Wang X et al.

SUPPLEMENTARY INFORMATION

Supplementary Materials and Methods

Construction of Recombinant Plasmids

Generation of I2CTF construct, plasmid in pTRUF12 vector and packaging of this vector in AAV were the same as described previously [15]. Employing pEGFP-N3/I2PP2A (wt) previously generated by us [14] as a template, I2CTF cDNA was obtained by PCR with primer 1 (5’-GATGGATCC AAAGCCAGCAGGAAGA) and primer 2 (5’-GATCTCGAGTTAGTCATCTTCTC). The BamHI site underlined in primer 1 and the XhoI site underlined in primer 2 were used to clone the fragment into pcDNA3.1 vector (Invitrogen, Carlsbad, CA). The plasmid was verified by DNA sequencing. The construction of pcDNA 3.1- I2CTF plasmid was performed as previously described [15].

Vector Packaging and Titering

Recombinant AAV serotype 1 was generated as described previously [7]. Briefly, 293-T cells were co-transfected with pTRUF plasmids and helper plasmid pXYZ1 [16]. The virus was purified by using iodixanol density gradient (Optiprep; Greiner Bio-One Inc.). Virus samples were concentrated and formulated into lactated Ringer’s solution (Baxter Healthcare). Genome containing particles (gcp) were determined by real-time PCR (LightCycler; Roche Diagnostics) and SYBR Green Taq ReadyMix (Sigma–Aldrich). Titers were calculated from a standard curve generated from pTRUF.

Western Blots

Frozen autopsied spinal cords from ten cases of sporadic ALS and three age-matched control cases and rat brain tissue were used for Western blots. The tissue was homogenized on ice to generate 12% (w/v) homogenate in buffer containing 50 mM Tris.HCl (pH 7.4), 8.5% sucrose, 2 mM EDTA, 10 mM b-mercaptoethanol, 0.2 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin and 2 µg/ml each of aprotinin and pepstatin A. The tissue homogenate used for Western blots also contained 20 mM b-glycerophosphate, 50 mM NaF, and 1 mM Na3VO4 to inhibit phosphatase activities. The protein concentration of each tissue homogenate was measured by modified Lowry assay [1]. The tissue samples were then boiled in Laemmli’s buffer in a water bath for 5 mins, subjected to 12% (for I2CTF) or 10% SDS-PAGE and Western blots. Immunoreactive protein bands were visualized with enhanced chemiluminescence (ECL) reagents (Pierce). For Western blots, the primary antibodies used were: I2CTF oAb (2 mg/ml), I2NTF mAb (5.2 mg/ml), mAb b-actin (1:2000, Sigma), anti-PP2Ac mouse monoclonal antibody (1:10,000, BD Transduction Laboratoriesä), pan tau antibody, rabbit polyclonal antibody (pAb) 92e (1:5000) [5], phospho-specific tau antibodies Tau pAb pS199 (1:1000, BioSource), Tau pAb pT205 (1:1000, BioSource), Tau pAb pT212 (1:500, BioSource), Tau pAb pS214 (1:500, BioSource), Tau monoclonal antibody (mAb) M4 to phosphorylated Thr 231/Ser 235 (1:1500) [6], 12E8 to phosphorylated Ser 262/356 (1:500) [11], Tau pAb pS262 (1:1000, BioSource), Tau pAb pT231 (1:1000, BioSource), Tau pAb pS396 (1:1000, BioSource), Tau pAb R145 to pS422 (1:3000) [12], mAb GFP (1:1000, Rockland), mAb β-actin (1:2000, Sigma), mAb DM1A to a-tubulin (1:1000, Sigma), rabbit pAb bIII tubulin (1:5000, Covance), mAb SMI52 to MAP2a,b (1:5000, Covance), mAb to synaptophysin (1:500, Chemicon), and rabbit pAb to synapsin-1 (1:1000, Stressgen, Ann Arbor, MI).

Extraction of sarkosyl insoluble tau

Sarkosyl insoluble tau was isolated from brain tissues as described previously [3]. In brief, 0.1-0.5 g of tissue samples from the cerebellum, the cerebral cortex, the subcortical area, and the hippocampus were homogenized in 10 volumes of 10 mM Tris, pH 7.4, 0.8 M NaCl, 1 mM EGTA and 10% sucrose and centrifuged at 27, 200 ´ g for 20 min. Then, cleared supernatant was adjusted to 1% (w/v) N-lauroylsarcosine (Sigma, Germany) and incubated 1 h at room temperature. After the incubation, the extract was spun at 12,300 ´ g for 1 h at RT. The pellet was resuspended in 60 ml of 10 mM sodium phosphate, pH 7.4, 150 mM NaCl and analyzed by Western blots.

Immunoprecipitation

Immunoprecipitation was carried out on 16,000 ´ g extracts of tissue from the cerebellum, the cerebral cortex, and the hippocampus from control AAV1-GFP and experimental (AAV1-I2CTF) rats at 14 months post-AAV infection. Briefly, tissues were homogenized in lysis buffer containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1 mM EGTA, 2 mM EDTA, 1% Triton X-100, 0.2 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin and 2 µg/ml each of aprotinin and pepstatin A. After centrifugation at 16,000 g for 15 min, the lysates (300 mg of protein) were immunoprecipitated with anti-PP2Ac rabbit polyclonal antibody R123d [9], followed by protein G Sepharose (Pierce, 20399, Rockford, IL). Eluted proteins were divided into two parts: one part was analyzed by Western blots developed with anti-PP2Ac mouse monoclonal antibody 1D6 (Millipore, Billerica, MA), the other part was used for the PP2Ac activity assay.

PP2A activity assay

An ELISA was used to assay the PP2A activity in the proteins immunoprecipitated by anti-PP2Ac rabbit polyclonal antibody R123d [9]. Briefly, a 96 well plate was coated at 4°C overnight with 30 ml of 8.0 µg/ml solution of synthetic tau phosphopeptide p [13] corresponding to tau194–207 to which three lysines were added at the C-terminal end to enhance its binding to the microtiter plate (Ac-Arg-Ser-Gly-Tyr-Ser-Ser(OPO32-)-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Lys-Lys-Lys-NH2). For blocking, the coating solution was removed and the wells were blocked with 190 µl of protein-free blocking buffer (Pierce, type 37571) for 1 hour at room temperature. The plates were then washed four times for 30 mins with 190 µl/well Tris.HCl (pH 7). The enzymatic reaction was performed with 34 µl/well of the immunoprecipitated protein solution resuspended with reaction buffer containing 50 mM Tris.HCl (pH 7), 20 mM b-mercaptoethanol, 2 mM MnCl2 and 0.1 mg/ml BSA. The plates were incubated in a moist chamber for 90 mins at 30°C. To stop the enzyme reaction, the mixture was immediately removed and the wells were washed with blocking buffer containing 50 mmol/l NaF. The plates were washed four times for 5 mins with 190 µl/well Tris, 0.05% Tween-20 (pH 7.6) and each well was then incubated with 37 µl of the monoclonal antibody Tau-1 to tau unphosphorylated at Ser198/199/202 [2,4] overnight at 4°C. The plates were washed four times and incubated with the secondary antibody (peroxidase linked goat anti-mouse IgG, 1:1,200) for 1 hour at room temperature. Finally, after the plates were washed five times with TBS (pH 7.6), the color reaction was performed using 50 µl of tetramethylbenzidine (TMB) per well and the development was monitored at 650 nm in a microtiter plate reader.

Immunohistochemistry and toluidine blue staining

Rats were anesthetized with intraperitoneal injection of 50 mg Nembutal/kg body weight, and then perfused through the aorta with 100 ml 0.9% NaCl followed by 100 ml phosphate buffer containing 4% paraformaldehyde. Brains and spinal cords were removed and post-fixed in 4% paraformaldehyde overnight then equilibrated in a cryoprotectant solution of 30% sucrose/PBS and stored at 4°C. Coronal sections (40 µm thick) were cut using a freezing microtome. Spinal cord tissue blocks were cut into 5 mm sections on a cryostat, mounted onto gelatin-coated slides and kept at 4°C until used.

For immunofluorescence staining, after incubation in 0.3% Triton-X100-PBS for 30 mins, free floating cryostat sections were blocked with 5% goat serum containing 0.1% Tween 20 in PBS for 30 mins at room temperature. Sections were then incubated overnight at 4°C with primary antibodies. The following primary antibodies were used: pAb GFP (1:500, Rockland), Tau mAb 12E8 to phosphorylated Ser 262/356 (1:500) [11], mAb SMI52 to MAP2a,b (1: 1,000; Covance), mAb anti-synaptophysin (1:200; Chemicon), rabbit polyclonal anti-synapsin-1 (1:500; Stressgen, Ann Arbor, Michigan). Treatment with primary antibodies was followed by incubation with secondary antibodies: Alexa 488-conjugated goat anti-rabbit antibody (1:1,000) and Alexa Fluor 555 goat anti-mouse antibody (1:1,000; Invitrogen). Finally, the sections were washed in TBS and mounted on glass slides with ProLong® Gold anti-fade reagent (Invitrogen).

To reduce endogenous peroxidase activity and to prevent nonspecific antibody binding, selected sections were treated in 1% hydrogen peroxide for 2 hr and 5% normal goat serum (NGS) for 30 min in PBS after incubation in PBS containing 1% Triton X-100. Thereafter, sections were incubated with primary antibodies mAb anti-Ab17–24 (4G8, 1:300; Covance), rabbit polyclonal anti-Ab34–40 (1:500; Invitrogen), rabbit polyclonal anti-Ab36–42 (1:500; Invitrogen), mouse monoclonal SMI33 and SMI34 to non-phosphorylated (np) and phosphorylated (p) neurofilament-heavy subunit (NF-H), respectively (Sternberger Monoclonals, Baltimore, MD, USA; 1:5000), at 4°C for 35–40 hr, followed by incubation with biotinylated goat anti- mouse secondary antibody at room temperature for 2 hr and avidin–biotin complex (1:100, Vector ABC Elite; Vector Labs) at room temperature for 2 hr and developed with diaminobenzidine tetrachloride (5 g/l) and 0.005% hydrogen peroxide in 0.05 M Tris-HCl buffer for 10–15 min. Sections were then dehydrated, coverslipped, and examined using a light microscope.

For toluidine blue staining, spinal cord sections were deparaffinized and rehydrated by 5 min incubation in xylene twice, 2 min in 100% alcohol twice, 2 min in 95% alcohol, 2 min in 80% alcohol, 2 min in 70% alcohol and a 1 min rinse in distilled water. The sections were then treated with a staining solution of 1% toluidine blue for 15 min and rinsed in distilled water for 1 min. The sections were dehydrated in ascending ethanol concentrations, cleared in xylene, and mounted.

Fluoro-Jade B labeling

Fluoro-Jade staining was carried out according to Schmued et al.[10]. Tissue sections were mounted on 2% gelatin-coated slides and then dried at room temperature overnight. Slides were briefly rinsed in distilled water followed by three min incubation in 100% alcohol, 1 min in 70% alcohol, 1 min in 30% alcohol, and a 1 min rinse in distilled water. The sections were then treated with a solution of 0.06% potassium permanganate for 15 mins with gentle shaking and rinsed in distilled water for 1 min. The staining solution contained a 0.001% Fluoro-Jade B (Chemicon, Temecula, California) in 0.1% acetic acid. After incubating for 30 mins in the staining solution, the sections were washed three times for 1 min in distilled water and dried. The dry sections were washed 3 times in xylene two mins each before mounting with DPX Fluka (Milwaukee, WI). Images were captured using a Nikon 90i microscope equipped with epifluorescence optics. The fluoro-Jade B positive signal of a given area from 3 animals/group was measured using NIH Image J software. The average of the fluorescent relative density was expressed as percentage of the control.

Morris water maze spatial reference memory task

Spatial reference memory was evaluated in a Morris water maze task. The test requires that rats use a spatial navigational strategy based on a spatial representation of the environment to find a fixed submerged escape platform. In the present procedure, rats were submitted to 3 days of training. All animals for behavioral testing were coded such that the experimentator was blind to the assignment of the animals to specific treatment groups. Before training, the rats were handled gently for 2-3 min/day during 3 days to minimize non-specific stress. The Morris water maze procedure was performed using a 180-cm diameter circular tank filled with water (±21°C) made opaque by addition of white non-toxic paint. The pool was placed in a part of the room separated from the experimentator and the control station with a black opaque curtain. Acquisition was started with the submerged escape platform (1.5 cm below the water surface) in the North-East quadrant and each animal was given 90 sec to find the submerged escape platform. If the rat did not find the platform in 90 sec, it was guided to it. Four such acquisition trials were given on each day, for three consecutive days. Three months later (i.e., 8 months of age), the rats were tested in the same procedure, but the environment of the room was modified and the platform located in a different quadrant. One month later (i.e., 9 months of age) rats performed a transfer task. The basic rule of this task is similar to the training procedure except that the submerged platform was located in another quadrant of the latest environment. During the transfer test, rats where submitted to three trials of 90 s each. The measures of learning were the distances swum to reach the escape platform. Rat behavior in the water maze was monitored by a Samsung Digital Camera (SDC 4304) mounted to the ceiling and tracked and timed by a SMART (Pan Lab/San Diego Instruments) version 2.0.14 software.

Neurological examination

Neurological status of the animals was evaluated using a scoring system adapted from Korenova and collaborators [8] (see sTable 2). This examination provided a quantitative reflection of sensorimotor, neurological and neuromuscular alterations.

General observation: The observation involved assessment of posture and limb functions.

Hind-limb extension reflex: The hind-limb escape reflex response evaluates the distance between the hind limbs after elevation by the tail. It is reported that muscle atrophy correlates with impaired extension reflex response [8].

Beam walking test: For the beam-walking test, three types of beams were used (3 cm x 3 cm; 4 cm x 2 cm; a round beam of 3.5 cm in diameter). The beam lengths were 180 cm and they were placed 75 cm above the floor. Two training and 2 testing trials were performed. Traversing latency and the number of hind-limb slips made during the test performance were measured and scored according to a defined rating scale (sTable 2). The task was performed using (1) a beam with a cross-section of 3 cm x 3 cm (1st day); (2) a beam with a rectangular cross-section of 4 cm x 2 cm (2nd day); and (3) a beam with a round cross-section of 3.5 cm diameter (3rd day).

Prehensile traction test: With its forepaws, the rat was allowed to grasp a horizontal steel wire (3 mm in diameter) suspended 75 cm above a padded surface. The latency for the rat to fall from the wire was measured. The scoring conditions are described in sTable 2.

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Wang X et al.

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