α-Synuclein is the major platelet isoform but is dispensable for activation, secretion, and thrombosis

Abstract Platelets play many roles in the vasculature ensuring proper hemostasis and maintaining integrity. These roles are facilitated, in part, by cargo molecules released from platelet granules via Soluble NSF Attachment Protein Receptor (SNARE) mediated membrane fusion, which is controlled by several protein-protein interactions. Chaperones have been characterized for t-SNAREs (i.e. Munc18b for Syntaxin-11), but none have been clearly identified for v-SNAREs. α-Synuclein has been proposed as a v-SNARE chaperone which may affect SNARE-complex assembly, fusion pore opening, and thus secretion. Despite its abundance and that it is the only isoform present, α-synuclein’s role in platelet secretion is uncharacterized. In this study, immunofluorescence showed that α-synuclein was present on punctate structures that co-stained with markers for α-granules and lysosomes and in a cytoplasmic pool. We analyzed the phenotype of α-synuclein−/− mice and their platelets. Platelets from knockout mice had a mild, agonist-dependent secretion defect but aggregation and spreading in vitro were unaffected. Consistently, thrombosis/hemostasis were unaffected in the tail-bleeding, FeCl3 carotid injury and jugular vein puncture models. None of the platelet secretory machinery examined, e.g. the v-SNAREs, were affected by α-synuclein’s loss. The results indicate that, despite its abundance, α-synuclein has only a limited role in platelet function and thrombosis. Plain Language Summary What did we know? The N-terminus of α-Synuclein affects SNARE-complex assembly, fusion pore opening, and granule docking. Microvascular bleeding is seen in Parkinson Disease patients where α-synuclein has a pathological role. What did we discover? α-Synuclein colocalizes with P-selectin (α-granules) and LAMP-1 (lysosomes) in platelets. The loss of α-synuclein has only a mild, agonist-dependent effect on platelet secretion. The loss of α-synuclein had no effect on thrombosis/hemostasis in 3 injury models. What is the impact? Despite its abundance, α-synuclein is not required for platelet secretion. α-Synuclein is not required for hemostasis or thrombosis.


Introduction
Platelets play many roles in the vasculature ensuring hemostasis and maintaining vascular integrity.These processes are supported by the various cargo molecules released from the three types of granules in platelets: α, dense, and lysosomes. 1,2ranule cargo release is mediated by a family of membrane proteins called Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptors (SNAREs).SNAREs are classified based on their subcellular localization and a charged amino acid at their SNARE domain's center: (v/R, Arg) SNAREs located on the vesicles and (t/Q, Glu) SNAREs located on target membranes. 3,4These proteins (v-SNAREs and t-SNAREs) form a transmembrane complex that spans the two bilayers to mediate granule-membrane fusion for cargo release. 5,6[9][10][11][12] Several SNARE regulators have also been identified in platelets.4][15] Others, such as Munc18b and STXBP5, serve as chaperones that control when and where the SNAREs interact.Dysfunctions in these regulators have variable effects.Some are essential i.e., Munc13-4, while the loss of others has only a modest effect on release, i.e., Exocyst. 13,15Taken together, these data imply that platelet secretion is controlled by sequential protein-protein interactions culminating in membrane fusion and cargo release.
The chaperone mode of SNARE regulation is exemplified by Munc18b and its interaction with Syntaxin-11.Sec1/Munc18 (SM) proteins are needed for cognate syntaxin sorting and serve as a template for the formation of the transbilayer SNARE complex. 14,16Chaperones for other classes of SNAREs (i.e., VAMPs and SNAP-23/25s) have been reported but not as definitively characterized.Proteins of interest as potential chaperones are the synucleins.1][32][33][34] Studies have shown that α-synuclein enhances fusion pore opening and docking, and that the N-terminal region is critical for these functions. 22,24,33,35][37] Most cases of Parkinson disease are idiopathic, but there are some rare familial cases caused by mutations in α-synuclein including A30P, A53T, E46K, H50Q, and G51D.9][40][41][42] Studies show that cerebral microbleeds occur more often in Parkinson disease patients that have developed dementia than patients without dementia.This increased risk is associated with cerebral amyloid angiopathy, which affects vascular integrity potentially leading to the development of microbleeds.Data are inconsistent regarding whether microbleeds occur more often in either the deep or lobar regions of the brain, but there is a clear association between microbleeds and cognitive decline.Whether this bleeding risk is due to vascular defects or hemostatic dysfunction is unclear.Since platelet function is important for controlling microvessel integrity, we sought to determine if α-synuclein contributes to platelet function, specifically in secretion and thrombosis. 30,33,34,43lthough α-synuclein has been suggested to have a role in SNARE-complex assembly and fusion pore opening, its exact molecular role in these processes is unknown.Platelets are a good model to study the function of α-synuclein since it is abundant and the only isoform present. 31,32In this manuscript, we analyze the role of α-synuclein using platelets from α-synuclein −/ − mice.We found that its deletion caused a modest defect in activation-dependent secretion.Consistent with this modest secretion defect, the α-synuclein −/− mice had no significant bleeding diathesis in the three models tested.Our data suggest that α-synuclein does not play a central role in platelet secretion and is thus either not required for SNARE-complex assembly or is compensated for by another protein or pathway.

Mouse strains
C57BL/6 mice were purchased from Jackson Laboratory and bred in our animal vivarium.The α-Synuclein −/− mice (B6; 129X1-Scna tm1Rosl /J; Stock # 003692) were purchased from Jackson Laboratory and were bred using heterozygous crosses.Genotyping strategies are described in Supplemental Methods.

Platelet preparation from mouse blood
Mice were euthanized by CO 2 inhalation.The thoracic region was then exposed, and blood was collected from the right ventricle via a syringe with approximately 120 μL of 3.8% sodium citrate with apyrase (0.2 U/mL) and prostaglandin I 2 (PGI 2 ; 1 µg/mL).The platelets were prepared from pooled C57BL/6 wild-type mice and αsynuclein −/− mice as described. 2,44Platelet concentration was calculated using a Z2 Counter (Beckham Coulter, Inc. Brea, California).

Granule secretion measurements
Washed mouse platelets were collected and isolated as described above and incubated with 0.4 µCi/mL [ 3 H] serotonin (Perkin-Elmer Cetus Life Sciences, Boston, MA) for 30 min at 37°C.Final platelet concentrations were adjusted to 2.5 × 10 8 /mL and CaCl 2 was added to a final concentration of 1 mM.Platelets were either stimulated with various thrombin concentrations (0.005, 0.01, 0.05, 0.1, or 0.5 U/mL) or for different time points (15,  30, 45, 60, 90, 120, and 300 sec).Hirudin (2X the concentration of thrombin) was used to stop the reactions.Samples were then placed on ice.All samples were centrifuged at 16 200 × g for 2 min in a microfuge.The supernatants were transferred to another tube, and the pellets were lysed with 60 μL of lysis buffer (1% Triton X-100 in 1X PBS, pH 7.4) for 45 min on ice.To measure the secretion kinetics from the three granules equal volumes of the supernatant and the solubilized pellet were taken, [ 3 H] serotonin was measured from dense granules, Platelet Factor 4 (PF4) from α-granules, and β-hexosaminidase from lysosomal granules.Release was calculated as percent release = [(amount in supernatant)/(amount in supernatant + amount in pellet)] * 100.

Lumi-aggregometry
Washed platelets were prepared as described above.ATP release and aggregation were measured using a Chrono-Log Model 700 Lumi-aggregometer (Havertown, PA).Mouse platelets (250 μL of 3 × 10 8 /mL) were placed in a siliconized glass cuvette (Chrono-Log) with a metal stirring bar (Chrono-Log) spinning at 1,200 RPM for 3 min at 37°C.To measure ATP release, 10 μL of Chrono-Lume reagent was also added (Chrono-Log).Agonists (thrombin or collagen as indicated) were added to initiate platelet activation as indicated.The aggregation traces were monitored by turbidity and ATP release was measured by luminescence using AGGRO/Link8 software (Chrono-Log) as described. 45

Flow cytometry
Washed mouse platelets (20 μL of 5 × 10 7 /mL) were either held in a resting state (no agonist) or stimulated with various concentrations of thrombin, convulxin, CRP, U46619 for 2 min at RT. Platelets then were incubated with 2.5 μL of FITC-conjugated or PE-conjugated antibodies for 20 min at 37°C (see Supplemental Methods).The samples were diluted 10-fold with HEPES Tyrode buffer pH 6.5 to stop the reaction and transferred to polystyrene Falcon TM tubes (BD Biosciences, San Jose, CA).Fluorescent intensity was measured using BD FACSymphony TM flow cytometer (BD Biosciences, San Jose, CA).The platelet populations were detected by adjusting the voltages for forward light scattering (FSC) and side light scattering (SSC).Fluorescent intensity was optimized by adjusting the voltage for excitation of green (FITC) and yellow (PE) channels.Platelet fluorescent intensities were analyzed using FlowJo TM software v10.8.0 (BD Biosciences, San Jose, CA).Fifty thousand events were analyzed for each sample, and geometric mean fluorescent intensities (GFMI) were calculated and plotted with statistical values.

Tail bleeding assay
Mice (6-8 weeks old of both sexes) were anesthetized using ketamine hydrochloride 75 mg/kg i.p.The tails were transected 3 mm from the tip and immediately placed into 37°C normal saline.The time from the transection until cessation of bleeding was recorded.After initial cessation of bleeding, the mice were observed for an additional minute to exclude re-bleeding.Bleeding was manually stopped in mice after 10 min.

FeCl 3 carotid injury model
This FeCl 3 -induced carotid injury model was performed as described. 46Mice (8-12 weeks old of both sexes) were anesthetized using Avertin (tribromoethanol; 0.2 g/kg, i.p.).The left carotid artery was exposed using blunt dissection under a dissecting microscope.A miniature Doppler flow probe (0.5VB, Transonic Systems Inc., Ithaca, NY, USA) was placed under the carotid artery to monitor blood flow.After baseline readings, thrombus formation was induced by placing a round filter paper (1 mm diameter) saturated with 7.5% FeCl 3 solution on top of the carotid artery for 3 min.The filter paper was removed and the time from the removal of the filter paper until cessation of blood flow was recorded.After recording for 30 min, the animal was euthanized.

Jugular vein puncture model
Mice (8-12 weeks old of both sexes) were anesthetized using Avertin (tribromoethanol; 0.2 g/kg, i.p.).The left external jugular vein was exposed using blunt dissection under a dissecting microscope.Venous injury was inflicted by puncturing the jugular vein with a 30 G needle.Normal saline is then pumped into the area to remove extravasating blood and help determine cessation of blood flow.Time from injury to the cessation of blood flow was recorded.

Data processing
GraphPad Prism v10.0.2 was used to generate secretion assay plots, FACS, aggregation, thrombosis models survival curves, and western blot quantifications.Statistical tests used were: two-way ANOVA multiple comparisons, unpaired Student t test, Kaplan-Meier method using the log-rank comparison test, and paired Student t test.Statistical analyses were computed by GraphPad Prism v10.0.2.In all studies, the p values are as indicated and values less than 0.05 were considered significant.

Study approval
All animal work was approved by the Institutional Animal Care and Use Committee at the University of Kentucky.Protocol #2019-3384.
Additional methods are described in detail in the Supplemental Methods.

Hematological profile of α-synuclein −/− mice
To probe the role of α-synuclein in thrombosis and hemostasis, we used global α-synuclein −/− mice from Jackson Laboratory and confirmed that α-synuclein was deleted from platelets via western blotting (for example see Figure 6).Platelet biogenesis appeared unaffected since platelet counts and sizes were similar in knockouts and WT littermate controls (Table I).Although platelets were normal in the α-synuclein −/− mice there was a statistically significant decrease in erythrocyte (RBC) volume 51.7 ± 1.35 fL versus 48.7 ± 1.22 fL (p value < 0.0001).There was no significant difference between leukocyte or RBC counts.The differences in erythrocytes were not pursued in this manuscript.While the values in literature are inconclusive and differ, potentially due to the use of different hematological analyzers, there have been reports that α-synuclein is abundant in RBCs.[49]

Co-localization of α-synuclein with α and lysosomal granules
To determine the localization of α-synuclein in platelets, we used 3-Dimensional Structured Illumination Microscopy (3D-SIM) to examine immunofluorescence staining (Figure 1A,B).The staining pattern of α-synuclein suggests both a diffuse cytoplasmic and a punctate, potentially granular distribution.To assess whether α-synuclein is on a granule, we performed colocalization studies with both P-selectin (membrane marker for α-granules) and LAMP-1 (membrane marker for lysosomes).In resting platelets, there was some overlap between LAMP-1 and α-synuclein though incomplete (Figure 1A; Pearson correlation coefficient: 0.345 and Mander's overlap: 0.675).Similarly, there was also some colocalization with P-selectin (Figure 1A; Pearson correlation coefficient: 0.408 and Mander's overlap: 0.680).Absence of nonspecific  antibody staining was confirmed in (Supplemental Figure S1A  and S1B).Colocalization studies with endosomal markers (i.e., Rabs) were not done, however, it is possible that αsynuclein could be present on those compartments based on the punctate staining pattern and the lack of complete colocalization with standard granule markers.We further addressed whether loss of α-synuclein affected dense granules.Given that methods for imaging dense granules are limited, we evaluated dense granule numbers per platelets by vital staining with mepacrine. 50The average numbers of mepacrine-positive structures were similar in wild-type (4.73 ± 1.74/platelet; n = 40 platelets) vs. α-synuclein −/− (4.93 ± 1.86/platelet; n = 27 platelets) platelets.Localization of αsynuclein in platelets was further assessed to determine the extent to which it associated with platelet membranes in either a resting or activated state (Figure 1C).Platelets were disrupted by freeze-thaw cycles and cytosolic and membrane fractions were generated by ultracentrifugation.In resting and activated platelets, α-synuclein is present mostly in the supernatant 70% (S1) and 30% in the pelleted membranes treated with Triton X-100 (S TX ).The ratio of α-synuclein in the membrane fractions did not change upon activation with 0.1 U/mL thrombin for 10 min.These data correlate with the immunofluorescence staining (Figures 1A,B) where we see a diffuse cytoplasmic staining of α-synuclein.
α-Synuclein −/− platelets have a mild secretion defect Surface exposure of P-selectin (α-granules), LAMP-1 (lysosomal granules), and α IIb β 3 integrin activation expression were assessed in α-synuclein −/− platelets by flow cytometry (Figure 2).Platelets were stimulated with thrombin (Figure 2A,E,I), convulxin (Figure 2B,F,J), CRP (Figure 2C,G) or U46619 (Figure 2D,H) for 2 min at various concentrations.In response to 0.1 U/mL thrombin, P-selectin, Jon/A and LAMP-1 expression were unaffected (Figure 2A,E,I).In response to both concentrations of convulxin (100 and 200 ng/mL), there was a slight reduction in P-selectin exposure (Figure 2B) and in Jon/A binding that did not reach statistical significance (Figure 2F).There was no significant difference in LAMP-1 exposure (Figure 2J).In response to both concentrations of CRP (1 and 10 µg/mL) and U46619 (30 and 1000 nM), there were slight reductions in both P-selectin exposure and Jon/A binding but these did not reach statistical significance (Figure 2C,D,G,H).We further addressed whether CD63 exposure was affected and there was no change in CD63 exposure upon thrombin, convulxin, CRP, U46619 stimulation (data not shown).These data imply that α-synuclein plays a minor role in granule secretion.To further define the role of α-synuclein in granule secretion, we examined agonist-and time-dependent release of cargo from all three granules: dense, α, and lysosomes (Figure 3).Dense granule secretion from α-synuclein −/− platelets, when stimulated for 2 min with thrombin was slightly decreased ~ α-Synuclein is dispensable for thrombosis 5 10% at the various thrombin concentrations (Figure 3A).Alpha and lysosomal secretion were comparable to WT platelets (Figure 3B,C).When α-synuclein −/− platelets were stimulated with 0.1 U/mL thrombin at various time points there was no significant difference in secretion from the dense, α, or lysosomal granules (Figure 3D,E,F).There were no defects in serotonin uptake (dense granule cargo), or the levels of PF4 (α-granule cargo) and β-hexosaminidase (lysosome cargo) in the αsynuclein −/− platelets (Supplemental Figure S3).These data suggest that α-synuclein plays only a minor role in platelet secretion and that it may play a subtle role in ITAM signaling pathways leading to integrin activation.

α-Synuclein −/− platelets have no aggregation or spreading defect
Dense granule release is extremely sensitive and has the fastest release kinetics compared to the other granules.To confirm the slight defect from the secretion assay, we used lumi-aggregometry to analyze aggregation and ATP/ADP release in response to 0.05, 0.025, and 0.0125 U/mL thrombin, and 5 μg/μL collagen.Aggregation was unaffected (Figure 4A-D).ATP/ADP release was minimally affected in α-synuclein −/− platelets (Figure 4E-H).
We also evaluated whether the deletion of α-synuclein could affect spreading on fibrinogen-coated surfaces (Supplemental Figure S4).α-Synuclein −/− platelets spread at similar rates when compared to WT platelets at the respective time points (30, 60, 90, and 120 min).These data imply that the loss of α-synuclein has no significant effects on specific platelet activities.

Loss of α-synuclein did not affect the platelet secretory machinery
Semiquantitative western blotting was used to assess the levels of other proteins in the platelet secretory machinery (Figure 6).Expression of the v-SNAREs VAMP-2, VAMP-3, VAMP-7, and VAMP-8 were unaffected.The t-SNAREs Syntaxin-11 and SNAP-23 were also unaffected.We examined the t-SNARE chaperone, Munc18b, and Cysteine String Protein-α (CSPα), which interacts with α-synuclein, and both were unaffected.
Thus, the loss of α-synuclein had no effect on any of the platelet secretory machinery examined.

Discussion
40][41][42]51 We confirmed the deletion of the protein in platelets, but its deletion had no significant effects on the other SNARE proteins examined.Secretion from platelets lacking αsynuclein was modestly defective.Consistently, hemostasis was unaffected in the tail-bleeding model and thrombosis was unaffected when examined on both the arterial (FeCl 3 carotid artery injury model) and venous (jugular vein puncture model) vasculature.From our data, α-synuclein plays only a limited role in platelet secretion and hemostasis.Previous studies suggested roles for α-synuclein in platelet activation and in endothelial cell release of von Willebrand Factor (vWF). 43,52Addition of exogenous α-synuclein to platelets inhibited ionomycin-and thrombin-induced α-granule release but had no effect on dense granule or lysosome release. 43This inhibition was dose dependent but not time or temperature dependent (working at 4, 25, and 37°C).Mutant forms of α-synuclein, lacking N or C-terminal domains were without effect.In contrast to our data in Figure 1, exogenous αsynuclein was found associated with the platelet cytoskeleton and there was no staining of granules.In HUVEC cells exogenous α-synuclein and its overexpression inhibited release suggesting that α-synuclein was a negative regulator of Weibel-Palade Bodies secretion. 52In other studies, platelet function was examined in a multimerin 1/α-synuclein doubledeficient strain. 53Platelet adhesion and thrombus formation were reduced and partially rescued when multimerin was added back.The authors did not fully determine if the defects were due to the loss of multimerin or α-synuclein; however, given the multiple roles of multimerin in promoting collagen and von Willebrand factor binding, it is difficult to discern whether α-synuclein has any independent role in platelets.
The molecular mechanisms of α-synuclein's physiologic function remain unclear.8][19][20][21][22][23][24][25][26][27][28][29] Platelets offer a unique system to probe the role of α-synuclein since they have activation-dependent secretion from three different compartments and there are clear in vitro and in vivo outcomes (e.g., bleeding, Figure 5) if secretion is defective.1][32] Overall, our data suggest only a minimal role for α-synuclein; however, our experiments do have limitations.In neurons, α-synuclein acts as a chaperone for VAMP-2 and when deleted neuronal SNARE-complex assembly is reduced. 17If α-synuclein is a v-SNARE chaperone, specific for a single VAMP, its importance may be masked by the fact that platelets contain at least five different VAMPs, which are functional in platelet secretion (VAMP-2, −3, −7, −8, Ykt6; Joshi et al in preparation). 1,2Thus, a VAMP-specific role might be compensated for by a different VAMP that does not need its chaperone activity.It should be noted that loss of αsynuclein did not affect the levels of any of the VAMPs examined (Figure 6).α-Synuclein is highly abundant in both the brain and platelets suggesting it plays a critical role, but its function is still unknown.Mutant forms of the protein have been linked to neurodegenerative diseases, e.g., Parkinson disease, due in part to their ability to form potentially toxic aggregates.9][40][41][42] These risks also correlate with dementia.Together these symptoms and data might suggest a physiological role for α-synuclein in platelet function that is dysregulated by the mutant forms.Our data show that the complete loss of the protein does not cause significant platelet dysfunction.However, α-synuclein could play a role in endothelial cells and when mutated could cause a defect in vascular integrity that precipitates bleeding or DVT risks. 34,52Further analysis with patient samples will be needed to fully assess this possibility.
The importance of the different v-SNAREs to platelets has been assessed by manipulating them in various mouse strains and measuring how they contribute individually or in combination to platelet granule secretion. 2In this study we have used the same approach to assess whether a purported v-SNARE regulator/chaperone, α-synuclein, is critical for platelet secretion and function.Overall, our data suggest that this protein is not essential for platelet secretion or function.Loss of α-synuclein did not affect hemostasis in three injury models and had only a modest effect of dense granule secretion.Thus, our data do not directly support a role for α-synuclein in platelets.Figure 6.Platelet secretory machinery protein levels in α-synuclein −/− platelets are normal.(A) Washed platelets (5 × 10 7 platelets per lane) were prepared from α-synuclein −/− and WT mice (n = 3), and the indicated proteins were probed by western blotting.Data are representative of the three independent experiments and three individual mice were used for biological replicates for both WT and α-synuclein −/− mice.α-Synuclein, CSPα (CSP), SNAP-23, and VAMP-8 were normalized using Rab GDI a loading control.VMAT and VAMP-3 were normalized using Syntaxin-11 a loading control.Munc18b, VAMP-7, and VAMP-2 were normalized using actin a loading control.(B) Quantification of protein levels was performed using ImageQuantTL, and data was plotted as the ratio of α-synuclein −/− mice over WT mice.Statistical analyses were done using the averages of the technical replicates and performed using the paired Student t test.

Figure 1 .
Figure 1.α-Synuclein is present on both lysosomes and α-granules.(A) WT platelets were immunostained for α-synuclein (red) and LAMP-1 (green) and imaged by using 3-dimensional structured illumination microscopy (3D-SIM).The white lines in the merged images indicate where the profile line analyses were performed.Profile line analyses are shown below the images.(B) WT platelets were immunostained for α-synuclein (red) and P-selectin (green).Scale bar: 10 μm for widefield merge images; 5 μm for cropped images.(C) Samples were prepared from washed human platelets subjected to five thaw-freeze cycles and centrifuged to separate membrane and cytosol (S1) fractions.The pellet fraction was treated with 1% Triton X-100 to obtain Triton X-100 soluble (S TX ) and Triton X-insoluble (I TX ) fractions, which were separated by ultracentrifugation.The fractions were analyzed by SDS-PAGE and probed by western blotting with the indicated antibodies.All data are representative of two independent experiments.

Figure 2 .
Figure 2. α-Synuclein −/− platelets have near normal platelet receptor levels and activation.Washed platelets (5 × 10 7 /mL) from WT and α-synuclein −/− mice were stimulated with various agonists (thrombin, onvulxin, CRP, and U46619, see text) for 2 min and then incubated with FITC anti-P-selectin (A-D), PE-conjugated Jon-A (E-H), or PE-conjugated LAMP-1 (I-J) antibodies for 20 min at 37°C.Fluorescent intensities were measured by flow cytometry.Shown are representative data and geometric mean fluorescent intensity (GFMI) (mean ± standard error of the mean) of three independent experiments.Statistical analyses were performed using the unpaired Student t test.

Figure 3 .
Figure3.α-Synuclein −/− platelets have a mild serotonin secretion defect.Washed platelets (2.5 × 10 8 /mL) were either stimulated with various thrombin concentrations or for different time points and secretion was measured from each granule type in WT and α-synuclein −/− platelets: (A, D) dense granule release, (B, E) αgranule release, and (C, F) lysosomal release; (A-C) for the thrombin dose-response experiment, platelets were stimulated for 2 min with the indicated concentrations of thrombin; (D-F) for the time-course experiments, platelets were stimulated with 0.1 U/mL thrombin for the indicated times.Data are mean ± standard error of the mean of triplicate measurements and are representative of ≥ 4 independent experiments.Statistical analyses were performed using twoway ANOVA multiple comparisons.

Table I .
Hematological parameters for α-synuclein −/− mice.Results are the means ± SD and are compared by using the unpaired student t test (complete blood count: WT n = 15 and α-synuclein −/− n = 13).