Materials
Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Isopropyl β-D-1-thiogalactopyranoside (IPTG), ampicillin, and phenylmethylsulfonyl fluoride (PMSF) 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). Dulbecco’s minimal essential media (DMEM), penicillin-streptomycin, the ViraPower Adenoviral Expression System, and LR clonase II were obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Corning Life Sciences (Corning, NY). Iodixanol and ECF substrate were obtained from GE Life Sciences (Pittsburgh, PA). 100 kDa spin filters were purchased from Millipore (Billerica, MA). Bicinchoninic acid (BCA) assay reagents and Coomassie Brilliant Blue were purchased from Thermo Scientific (Rockford, IL).
Antibodies
The following antibodies were used in these studies: mouse anti-aSyn (#610787, BD Biosciences, San Jose, CA); rabbit anti-ENSA (#D5Z1U), rabbit anti-β3-tubulin (#D65A4), and AP-linked anti-mouse and anti-rabbit IgG (Cell Signaling Technology, Danvers, MA); chicken anti-MAP2 (#CPCA-MAP2, EnCor Biotechnology, Gainesville, FL); rabbit anti-TH (#AB152, Millipore, Billerica, MA); mouse anti-β-actin (#A5316 or #A5441, Sigma-Aldrich); and anti-rabbit IgG-Alexa Fluor 488 and anti-chicken IgG-Alexa Fluor 594 (Invitrogen, Carlsbad, CA).
Protein purification
Recombinant aSyn was purified from BL21 (DE3) cells transformed with pT7-7 constructs encoding A30P or G51D as described [58]. A cDNA encoding S109E was generated via overlap-extension PCR using a pET21-ENSA construct as the DNA template and cloned as an NdeI-BamHI fragment into the pT7-7 expression vector. Protein expression was induced by adding IPTG (final concentration, 1 mM) to an E. coli culture pre-grown to an OD600 of 0.6, and the culture was incubated at 37 °C for 3 h. The cells were harvested by centrifugation, suspended in a buffer consisting of 10 mM Tris HCl, pH 7.4, 1 mM PMSF, and 1 mM EDTA, and lysed with a French pressure cell (psi 1000). After lysis, 0.1% (w/v) streptomycin sulfate was added to the lysate, which was then cleared by centrifugation. ENSA was partially purified from the resulting supernatant by ammonium sulfate precipitation (30% saturation). The pellet was re-suspended in 10 mM Tris HCl, pH 7.4, and loaded on a DEAE-Sepharose anion exchange column. The column flow-through was analyzed by SDS-PAGE, and fractions containing ENSA were loaded onto a Sephacryl S-100 column. Fractions enriched with ENSA to a purity of >98%, as determined by SDS-PAGE with Coomassie Blue staining, were pooled, concentrated, aliquoted, and stored at −80 °C until use.
Lipid vesicle preparation
Small unilamellar vesicles (SUVs) were prepared as described [58]. Egg PG and egg PC suspended in chloroform were mixed at an equimolar ratio 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 (10 mM phosphate buffer, 2.7 mM KCl, and 137 mM NaCl, pH 7.4), 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.
Lipid flotation assay
Lipid flotation analyses were carried out as described [58]. aSyn was dialyzed overnight into PBS with 0.02% (w/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. aSyn (40 μM) was incubated with PG:PC SUVs (protein-to-lipid ratio, 1:20 mol/mol) in the absence or presence of ENSA (10–40 μM) 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 × 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 stored at −20 °C prior to Western blot analysis.
Membrane-bound proteins isolated by lipid flotation were analyzed by Western blotting as described [58]. Protein samples were combined with Laemmli buffer (final concentration, 60 mM Tris HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.01% (w/v) bromophenol blue) and boiled for 2 min. Denatured proteins were separated via SDS-PAGE on a 4–20% (w/v) polyacrylamide gradient gel (BioRad, Hercules, CA) and transferred to a polyvinylidene difluoride (PVDF) membrane (pore size, 0.4 μm). After blocking in 10% (w/v) non-fat milk, the membrane was washed with PBS + 0.5% Tween 20 (w/v) (PBS-T) and probed for aSyn with the Syn-1 primary antibody, diluted 1/2000 in PBS-T with 1% (w/v) BSA. After washing in PBS-T, the membrane was probed with a secondary anti-mouse antibody conjugated to alkaline phosphatase (diluted 1/3000). To visualize the bands, the membrane was exposed to ECF substrate for 30 s, and images were acquired using a Typhoon imaging system (GE Life Sciences, Pittsburgh, PA). Band densities were quantified using ImageJ software (NIH, Bethesda, MD). Oligomer band intensities were determined by carrying out a densitometric analysis of the region of the gel spanning predicted molecular weights of 30 kDa through 250 kDa. We verified using ImageJ software that the band intensities were below the saturation range. 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 [58].
Membrane disruption assay
Calcein-loaded egg PG:PC SUVs were prepared as described [3] 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 was isolated as described above (see ‘Lipid Flotation Assay’) and dialyzed into PBS with 0.02% (w/v) NaN3. aSyn (40 μM) was incubated with calcein-loaded PG:PC SUVs (protein-to-lipid ratio, 1:20 mol/mol) in the absence or presence of ENSA (10–40 μM) at 37 °C in a total volume of 50 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:
$$ \%\ leakage=\frac{I_t-{I}_0}{I_{max}-{I}_0}\times 100\% $$
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).
aSyn fibrillization
aSyn fibrillization rates were assessed using a thioflavin T fluorescence assay as described [59], with some modifications. Monomeric aSyn prepared as outlined above (see ‘Lipid Flotation Assay’) was diluted to a final concentration of 40 μM in a solution of PBS, pH 7.4, 0.02% (w/v) NaN3, and 20 μM thioflavin T, and the diluted protein solution was aliquoted in triplicate in a 96-well microtiter plate (200 μL per well). A Teflon ball was added to each well to enhance mixing. The plate was incubated at 37 °C with constant shaking in a Genios plate reader (Tecan, Uppsala, Sweden). The formation of amyloid-like fibrils was monitored by measuring thioflavin T fluorescence (excitation wavelength, 440 nm; emission wavelength, 480 nm), with one reading taken every 15 min.
Preparation of adenoviral constructs
Adenoviruses encoding aSyn and ENSA variants were produced using the ViraPower Adenoviral Expression System. Methods used to generate aSyn-encoding adenoviral DNAs were described previously [58]. A cDNA encoding ENSA was cloned as a KpnI-XhoI fragment into the pENTR1A entry plasmid, and the insert from the pENTR1A construct was transferred into the pAd/CMV/V5 adenoviral expression vector via recombination using Gateway LR Clonase. The sequence of the DNA insert in the resulting adenoviral construct was verified using an Applied Biosystems (ABI3700) DNA sequencer (Purdue Genomics Core Facility).
A low-titer preparation of each adenovirus was produced by lipid-mediated transfection of the HEK 293A packaging cell line with adenoviral DNA. The low-titer preparation was then applied to a new batch of HEK 293A cells to generate an amplified virus preparation. Adenovirus packaged with the plasmid pAd/V5-DEST/LacZ (Invitrogen), encoding the control protein β-galactosidase (β-gal) fused to the V5 epitope, was also prepared to control for non-specific effects of viral transduction.
Preparation and treatment of primary mesencephalic cultures
Primary midbrain cultures were prepared via dissection of day 17 embryos obtained from pregnant Sprague-Dawley rats (Envigo (Harlan), Indianapolis, IN) using methods approved by the Purdue Animal Care and Use Committee, as described [39, 58]. The mesencephalic region containing the substantia nigra and ventral tegmental area was isolated stereoscopically, and the tissue was treated with trypsin (final concentration, 26 μg/mL in 0.9% [w/v] NaCl). Dissociated cells were plated on 96-well plates (pretreated with poly-L-lysine, 5 μg/mL) at a density of 82,000 cells per well in media consisting of DMEM, 10% (v/v) FBS, 10% (v/v) horse serum, penicillin (10 U/mL), and streptomycin (10 μg/mL). Five days after plating, the cells were treated for 48 h with cytosine arabinofuranoside (20 μM) to inhibit the growth of glial cells. At this stage (7 days in vitro), the cultures were transduced for 72 h with adenoviruses encoding aSyn variants at a multiplicity of infection (MOI) of 10, with or without adenovirus encoding ENSA at an MOI of 5. Adenovirus encoding β-gal was used as a control for viral transduction. The cultures were then incubated in fresh media for an additional 24 h, and immunocytochemistry was performed as described [58]. The cells were fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.4) and subsequently permeabilized for 1 h with PBS, pH 7.4, 0.3% (v/v) Triton X-100, 1% (w/v) BSA, and 10% (v/v) FBS. After washing with PBS, the cells were treated for 48 h at 4 °C with chicken anti-MAP2 (1:2000) and rabbit anti-TH (1:500) primary antibodies. The cells were then washed with PBS, treated with AlexaFluor 594-conjugated goat anti-chicken and AlexaFluor 488-conjugated goat anti-rabbit secondary antibodies (both at 1:1000) for 1 h at 22 °C, and washed a final time with PBS before imaging.
Measurement of neuronal viability
Relative dopaminergic cell viability was assessed by counting MAP2- and TH-immunoreactive primary neurons in a blinded manner [58]. The neurons were imaged using an automated Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT) equipped with a 4× objective. A total of 9 images were taken to provide representation of the whole well, and at least 900 MAP2+ neurons were counted per experiment for each treatment. Each experiment was repeated using cultures prepared from at least 3 different pregnant rats. The data are expressed as a percentage of MAP2+ neurons that were also TH+ to correct for variations in cell plating density.
Neurite length measurements
Neurite length measurements were carried out on the same images generated by the Cytation 3 cell imager for neuron viability analysis. Lengths of MAP2+ processes extending from TH+/MAP2+ neurons with an intact cell body (>40 neurons and >70 neurites per experiment for each treatment) were assessed in a blinded manner using the Nikon NIS Elements analysis software (Nikon Instruments, Melville, NY) [58].
Analysis of brain tissue homogenates
Research involving analyses of coded human brain samples was granted an exemption from requiring ethics approval by the Purdue University and Indiana University Institutional Review Boards. The French brain samples were obtained from brains collected in a Brain Donation Program of the Brain Bank “GIE NeuroCEB” run by a consortium of Patients Associations: ARSEP (association for research on multiple sclerosis), CSC (cerebellar ataxias), France Alzheimer and France Parkinson. The consents were signed by the patients themselves or their next of kin in their name, in accordance with the French Bioethical Laws. The Brain Bank GIE NeuroCEB (Bioresource Research Impact Factor number BB-0033-00011) has been declared at the Ministry of Higher Education and Research and has received approval to distribute samples (agreement AC-2013-1887). Samples of human cortex and substantia nigra were resuspended at a concentration of 0.1 mg/mL in RIPA buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Triton X 100) supplemented with protease inhibitor cocktail (Sigma P8340) and PMSF (1 mM). The tissue was homogenized by cup sonication at 4 °C using a Digital Sonifier (Branson, Danbury, CT). The homogenate was cleared by centrifugation at 13,000 × g, and the supernatant was fractionated via SDS-PAGE (20 μg of protein loaded in each lane of a 16% (w/v) polyacrylamide gel) and analyzed via Western blotting (primary antibody: rabbit anti-ENSA, 1:1,000 or 1:2,000; rabbit anti-β3-tubulin, 1:5,000; or mouse anti-β-actin, 1:10,000 (Sigma-Aldrich #A5316) or 1:1,000 (Sigma-Aldrich #A5441); secondary antibody: AP-conjugated anti-rabbit IgG or anti-mouse IgG, 1:3000). ENSA band intensities were determined via densitometric analysis using the ImageJ software and normalized to their respective β-3-tubulin band intensities. In turn, these ratios were normalized to the control ENSA/β-3-tubulin ratio.
Statistical analysis
Densitometry data from Western blots, calcein dye release data, and primary neuron viability data were analyzed via two-tailed t-test (when comparing 2 groups) or ANOVA followed by Tukey’s multiple comparisons post hoc test (when comparing >2 groups) using GraphPad Prism 6.0 (La Jolla, CA). In analyzing percentage dye release data and percentage cell viability data by ANOVA, square root transformations were carried out to conform to ANOVA assumptions. Neurite length data were analyzed using an approach that accounts for (i) the possibility of multiple neurites arising from a single cell, and (ii) comparison across experiments conducted on different days [58]. Neurite lengths for multiple treatment groups were compared using a general linear model implemented in the GLM procedure of SAS Version 9.3 followed by the Tukey’s multiple comparisons post hoc test (Cary, NC).