Materials
Unless otherwise stated all chemicals were purchased from Sigma-Aldrich (St. Louis, MO or Stockholm, Sweden). DMEM, FBS, penicillin-streptomycin, Lipofectamine 2000, Optimem, Trypsin-EDTA, ProLong Gold Antifade Reagent with DAPI, Proteinase K (25530–049), and the Virapower Adenoviral Expression System were obtained from Invitrogen (Carlsbad, CA). Plus slides (J3800AMNZ), Coomassie Brilliant Blue, and the Immobilon-FL PVDF membrane were purchased from Thermo Fisher Scientific (Rockford, IL). The QuickTiter Adenovirus Titer Elisa kit was purchased from Cell Biolabs (San Diego, CA). Antisedan, Temgesic, and the Fentanyl/Dormitor mixture were obtained from Apoteksbolaget, (Stockholm, Sweden). The Vectastain ABC kit was purchased from Vector Laboratories, Inc. (Burlingame, CA), and 3,3′-diaminobenzidine (DAB Safe) was obtained from Saveen Werner (Limhamnsvägen, Sweden). Isopropyl β-D-1-thiogalactopyranoside and ampicillin were purchased from Gold Biotechnology (St. Louis, MO). L-α-phosphatidylcholine (egg PC), L-α-phosphatidylglycerol (egg PG), and phospholipid extrusion membranes were purchased from Avanti Polar Lipids (Alabaster, AL). Iodixanol and the enhanced chemifluorescence (ECF) substrate were obtained from GE Life Sciences (Pittsburgh, PA). 100 kDa spin filters and 10 kDa concentration filters were obtained from Millipore (Billerica, MA or Solna, Sweden).
Antibodies
The following antibodies were used in these studies:
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Primary cell culture – chicken anti-microtubule associated protein 2 (MAP2) (CPCA-MAP2, RRID:AB_2138173, EnCor Biotechnology, Gainesville, FL); rabbit anti-tyrosine hydroxylase (TH) (AB152, RRID:AB_390204, Millipore, Bellerica, MA); anti-rabbit IgG-Alexa Fluor 488 (R37116, RRID:AB_2556544) and anti-chicken IgG-Alexa Fluor 594 (A11042, RRID:AB_142803) (Thermo Fisher Scientific, Rockford, IL).
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In vivo experiments – human-specific syn211 (36–008, RRID:AB_310817, Millipore, Germany); mouse anti-aSyn, clone 42 (Syn-1) (610,787, RRID:AB_398108, BD Biosciences, UK); rabbit anti-TH (P40101–0, RRID:AB_461064, Pel-freeze, USA); rabbit anti-VMAT2 (ab81855, RRID:AB_2188123, Abcam, UK or 20,042, RRID:AB_10013884, Immunostar, USA); mouse anti-HuC/HuD, clone 16A11 (A21271, RRID:AB_221448, Thermo Scientific, USA); biotinylated secondary antibodies (Vector Laboratories Inc., USA).
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Western blotting – mouse anti-aSyn, clone 42 (Syn-1) (610,787, RRID:AB_398108, BD Biosciences, San Jose, CA); AP-linked anti-mouse IgG (7056, RRID:AB_330921) (Cell Signaling Technology, Danvers, MA).
Preparation of bacterial expression constructs
Bacterial expression constructs encoding h-aSyn WT (pT7–7-h-aSyn), h-aSyn A53T (pT7–7-h-aSyn-A53T), and m-aSyn (pT7–7-m-aSyn) were described previously [26]. A construct encoding h-aSyn Chimera was generated by replacing the BamHI-HindIII fragment of pT7–7-h-aSyn-A53T (encompassing base pairs 354–423 of the h-aSyn A53T cDNA) with the equivalent fragment from pT7–7-m-aSyn. A construct encoding m-aSyn Chimera was generated by replacing the BamHI-HindIII fragment of pT7–7-m-aSyn (encompassing base pairs 354–423 of the m-aSyn cDNA) with the equivalent fragment from pT7–7-h-aSyn-A53T. pT7–7 constructs encoding A53T/D121G, A53T/N122S, A53T/S87N, h-aSyn Chimera S87N, m-aSyn N87S, and m-aSyn Chimera N87S were generated via site-directed mutagenesis using the QuikChange method (Stratagene). The sequence of the DNA insert in each bacterial expression construct was verified using an Applied Biosystems (ABI3700) DNA sequencer (Purdue Genomics Core Facility).
Adenoviral vector design and virus production
Adenoviral constructs encoding h-aSyn WT and h-aSyn A53T were described previously [16, 30]. cDNAs encoding h-aSyn Chimera, A53T/D121G, A53T/N122S, A53T/S87N, and h-aSyn Chimera S87N (obtained using the pT7–7 constructs outlined above as PCR templates) were subcloned as KpnI-XhoI fragments into the entry vector pENTR1A. cDNAs encoding m-aSyn, m-aSyn Chimera, and m-aSyn Chimera N87S were subcloned as SalI-XhoI fragments into pENTR1A. Inserts from the pENTR1A constructs were then transferred into the pAd/CMV/V5 adenoviral expression vector via recombination using Gateway LR Clonase. The sequence of the DNA insert in each adenoviral construct was verified using an Applied Biosystems (ABI3700) DNA sequencer (Purdue Genomics Core Facility). Adenoviral constructs were packaged into virus via lipid-mediated transient transfection of the HEK 293A packaging cell line. Purified adenoviral particles were titered using the QuickTiter Adenovirus Titer Elisa kit.
AAV vector design and virus production
Adeno-associated viral vectors of serotype 5 (AAV5) were comprised of a genome cassette flanked by inverted terminal repeats (ITR2). The cassette contained a chicken beta-actin promoter in combination with a CMV enhancer element (CBA) leading to expression of one of the four different transgenes. Genes were PCR-amplified with the plasmids pENTR1A h-aSyn A53T, pENTR1A h-aSyn Chimera, pENTR1A m-aSyn, and pENTR1A m-aSyn Chimera as templates and subcloned as HindIII-SpeI fragments in the pTR-UF20 plasmid. The validity of the constructs was confirmed by restriction analysis and Sanger sequencing. Downstream of each cDNA insert was an h-SV40 polyA transcription termination sequence. HEK293 cells were co-transfected at a confluency of 70–80% using the calcium-phosphate precipitation method. Plasmids used here encode essential adenoviral packaging and AAV5 capsid genes as previously described [31]. Three days after transfection, cells were harvested in PBS and lysed by performing three freeze-thaw cycles in a dry ice/ethanol bath. The lysate was then treated with benzonase and purified using a discontinuous iodixanol gradient followed by Sepharose Q column chromatography [32]. Vectors were concentrated using a 100 kDa molecular weight cut-off column, and titers of the stock solution were determined by qPCR using primers and probes targeting the ITR sequence. Before being used in an experiment, vectors were diluted in PBS, pH 7.4 and re-titered, yielding the values reported in the Results and in Fig. 3.
Preparation and treatment of primary mesencephalic cultures
Primary midbrain cultures were prepared via dissection of day 17 embryos obtained from pregnant Sprague-Dawley rats (Harlan, Indianapolis, IN, USA) as described [16, 30]. The cells were plated on poly-L-lysine-treated 48-well plates at a density of 163,500 cells per well. Five days after plating, the cells were treated with cytosine arabinofuranoside (AraC) (20 μM, 48 h) to inhibit the growth of glial cells. At this stage (i.e., 7 days in vitro (DIV)), the neurons appeared differentiated with extended processes. The cultures were transduced with adenoviruses encoding the aSyn variants for 72 h at a multiplicity of infection (MOI) ranging from 7 to 15 to ensure equal expression levels as determined via Western blotting. After incubation for an additional 24 h in fresh medium without virus, the cells were fixed in 4% (w/v) PFA in PBS (pH 7.4) and permeabilized and blocked simultaneously for 30 min with PBS containing 1% (w/v) BSA, 10% (v/v) FBS, and 0.3% (v/v) Triton X-100. The cells were then washed in PBS and incubated with chicken anti-MAP2 (1:2000) and rabbit anti-TH (1:500) for 24 h at 4 °C. After an additional wash in PBS, the cultures were incubated with AlexaFluor 594-conjugated goat anti-chicken and AlexaFluor 488-conjugated goat anti-rabbit secondary antibodies (1:1000) for 1 h at 22 °C. ProLong Gold Antifade Reagent with DAPI was applied to each well, and a coverslip was added.
Measurement of neuronal viability in midbrain cultures
Relative dopaminergic cell viability was evaluated as described [16, 30]. MAP2+ and TH+ cells were counted in a blinded manner in at least 10 randomly chosen observational fields (approximately 500–1000 neurons total) for each experimental condition using a Nikon TE2000-U inverted fluorescence microscope (Nikon Instruments, Melville, NY) equipped with a 20x objective. The data are expressed as the percentage of MAP2+ neurons that are also TH+ to correct for variation in cell density. Each experiment was repeated a minimum of 3 times with cultures isolated from different pregnant rats.
Animals for in vivo experiments
Young adult Sprague-Dawley rats received from Charles River (Kisslegg, Germany) were housed on a 12 h light-dark cycle with ad libitum access to water and food.
Stereotaxic surgery
Rats were anesthetized via i.p. injection of a 20:1 mixture of Fentanyl and Dormitor (6 mL/kg). After placing each animal into a stereotaxic frame (Stoelting, Wood Dale, USA), the SN was targeted unilaterally using the following coordinates: anteroposterior (AP), − 5.0 mm; mediolateral (ML), − 2.0 mm from bregma; and dorsoventral (DV), − 7.2 mm from the dura. The tooth bar was adjusted to − 2.3 mm. A pulled glass capillary (about 60–80 μm in diameter) was attached to a 5 μL Hamilton syringe equipped with a 22 s gauge needle and used to deliver 2 μL of rAAV5 vector solution at a pulsed injection of 0.1 μL every 15 s. After delivery of the vector, the capillary was left in place for 5 min, retracted 0.1 mm, and after 1 min it was slowly removed from the brain. After closing the wound with clips, Antisedan and Temgesic were administered s.c. as an analgesic treatment and to reverse the anesthesia.
Histology
An overdose of sodium pentobarbital was used to kill rats 8 weeks after vector delivery. Animals were perfused via the ascending aorta first with 50 mL of 0.9% (w/v) NaCl followed by 250 mL of ice-cold 4% (w/v) PFA in 0.1 M phosphate buffer, pH 7.4, for 5 min. Brains were taken out and post-fixed in 4% (w/v) PFA for 24 h and then incubated in 25% (w/v) sucrose for cryoprotection. Brains were cut on a microtome (Microm HM450, Thermo Scientific, USA) in 35 μm-thick coronal sections and placed into antifreeze solution (0.5 M phosphate buffer, 30% (v/v) glycerol, 30% (v/v) ethylene glycol) at − 20 °C for long term storage until further processed.
Immunohistochemical staining was performed on free-floating brain sections. Specimens were rinsed with Tris-buffered saline (TBS) buffer (5 mM Tris-HCl, 15 mM NaCl, pH 7.6). Only TH staining required an antigen retrieval step carried out for 30 min at 80 °C using Tris/EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 9.1). This step was terminated by repeated washes in TBS at 22 °C. Endogenous peroxidase activity was quenched using a solution of 3% (v/v) H2O2 and 10% (v/v) methanol in TBS buffer for 30 min, and the reaction was stopped by washing sections 3 times with TBS. To block unspecific binding sites, sections were incubated for 30 min in 0.05% (v/v) Triton X-100 in TBS buffer (TBS-T) containing 5% (v/v) normal serum from the same species as that in which the secondary antibody was raised. The primary antibodies were then applied in 1% (w/v) BSA in TBS-T overnight at 22 °C: human-specific syn211 at 1:100,000; Syn-1 at 1:10,000; TH at 1:5000; VMAT2 ab81855 at 1:4000, or 20,042 at 1:10,000; HuC/HuD 1:200. The next day sections were washed in TBS-T, and the appropriate biotinylated secondary antibody (1:200) was applied in 1% (w/v) BSA in TBS-T for 1 h. Specimens were rinsed once more with TBS-T and further incubated in a solution containing avidin-biotin-peroxidase complexes (Vectastain ABC kit, Vector Laboratories Inc., USA) for 1 h. Staining was visualized using 3,3′-diaminobenzidine (DAB Safe) and 0.01% (v/v) H2O2. Sections were then mounted on chromatin-gelatin coated glass slides, dehydrated in increasing alcohol solutions, cleared with xylene, and applied to coverslips using DPX (06522, Sigma-Aldrich, Sweden).
For cresyl violet staining, sections were mounted on chromatin-gelatin coated glass slides and dried overnight. Sections were then hydrated in decreasing alcohol solutions and stained for 30 s in 0.5% (w/v) cresyl violet + 0.1% (v/v) acetic acid. After rinsing specimens with H2O, sections were dehydrated in increasing alcohol solutions, cleared in xylene, and coverslipped using DPX.
To visualize insoluble aggregates, sections were treated with Proteinase K. For this purpose, specimens were first rinsed with potassium-containing phosphate-buffered saline (KPBS) and then heat-treated at 80 °C for 30 min in KPBS. Sections were then quenched using 3% (v/v) H2O2 and 10% (v/v) methanol in KPBS for 30 min. After mounting sections on plus slides, the sections were incubated in a KPBS solution containing 10 μg/mL Proteinase K for either 5 or 45 min. The same staining protocol was applied as described above using the aSyn-specific primary antibody Syn-1 at 1:1000.
Stereological analysis
In order to estimate surviving TH+ nigral neurons, an unbiased stereological quantification using the optical fractionator method [33, 34] was applied. The implementation of a random starting position and systematic sampling was done using the Visiopharm VIS software (version 4.5.5.433 Visiopharm A/S, Denmark) and a Nikon Eclipse 80i light microscope fitted with an xy motorized stage, a motorized z-axis (Prior, UK), and a microcator (Heidenheim, Germany). Through the rostro-caudal axis of the SN, every 6th section was stained for TH. Regions of interest were outlined with a 4x objective, and counting was executed using a 60x oil-immersion objective with a numerical aperture of 1.4. Counting parameters were set to reach at least 100 cell counts per hemisphere. Estimation of the total number of TH+ cells was performed by a single blinded investigator. The coefficient of error due to the sampling was calculated based on Gundersen and Jensen [35], and values lower than 0.1 were considered acceptable. The contralateral uninjected side of each animal served as an internal control, and values were expressed as a percentage of cell counts on the injected side relative to the intact side (mean ± SEM).
Densitometry
The optical density of TH+ and VMAT2+ fibers was measured on digital images of coronal striatal sections using the Zeiss microscope (Axio Zoom.V16, Zeiss, Germany). The striatum of every 24th section in the rostro-caudal axis – in total 6 sections per brain – was outlined using ImageJ, and optical density readings were corrected for non-specific background using density measurements from the corpus callosum of each animal. Data consisting of means ± SEM from all brains are expressed as a percentage of contralateral side values.
Protein purification
Recombinant aSyn variants were purified from BL21 (DE3) cells transformed with pT7–7 constructs as described [16]. The final purity of each protein was found to be > 98% as determined by SDS-PAGE with Coomassie Blue staining.
aSyn fibrillization
Lyophilized aSyn was dissolved in PBS (pH 7.4) with 0.02% [v/v] NaN3, and the solution was filtered by successive centrifugation steps through a 0.22 μm spin filter and a 100 kDa centrifugal filter to isolate monomeric protein. The stock solution was dialyzed against the same buffer (24 h at 4 °C) to remove excess salt. Fibrillization solutions of aSyn were prepared by diluting the appropriate stock solutions of dialyzed protein with buffer to a final concentration of 35 μM in the wells of a 96-well plate. To determine the extent of aSyn fibrillization, thioflavin T (ThioT, final concentration, 20 μM) was added to the fibrillization solutions, which were incubated at 37 °C with shaking at the ‘normal’ setting in a Spectra Fluor Plus or Genios plate reader (Tecan, Upsala, Sweden). ThioT fluorescence was measured with excitation at 440 nm and emission at 490 nm. Mean ThioT fluorescence data determined from 3 or 4 technical replicates (defined here as samples treated identically in a single experiment) were normalized using the following equation:
$$ normalized\ fluorescence=\frac{F_t-{F}_{min}}{F_{max}-{F}_{min}} $$
(1)
where Ft is the ThioT fluorescence emission intensity at time t, and Fmin and Fmax are the minimum and maximum fluorescence intensities during the incubation, respectively.
Lipid vesicle preparation
Small unilamellar vesicles (SUVs) (diameter = 50 nm) were prepared as described [16]. We opted to examine SUVs composed of egg PG and egg PC (1:1 mol/mol) because they contain anionic lipids necessary for aSyn membrane interactions [12] and this lipid composition is compatible with producing stable 50 nm vesicles [16]. Egg PG and egg PC suspended in chloroform were mixed in a round bottom flask. The chloroform was evaporated under a nitrogen stream and further dried under vacuum for 1 h. Dried lipids were suspended in PBS, and 50 nm SUVs were prepared by extruding the suspension through a Whatman membrane. The size of the vesicles was confirmed by dynamic light scattering. Lipid vesicles were stored at 4 °C prior to use.
Circular dichroism
Far-UV CD measurements were performed as described [16] using a Chirascan SC spectrometer (Applied Photophysics, Leatherhead, UK). Solutions of recombinant aSyn (5 μM in 20 mM KPi, pH 7.4) in the absence or presence of SUVs (protein-to-lipid ratio, 1:50 to 1:1600, mol/mol) were analyzed in a 1 mm quartz cuvette at 25 °C. The ellipticity at 222 nm was recorded and background corrected to eliminate scattering signals arising from the SUVs. The mean residue molar ellipticity at 222 nm ([θ]MR, 222) was calculated using the following equation:
$$ {\left[\theta \right]}_{MR,222}={\theta}_{222}/10 Cnl $$
(2)
where θ222 is the observed (background corrected) ellipticity at 222 nm in millidegrees, C is the protein concentration in M, n is the number of amino acid residues in the protein, and l is the path length of the cuvette in cm. Binding curves were generated by plotting [θ]MR, 222 versus the lipid concentration. Curves were analyzed by fitting to the following equation:
$$ R={R}_0-\left({R}_0-{R}_f\right)\frac{K_d+C+L/N-\sqrt{{\left({K}_d+C+L/N\right)}^2-4 CL/N}}{2C} $$
(3)
where R is the measured [θ]MR, 222 at a given lipid concentration, R0 is [θ]MR, 222 in the absence of lipid, Rf is [θ]MR, 222 in the presence of saturating lipid, L is the total lipid concentration, C is the total protein concentration, Kd is the apparent macroscopic dissociation equilibrium constant, and N is the binding stoichiometry (lipids/protein) [16, 36].
To determine the maximal helical content of each aSyn variant, we used the following equations:
$$ H=100\%\frac{\ \left(\theta -{\theta}_{coil}\right)}{\left({\theta}_{\alpha }-{\theta}_{coil}\right)} $$
(4)
$$ {\theta}_{\alpha }=-40000\left(1-\frac{2.5}{n}\right)+100t $$
(5)
$$ {\theta}_{coil}=640-45t $$
(6)
where H is the maximal % helicity, θ is [θ]MR, 222 in the presence of saturating lipid (Rf in Eq. 3), θα is the [θ]MR, 222 value of an idealized α-helical peptide, θcoil is the [θ]MR, 222 value of an idealized random coil peptide, n is the number of amino acid residues in the protein (= 140), and t is the temperature (25 °C) [16, 37, 38].
Lipid flotation assay
Lipid flotation analyses were carried out as described [16, 22]. Lyophilized aSyn was resuspended in PBS with 0.02% (w/v) NaN3, and the solution was dialyzed against the same buffer to remove salts and filtered by successive centrifugation steps through a 0.22 μm spin filter and a 100 kDa centrifugal filter to isolate monomeric protein. aSyn (40 μM) was incubated with PG:PC SUVs (protein-to-lipid ratio, 1:20 mol/mol) at 37 °C for 72 h in a total volume of 60 μL. After incubation, the sample was mixed with 4 mL of 30% (v/v) iodixanol solution and overlaid with 7.0 mL of 25% (v/v) iodixanol and 350 μL of 5% (v/v) iodixanol in a polyalomar tube (Beckman, Miami, FL). All of the above iodixanol solutions were prepared in lipid flotation buffer (10 mM HEPES, pH 7.4, 150 mM NaCl). The samples were spun at 200,000 x g in a Beckman SW 41 Ti rotor for 4 h. The membrane fraction was carefully collected from the 5% iodixanol fraction at the top of the gradient, concentrated using a 10 kDa spin filter, and analyzed via Western blotting using a primary antibody specific for aSyn (Syn-1) (1:1500) [16, 22]. In general, we found that the rate of aSyn aggregation increased in the presence of SUVs that had been stored for ~ 2–4 weeks at 4 °C [16].
Synthetic vesicle disruption assay
Calcein-loaded egg PG:PC SUVs were prepared as described [39] with some modifications. A solution of calcein (170 mM) was prepared in H2O using NaOH to adjust the pH to 7.4. The final osmolality was 280 mOsm/kg. The calcein solution was used to resuspend dried egg PG:PC lipids, and the suspension was extruded through a Whatman membrane to generate 50 nm SUVs. Calcein-containing vesicles were isolated from free calcein via gel filtration through a Sephadex G-50 column pre-equilibrated with PBS, pH 7.4, 0.02% (w/v) NaN3 (280 mOsm/kg). Fractions containing isolated vesicles were pooled and stored at 4 °C until use. Calcein-loaded vesicles were found to be very stable, with no spontaneous dye leakage observed over several weeks.
For membrane disruption experiments, monomeric aSyn variants were isolated as described above (see ‘Lipid flotation assay’). Each aSyn variant (40 μM) was incubated with calcein-loaded PG:PC SUVs (protein-to-lipid ratio, 1:20 mol/mol) at 37 °C in a total volume of 40 μL. At each time point, 3 μL of the reaction mixture was diluted into 180 μL of PBS, and the diluted samples were analyzed with a Fluoromax-3 spectrofluorometer (Horiba Scientific, Edison, New Jersey) (excitation wavelength, 485 nm; emission wavelengths, 505–530 nm; slit width, 1 nm). To determine the maximum dye release, vesicles were lysed by adding 5 μL of 1% (v/v) Triton X-100. The % leakage at time t was determined using the following equation:
$$ \% leakage=\frac{I_t-{I}_0}{I_{max}-{I}_0}\times 100\% $$
(7)
where It is the fluorescence emission intensity at time t, I0 is the intensity at time 0, and Imax is the maximal intensity determined after detergent lysis of the vesicles (intensity values were determined at 515 nm).
Atomic force microscopy analysis of aSyn amyloid-like fibrils
A mica surface functionalized with 1-(3-aminopropyl) silatrane (APS) was used to image aSyn amyloid-like fibrils by atomic force microscopy (AFM) [18, 40,41,42]. APS-mica was prepared by incubating freshly cleaved mica in a solution of APS (167 μM) for 30 min and then rinsed with deionized water and dried with an argon stream. A suspension of aSyn fibrils (10 μL, prepared as described above under ‘aSyn fibrillization’ with an incubation time of ~ 100 h and diluted 1/20 in deionized water) was deposited onto the APS-mica surface, and the sample was incubated for 2 min, rinsed with deionized water, and dried under an argon stream. The sample was imaged with an AFM Nanoscope VIII system (Bruker, Santa Barbara, CA) using MSNL probes (Cantilever F with spring constant 0.6 N/m), operating in air in peak force mode. Images were acquired over a few randomly selected locations. Images were analyzed using Gwyddion and FemtoScan online software (Advanced Technologies Center, Moscow, Russia) [43, 44].
TEM analysis of aSyn amyloid-like fibrils
The morphology of aSyn amyloid-like fibrils (prepared as described above under ‘aSyn fibrillization’ with an incubation time of ~ 100 h) was analyzed by negative stain biological TEM [45]. For biological sample preparation by negative staining, which is a sample preparation technique that imparts the necessary contrast for viewing during biological TEM imaging, 3 μL of aSyn sample solution (35 μM) was pipetted on a discharged carbon-coated copper TEM grid substrate. Subsequently, the sample was washed with deionized water carefully without letting it dry and stained with a 1% (w/v) phosphotungstic acid solution (3 μL), which was left in contact with the protein on the grid for 1 min. The excess solution was then removed by blotting with filter paper, and the sample was imaged using an FEI Tecnai G2 20 Transmission Electron Microscope operating at 200 KV.
Sample size estimation
Samples sizes for studies of primary neuron viability, membrane-induced aSyn aggregation, and aSyn-mediated membrane permeabilization were estimated via power analysis based on our previous studies [16, 22]. Power analyses were designed to determine the number of biological replicates required to detect a ~ 20% or ~ 30% difference from control, with α = 0.05 (type I error) and power = 0.80 (statistical power). Biological replicates are defined here as samples treated identically in independent experiments carried out on different days. For in vivo experiments, sample sizes were estimated based on previous data collected by our group and others for the A53T variant.
Statistical analysis
Statistical analyses were carried out using GraphPad Prism 6.0 (La Jolla, CA). Primary neuron viability data, in vivo stereology and densitometry data, densitometry data from Western blots, and calcein dye release data were analyzed via ANOVA followed by Tukey’s multiple comparisons post hoc test for normally distributed measurements. In analyzing percentage dye release data and percentage cell viability data by ANOVA, square root transformations were carried out to conform to ANOVA assumptions. The Kruskal-Wallis test followed by a Dunn’s multiple comparisons test or a two-tailed Mann-Whitney test was used as a non-parametric test. Differences were considered significant for p < 0.05. Unless otherwise stated, values of n indicated in the Figure Legends refer to the number of independent biological replicates.