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 cotransfected 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 -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 -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
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Wang X et al.
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: I 2CTF
oAb (2 g/ml), I2NTF mAb (5.2 g/ml), mAb -actin (1:2000, Sigma), anti-PP2Ac mouse monoclonal antibody
(1:10,000, BD Transduction Laboratories), 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 -tubulin
(1:1000, Sigma), rabbit pAb  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 l 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 g 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
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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 l 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-SerPro-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 -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
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Wang X et al.
freezing microtome. Spinal cord tissue blocks were cut into 5 m sections on a cryostat, mounted onto gelatincoated 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 antisynapsin-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-A17–24
(4G8, 1:300; Covance), rabbit polyclonal anti-A34–40 (1:500; Invitrogen), rabbit polyclonal anti-A36–42 (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
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Wang X et al.
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
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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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References
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(1):241-250
2. Binder LI, Frankfurter A, Rebhun LI (1985) The distribution of tau in the mammalian central nervous system.
The J Cell Biol 101 (4):1371-1378
3. Greenberg SG, Davies P (1990) A preparation of Alzheimer paired helical filaments that displays distinct tau
proteins by polyacrylamide gel electrophoresis. Proc Natl Acad Sci USA 87 (15):5827-5831
4. Grundke-Iqbal I, Iqbal K, Tung YC, Quinlan M, Wisniewski HM, Binder LI (1986) Abnormal phosphorylation of
the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci USA
83 (13):4913-4917
5. Grundke-Iqbal I, Vorbrodt AW, Iqbal K, Tung YC, Wang GP, Wisniewski HM (1988) Microtubule-associated
polypeptides tau are altered in Alzheimer paired helical filaments. Brain Res 464 (1):43-52
6. Hasegawa M, Watanabe A, Takio K, Suzuki M, Arai T, Titani K, Ihara Y (1993) Characterization of two distinct
monoclonal antibodies to paired helical filaments: further evidence for fetal-type phosphorylation of the tau
in paired helical filaments. J Neurochem 60 (6):2068-2077
7. Henckaerts E, Dutheil N, Zeltner N, Kattman S, Kohlbrenner E, Ward P, Clement N, Rebollo P, Kennedy M,
Keller GM, Linden RM (2009) Site-specific integration of adeno-associated virus involves partial
duplication of the target locus. Proc Natl Acad Sci USA 106 (18):7571-7576
8. Korenova M, Zilka N, Stozicka Z, Bugos O, Vanicky I, Novak M (2009) NeuroScale, the battery of behavioral
tests with novel scoring system for phenotyping of transgenic rat model of tauopathy. J Neurosci Methods
177 (1):108-114
9. Pei JJ, Gong CX, Iqbal K, Grundke-Iqbal I, Wu QL, Winblad B, Cowburn RF (1998) Subcellular distribution of
protein phosphatases and abnormally phosphorylated tau in the temporal cortex from Alzheimer's disease
and control brains. J Neural Transm 105 (1):69-83
10. Schmued LC, Albertson C, Slikker W, Jr. (1997) Fluoro-Jade: a novel fluorochrome for the sensitive and
reliable histochemical localization of neuronal degeneration. Brain Res 751 (1):37-46
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11. Seubert P, Mawal-Dewan M, Barbour R, Jakes R, Goedert M, Johnson GV, Litersky JM, Schenk D, Lieberburg
I, Trojanowski JQ, et al. (1995) Detection of phosphorylated Ser262 in fetal tau, adult tau, and paired
helical filament tau. J Biol Chem 270 (32):18917-18922
12. Tanaka T, Zhong J, Iqbal K, Trenkner E, Grundke-Iqbal I (1998) The regulation of phosphorylation of tau in
SY5Y neuroblastoma cells: the role of protein phosphatases. FEBS Lett 426 (2):248-254
13. Tanimukai H, Grundke-Iqbal I, Iqbal K (2004) Inhibitors of protein phosphatase-2A: topography and subcellular
localization. Brain Res Mol Brain Res 126 (2):146-156
14. Tsujio I, Zaidi T, Xu J, Kotula L, Grundke-Iqbal I, Iqbal K (2005) Inhibitors of protein phosphatase-2A from
human brain structures, immunocytological localization and activities towards dephosphorylation of the
Alzheimer type hyperphosphorylated tau. FEBS Lett 579 (2):363-372
15. Wang X, Blanchard J, Kohlbrenner E, Clement N, Linden RM, Radu A, Grundke-Iqbal I, Iqbal K (2010) The
carboxy-terminal fragment of inhibitor-2 of protein phosphatase-2A induces Alzheimer disease pathology
and cognitive impairment. FASEB J 24 (11):4420-4432
16. Zolotukhin S, Potter M, Zolotukhin I, Sakai Y, Loiler S, Fraites TJ, Jr., Chiodo VA, Phillipsberg T, Muzyczka
N, Hauswirth WW, Flotte TR, Byrne BJ, Snyder RO (2002) Production and purification of serotype 1, 2,
and 5 recombinant adeno-associated viral vectors. Methods 28 (2):158-167
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sTable 1: Characteristics of ALS and control cases
Dx
PMIa
Age/Gender
Age at
Symptom
Onset
Con 1
corticobasal ganglionic degeneration
15.5
74/F
69
65
Con2
McArdle disease
20
50/M
10
Con 3
mitochondrial neurogastrointestinal
encephalopathy
10
26/F
ALS1
sALS
unknown
ALS2
sALS
ALS3
Number in
Figure
Disease
Duration
(Months)
Site of Onset
Cause of Death
bulbar
Respiratory arrest
460
decreased exercise
tolerance
Myocardial infarction
15
129
GI
Myocardial infarction
70/F
69
8
lower extremity
Respiratory arrest
unknown
51/F
48
47
bulbar
Respiratory arrest
sALS
unknown
53/M
41
lower extremity
Myocardial infarction
ALS4
sALS
15
74/M
70
56
bulbar
Respiratory arrest
ALS5
sALS
8
55/F
51
48
lower extremity
Respiratory arrest
ALS6
sALS
6
53/M
45
100
lower extremity
Respiratory arrest
ALS7
sALS
8
62/M
58
50
lower extremity
Respiratory arrest
ALS8
sALS
8
57/M
52
66
lower extremity
Respiratory arrest
ALS9
sALS
unknown
69/F
65
40
upper extremity
Respiratory arrest
ALS10
sALS
24
63/F
61
27
upper extremity
Respiratory arrest
a
57
(till vented)
Lumbar spinal cord from all cases, except ALS8, which was thoracic spinal cord; sALS, sporadic ALS.
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sTable 2
Neuroscale scoring protocol (adapted from Korenova et al., 2009)
General observation (normal 0; maximum 4 points)
1 Abnormal posture
1 Forelimb paralysis
1 Hind-limb paralysis
1 Vocalization during examination
4
Hind-limb extension reflex (normal 0; maximum 3 points)
0 Hind-limb extended with spreading toes
Hind-limb extended with spreading toes, distance between limbs is shorter than
1
in previous scoring
2 Hind-limb almost in contact, toes spread (or flexed)
3 Hind-limb extended clasped or crossed with toes flexed
3
Beam walking test – escape latency – 3 testing conditions (normal 0-5; maximum 15
points)
0 Latency ≤2s
1 Latency 2.1-4.0s
2 Latency 4.1-6.0s
3 Latency 6.1-10.0s
4 Latency 10.1s and more
5 Animal not able to perform the task
Beam walking test – Number of hind-limb slips – 3 testing conditions (normal 0-5;
maximum 15 points)
0 Hind-limb slips ≤1
1 Hind-limb slips 1.1-2.0
2 Hind-limb slips 2.1-3.0
3 Hind-limb slips 3.1-5.0
4 Hind-limb slips5.1-8.0
5 More than 8 hind-limb slips; animal is not able to perform the task
15
15
Prehensile traction-test – latency to fall (normal 0; maximum 5 points)
5 Latency ≤2s
4 Latency 2.1-4.0s
3 Latency 4.1-6.0s
2 Latency 6.1-10.0s
1 Latency 10.1-15.0s
0 More than 15s
5
Maximum points
42
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Supplemental figure legends:
sFig. 1. Both pNF and ubiquitin stainings are markedly increased in axon of the lumbar level of spinal
cords of I2CTF rats. The data are presented as means ±S.D. of 21 images of tissue sections per group. **P<0.01
vs. GFP control.
Supplementary Movies
Movies of rat neurological evaluation, Movie S1, Movie S2, Movie S3 and Movie S4.
File contains four movies showing neurological evaluation in GFP (control) and I 2CTF rats. Movie S1 shows GFP
(control) rats hold themselves with their forelimbs on a rod (prehensile test) (MPEG; 5.7 mb). Movie S2 shows
inability of I2CTF rats to hold themselves with forelimbs on a rod (MPEG 3.28 mb). Movie S3 shows the ability of
GFP rats to work on an elevated beam (elevated beam test) (MPEG; 1.65 mb). Movie S4 shows the inability of I2CTF
rats to walk on an elevated bean (MPEG; 2.5 mb).
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