Recombinant adeno-associated virus 2 preparation
The AAV2-A53T-α-synuclein vector contains the coding sequence for the human α-synuclein gene under the control of a synapsin promoter. The virus was produced by Harvard Gene Therapy Initiative. The final titer for the vector encoding A53T-α-syn was 1.8 × 1012 genome copies/ml, and green fluorescent protein (GFP), 1.5 × 1012 as determined by dot blot.
AAV2 nigral injection
Female Sprague-Dawley rats weighing ~280 g (Charles River Laboratories) were used in the animal experiments. These experiments had previously been approved by the McLean Hospital Institutional Animal Care and Use Committee. Stereotaxic coordinates for the surgeries were taken from the “Rats Atlas” by Paxinos and Watson (1986)
. Before surgery, the animals were anesthetized with xylazine and ketamine (3 mg/kg and 60 mg/kg, respectively). The animals were placed in a stereotaxic frame (Stoelting), where a 10 μ
l Hamilton syringe and 31 gauge needles were used as a delivery system. All injections were made into the substantia nigra using the following anteroposterior (AP), mediolateral (ML), and dorsoventral (DV) coordinates: first site, AP = −4.8 mm; ML = −2.0 mm; DV = −7.2 mm relative to dura, second site, AP=−5.5 mm; ML= −1.9 mm, DV = 7.0 mm relative to dura; toothbar (TB) set at −3.3 mm. Two microliters of AAV GFP or A53T α
-synuclein was injected per site at a rate of 0.5 μ
l/min using microinfusion pumps (Stoelting), with a wait time of 10 min between injection.
Tissue preparation for immunostaining
Animals were terminally anesthetized with an overdose of sodium pentobarbital (150 mg/kg, i.p.), and perfused intracardially with heparinized saline (0.1% heparin in 0.9% saline) followed by paraformaldehyde (4% in PBS). The brains were removed and postfixed for 8 h in 4% paraformaldehyde solution. Following postfixation, the brains were equilibrated in 20% sucrose in PBS, sectioned at 40 μm on a freezing microtome, and serially collected in PBS.
All immunohistochemistry was performed on randomly selected series of sections. Sections were treated for 5 min in 3% hydrogen peroxide (Humco), washed three times in PBS, and incubated in 10% normal goat serum (NGS) and 0.2% Triton X-100 in PBS (PBS-T) for 1 h before overnight incubation at 4°C with the primary antibody diluted in 10% NGS and PBS-T. The primary antibodies used were rabbit anti-tyrosine hydroxylase (TH) (Pel Freez; 1:1000) and mouse anti-human α-synuclein (LB509, Zymed, 1:500). For light microscopy, biotinylated secondary antibodies (Vector Laboratories; 1:300) were used, followed by incubation in streptavidin-biotin complex (Vectastain ABC Kit Elite, Vector Laboratories) for 1 h at room temperature and visualized by incubation in 3,3′-diaminobenzidine (DAB) solution (Vector Laboratories). For immunofluorescence, actin (1:500, Sigma) or phalloidin (1:100, Molecular Probe) was used with Alexa 488, 564, and 647 as secondary antibodies (1:500, Invitrogen) for 1 h at room temperature. Confocal analysis was performed using a Zeiss LSM510/Meta station.
Tissue preparation for Western blot analysis
Animals were terminally anesthetized the same way described above and perfused intracardially with heparinized saline (0.1% heparin in 0.9% saline). Striatum and substantia nigra (SN) were hand-dissected on ice-cold surface, assisted by a tissue chopper. Striatal tissue samples were homogenized using hand-held Polytron homogenizer in ice-cold TEVP buffer, pH 7.4 [10 mM Tris–HCl, 5 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose and protease inhibitors (Sigma P8340, 1:100)]. The homogenate was centrifuged at 800 × g to separate P1 (nuclear and debris) and supernatant (S1) fraction. S1 fraction was centrifuged at 9200 × g to separate P2 (membranous fraction) and S2 (cytosolic fraction). Protein concentrations were determined by BCA assay (Pierce).
Western blot analysis
10~20 μg of protein from the tissue preparations were used to run a gel. The respective volumes were mixed 1:1 with sample buffer and then boiled for 5 min. After boiling, the samples were loaded into the Criterion precast 4~15%, 10%, or 12.5% SDS polyacryl-amide gel system (Bio-Rad). After the electrophoresis was completed, the proteins were transferred from the gel to a PVDF membrane electrically at 80 V for 1 h. Then, the membranes were washed in Tris-buffered saline with 0.1% Tween 20 (TBS-T). After at least 1 h of blocking in 5% nonfat dry milk, the membranes were incubated overnight at 4°C in various primary antibodies [α-synuclein (clone 42, BD Transduction Laboratories; 1:2000), GFP (Invitrogen; 1:5000) TH (Pel Freeze; 1:3000), dopamine transporter (DAT, Millipore Bioscience Research Reagents; 1:2000), Vesicular Monoamine Transporter 2 (VMAT2, Pel Freeze; 1:1000), SNAP-25 (Millipore Bioscience Research Reagents; 1:4000), Rabphilin3A (BD Transduction Laboratories; 1:2000), RAB3A (Affinity Bioreagent; 1:3000), syntaxin (Millipore Bioscience Research Reagents; 1:2000), synaptophysin (Santa Cruz; 1:500), synaptotagmin (BD Transduction Laboratories; 1:5000), synapsin (Millipore Bioscience Research Reagents; 1:5000), Munc-18 (Affinity Bioreagents; 1:3000), KIF1A (clone 16, BD Transduction Laboratories; 1:2000), KIF1B (Bethyl Laboratory; 1:2000), KIF2A (Abcam; 1:10,000), KIF3A (Abcam; 1:2000), KIF5 (Abcam; 1:1000), KIF17 (Abcam; 1:500), myosin Va (Sigma; 1:500), dynein (clone 74.1, Millipore Bioscience Research Reagents; 1:2000), dynamitin (Millipore Bioscience Research Reagents; 1:2000), dynactin1 (Novus, 1:1000), α-tubulin (clone DM1A, Upstate; 1:5000), β-tubulin (Upstate; 1:5000), γ-tubulin (clone GTU-88, Sigma; 1:5000), Actin (clone C-2, Santa Cruz; 1:500), neurofilament 160 (clone NN18, Sigma; 1:1000), neurofilament 200 (clone N52, Sigma; 1:1000), and GAPDH (Millipore Bioscience Research Reagents; 1:5000)]. Then, the membranes were incubated in HRP-conjugated secondary antibodies for 1 h at room temperature. After washing with TBS-T, ECL or ECL plus (Amersham Biosciences) was used to probe for immunoreactive bands and exposed to film using the Kodak Biomax film system. Optical density analysis (NIH ImageJ) was used to determine the relative abundance of protein in each sample.
Quantification of TH-positive neuronal number within the SN was performed using Stereo Investigator software (MBF Bioscience) and stereologic principles as previously described (Chung et al., 2007
). Briefly, the anterior and posterior boundaries of the SN included in the analysis were defined according to the area transduced by the rAAV-eGFP in preliminary experiments [~−4.80 mm through −6.00 mm from bregma (according to the rat brain atlas of Paxinos and Watson, 1986
)]. Stereology was performed using a Zeiss Axiovert microscope coupled to an Optronics Microfire digital camera for visualization of tissue sections. The coefficients of error were calculated according to the procedure of West (1993)
, and values <0.10 were accepted.
Animals were terminally anesthetized the same way as described above and perfused intracardially with heparinized saline (0.1% heparin in 0.9% saline). Small pieces of dorsolateral striatum were hand-dissected on ice-cold surface, assisted by a tissue chopper. Tissue pieces were sent to CMN/KC Neurochemistry Core Laboratory in Vanderbilt University for HPLC analysis. The brain sections were homogenized in 200–750 μl of 0.1 M TCA, which contains 10−2 M sodium acetate, 10−4 M EDTA and 10.5% methanol, pH 3.8, using a tissue dismembrator (Fisher Scientific). Samples were spun in a microcentrifuge at 10,000 ×g for 20 min. The supernatant was removed and stored at −80 degrees. The pellet was saved for protein analysis. Supernatant was then thawed and spun for 20 min. Samples of the supernatant were then analyzed for biogenic monoamines and/or amino acids. Biogenic amines were determined by a specific HPLC assay using an Antec Decade II (oxidation: 0.5) electrochemical detector operated at 33°C. Twenty microliter samples of the supernatant were injected using a Water 717+ autosampler onto a Phenomenex Nucleosil (5 μ, 100A) C18 HPLC column (150 × 4.60 mm). Biogenic amines were eluted with a mobile phase consisting of 89.5% 0.1 M TCA, 10−2 M sodium acetate, 10−4 M EDTA and 10.5% methanol, pH 3.8. Solvent was delivered at 0.8 ml/min using a Waters 515 HPLC pump. Using this HPLC solvent the following biogenic amines eluted in the following order: noradrenaline, Adrenaline, DOPAC, dopamine, 5-HIAA, HVA, 5-HT, and 3-MT. HPLC control and data acquisition were managed by Waters Empower software.
Tissue samples were collected and suspended in lysis buffer (TPER). In addition, phosphatase inhibitors I–II (1:100) and protease inhibitors (1:100) were added fresh before cell lysis (Sigma-Aldrich, P2850, P5276, and P8340 respectively). Following cell lysis, the homogenate was centrifuged, a portion of the supernatant was reserved for protein determination (BCA Assay, Pierce) and the remaining stored at −20 °C. Samples were analyzed for the simultaneous detection of cytokines, chemokines and receptors using a multiplex ELISA based format and performed in duplicate. Testing was performed independently through the Searchlight Testing Service (Pierce, ThermoFisher Scientific).