CCPs; α-Syn-CC-H: 0 ± 0 CCPs; Stage 2-Control: 0.3 ± 0.1 CCPs; α-Syn-CC-H: 0.3 ± 0.1 CCPs; Stage 3-Control: 2.2 ± 0.3 CCPs; α-Syn-CC-H: 5.4 ± 0.6 CCPs; Stage 4-Control: 0.9 ± 0.2 CCVs; α-Syn-CC-H: 1.4 ± 0.3 CCVs; n = 10–26 synapses per condition; ANOVA p < 0.00005, Tukey’s post hoc; NC dimer: Stage 1-Control: 0.1 ± 0.1 CCPs; α-Syn-NC-H: 0.1 ± 0.1 CCPs; Stage 2-Control: 0.4 ± 0.2 CCPs; α-Syn-NC-H: 0.5 ± 0.2 CCPs; Stage 3-Control: 1.1 ± 0.4 CCPs; α-Syn-NC-H: 5.3 ± 0.7 CCPs; Stage 4-Control: 0.4 ± 0.3 CCVs; α-Syn-CC-H: 1.4 ± 0.3 CCVs; n = 10–26 synapses per condition; ANOVA p = 0, Tukey’s post hoc).

To corroborate that α-synuclein dimers were indeed causing a bona fide fission defect, we also examined the effects of Dynasore (80 μM), a potent and selective inhibitor of dynamin GTPase activity that arrests late stages of budding prior to vesicle fission, leading to an increase in CCPs with constricted necks that are still attached to the membrane (Macia et al., 2006; Newton et al., 2006). We performed these experiments using the same injection and stimulation conditions as in the α-synuclein dimer experiments. After diffusion and dilution, we estimate that the final axonal concentration of Dynasore was ~4–8 μM. As with CC and NC dimers, Dynasore also selectively increased the number of stage 3 CCPs (Figure 6D; Stage 1-Control: 0.1 ± 0.1 CCPs; Dynasore: 0.1 ± 0.1 CCPs; Stage 2-Control: 0.4 ± 0.1 CCPs; Dynasore: 0.1 ± 0.1 CCPs; Stage 3-Control: 2.0 ± 0.2 CCPs; Dynasore: 3.5 ± 0.4 CCPs; Stage 4-Control: 1.1 ± 0.2 CCVs; Dynasore: 1.5 ± 0.4 CCVs; n = 23 control synapses, n = 19 Dynasore synapses; ANOVA p < 0.00005, Tukey’s post hoc). We also examined the effects of monomeric α-synuclein on each stage of clathrin coat formation, which we had not done in our prior study (Busch et al., 2014). Surprisingly, and in contrast to α-synuclein dimers and Dynasore, monomeric α-synuclein impaired a later stage of clathrin-mediated endocytosis during the uncoating step, as shown by a selective increase in stage 4, free CCVs (Figure 6E; Stage 1-Control: 0.1 ± 0.1 CCPs; Monomeric α-Syn: 0.2 ± 0.2 CCPs; Stage 2-Control: 0.3 ± 0.1 CCPs; Monomeric α-Syn: 0.8 ± 0.3 CCPs; Stage 3-Control: 1.4 ± 0.3 CCPs; Monomeric α-Syn: 1.0 ± 0.4 CCPs; Stage 4-Control: 0.6 ± 0.2 CCVs; Monomeric α-Syn: 3.7 ± 1.2 CCVs; n = 6–14 synapses per condition; ANOVA, p = 1.38 × 10⁻⁷, Tukey’s post hoc). Thus, α-synuclein CC and NC dimers selectively inhibited the vesicle fission step during clathrin-mediated SV endocytosis, while monomeric α-synuclein appeared to inhibit clathrin uncoating.

In order to better understand the fission defects, we further examined the morphologies of the CCPs observed in the presence of α-synuclein dimers. Free CCVs were excluded from these analyses. At stimulated control synapses, the vast majority of CCPs were single buds with short, constricted necks, and only a very small number of complex buds with multiple clathrin coats were observed (Figure 6F). However, in the presence of CC and NC dimers, individual CCPs often exhibited longer necks (Figure 6F, top, asterisks). Furthermore, with the α-synuclein dimers, there were larger numbers of complex buds comprising extended membrane tubules ending in multiple clathrin coats (Figure 6F, bottom). These complex budding structures typically emanated directly from the PM (Figure 6F, bottom, arrowheads). A clear example of this can be seen in Figure 6G, where a long, narrow tubule can be traced through two adjacent sections, showing its connection with the PM and a large number of budding CCPs attached (Figure 6G). The individual CCPs induced by α-synuclein dimers also appeared to be morphologically distinct from those observed in the presence of Dynasore or monomeric α-synuclein, which more closely resembled controls (Figure 6F, top). Complex buds and tubules were rarely if ever seen with Dynasore or monomeric α-synuclein treatment under these conditions (Figure 6F, bottom). Quantitatively, there were significantly more single and complex buds at synapses treated with CC and NC dimers, compared to the other experimental conditions (Figures 6H,I; Single buds—Control: 2.2 ± 0.2 CCPs; CC dimer: 4.8 ± 0.5 CCPs; NC dimer: 4.8 ± 0.5 CCPs; Dynasore: 3.4 ± 0.4 CCPs; Monomeric α-Syn; 2.1 ± 0.3 CCPs; p = 1.46 × 10⁻¹⁰; Tukey’s post hoc; Complex buds—Control: 0.1 ± 0.1 CCPs; CC dimer: 0.8 ± 0.3 CCPs; NC dimer: 1.0 ± 0.4 CCPs; Dynasore: 0.3 ± 0.2 CCPs; Monomeric α-Syn: 0 ± 0 CCPs; ANOVA p < 0.001; Tukey’s post hoc). When we further examined the necks of individual CCPs, we found no difference in the neck width at synapses treated with α-synuclein dimers, Dynasore, or monomeric α-synuclein, compared to controls (Figure 6J; Control: 31.5 ± 1.1 nm, n = 39 CCPs, 28 synapses; α-Syn CC: 28.9 ± 1.2 nm, n = 34 CCPs, 10 synapses; α-Syn NC: 35.4 ± 1.3 nm, n = 25 CCPs, 12 synapses; Dynasore: 33.3 ± 1.8 nm, n = 26 CCPs, 10 synapses; Monomeric α-Syn: 32.0 ± 1.6 nm, n = 16 CCPs, 6 synapses; ANOVA p = 0.01, Tukey’s post hoc). However, both CC and NC dimers significantly increased the neck length on CCPs, compared to all other conditions (Figure 6K; Control: 32.1 ± 1.5 nm, n = 39 CCPs, 28 synapses; α-Syn CC: 52.0 ± 2.7 nm, n = 34 CCPs, 10 synapses; α-Syn NC: 58.7 ± 3.6 nm, n = 25 CCPs, 12 synapses; Dynasore: 28.6 ± 1.8 nm, n = 26 CCPs, 10 synapses; Monomeric α-Syn: 27.3 ± 1.6 nm, n = 16 CCPs, 6 synapses; ANOVA p = 0; Tukey’s post hoc). The greater numbers of elaborated complex buds connected by membrane tubules, as well as CCPs with longer extended necks, provides further indication that α-synuclein dimers inhibit vesicle fission during clathrin-mediated SV recycling.

The analyses above suggested that monomeric and dimeric α-synuclein produced different effects on clathrin-mediated SV recycling, such that the dimeric α-synuclein inhibited an earlier step during vesicle fission while monomeric α-synuclein inhibited a later step during clathrin uncoating. To corroborate this, we further examined the endocytic phenotype produced by monomeric α-synuclein and directly compared it against that produced by dimers. Notably, in the presence of monomeric α-synuclein, free CCVs were abundant (Figure 7A, blue circles), whereas the budding CCPs were sparse (Figure 7A, red circles). This can be seen clearly in Figure 7C where a single CCP (red arrowhead) is surrounded by a cluster of CCVs (blue arrowheads). Clusters of CCVs are highly unusual and are typically due to clathrin uncoating defects, for example after perturbations of the clathrin uncoating ATPase, Hsc70 (Morgan et al., 2001). As reported in our previous study (Busch et al., 2014), monomeric α-synuclein inhibited SV recycling, as indicated by a loss of SVs, which was compensated by an expansion of PM and increased numbers of endocytic intermediates, including cisternae and total clathrin coats (Figures 7D–I; SVs: Control: 127.6 ± 12.1 SVs; α-Syn: 34.3 ± 5.0 SVs, Student’s t-test p = 0.00012; PM: Control: 1.8 ± 0.1 μm; α-Syn: 2.9 ± 0.5 μm, Student’s t-test p = 0.005; # Cist: Control: 2.9 ± 0.5 cisternae; α-Syn: 11.8 ± 3.3 cisternae, Student’s t-test p = 0.00082; Cist Size: Control: 0.40 ± 0.02 μm; α-Syn: 0.63 ± 0.06 μm, Student’s t-test p = 0.006; Clathrin coats: Control: 2.4 ± 0.4 coats; α-Syn: 5.7 ± 1.4 coats, Student’s t-test p = 0.005; Total membrane: Control: 1.47 ± 0.13 μm², α-Syn: 1.14 ± 0.21 μm², Student’s t-test p = 0.18; n = 14 Control synapses, n = 6 α-Syn synapses). While stimulated control, dimer- and Dynasore-treated synapses normally exhibited ~1 CCV per section (Figures 6B–D, Stage 4), in contrast, after treatment with monomeric α-synuclein, the free CCVs were most prevalent (Figure 6E, Stage 4; Figures 7A,C).

A direct comparison of the clathrin coat defects produced by monomeric and dimeric α-synuclein further demonstrated the differences in their phenotypes. We generated 3D reconstructions of synapses where the distributions of CCPs and CCVs are shown in the absence of the other synaptic organelles. These reconstructions clearly revealed that, in comparison to control synapses, monomeric α-synuclein caused an atypical build up of free CCVs that were dispersed throughout the synaptic area (Figures 7J,K, white spheres). Conversely, CC and NC dimer caused a buildup of CCPs at the PM (Figures 7L,M, yellow spheres), and very few CCVs were observed. High magnification electron micrographs clearly revealed the differences in the clathrin-coated structures. Monomeric α-synuclein induced atypical clusters of free CCVs that were separated from the PM (Figure 7N, blue arrowheads). α-Synuclein dimers induced CCPs with constricted necks that were still connected to the PM (Figures 7O,P, red arrowheads). These data provide evidence that monomeric and dimeric α-synuclein affect clathrin-mediated SV recycling at different stages during vesicle endocytosis.