N-terminal amino acid sequencing of secreted NTN

MTHE-D-06-00146-Supplemental information
N-terminal amino acid sequencing of secreted NTN
293 cells were transfected using the calcium phosphate method with CERE-120
vector genome plasmid described above. Sixteen to 18 hours post transfection, the
culture medium was replaced with fresh serum-free IMEM/F12 50/50mix medium
supplemented with 1mM sodium pyruvate, 1X non essential amino-acids, and 1X
essential amino-acids (Mediatech, Herndon, VA).
Forty-four to 48 hours post
transfection the conditioned medium was collected, filtered and purified by heparin
affinity column chromatography. Eluates were then pre-treated with reducing agent and
electrophoresed onto a 4-12% NuPAGE gel (Invitrogen, Carlsbad, CA) in order to isolate
NTN. Upon transfer onto a PVDF membrane the corresponding 11.8 kD NTN band,
whose location was deduced from a Western blot performed in parallel, was excised and
submitted to Midwest Analytical Incorporated (St. Louis, MO) for N-terminal amino acid
Ninety-six well plates were coated at 4°C overnight with 100µL recombinant GFR-2/Fc
chimera (R&D systems) at 200ng/mL in 25mM NaHCO3 pH 9.7. Wells were then
blocked with 2% BSA, 0.05% Tween-20,1XCasein blocking buffer (BioFX laboratories,
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Owing Mill, MD) in PBS for 2 hours at room temperature (RT). Samples containing
NTN or rhNTN standards (R&D systems, Minneapolis, MN) diluted in IMDM were then
added to wells and incubated overnight at 4°C. After each step, wells were washed four
times with TBST (Tris-HCl 25mM, 135 mM NaCl, 0.5% Tween-20, pH 7.6).
secondary biotinylated goat-anti human NTN polyclonal antibody (R&D systems) diluted
to 400 ng/mL in 1X TBS-Casein, (BioFx laboratories), 0.05%BSA was then added and
incubated for 2 hours at RT. After washing, wells were incubated with 100 L of
streptavidin-horseradish peroxydase conjugate diluted 1:1000 (Chemicon, Temecula,
CA) for 30 min at RT. Finally, NTN was detected by addition of TMB chromogenic
substrate (Promega, Madison, WI). Optical density (450 nm) was determined using a
VersaMax Plate reader (Molecular Devices). The amount of NTN present in samples
was inferred from a rhNTN standard curve.
Surgical Procedures
For all surgical procedures, animals were anesthetized using a cocktail of ketamine (62.5100 mg/kg), xylazine (3.25-5.2 mg/kg), and acepromazine (0.62-1 mg/kg). Each animal
was secured in a stereotaxic frame (Stoelting, Wood Dale, IL), and a midline scalp
incision was made.
The target anterior-posterior (AP) and medial-lateral (ML)
coordinates were measured in mm from bregma, and the dorsal-ventral (DV) coordinates
were measured in mm from dura.
Burr holes were drilled in the skull over the
appropriate injection coordinates. Injections were made using a 10-µL Hamilton syringe
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with a 26-gauge needle, and infusions were controlled using an infusion pump (Stoelting,
Wood Dale, IL).
Histology and immunohistochemistry analyses
At the appropriate time point, animals were deeply anesthetized with an anesthetic
cocktail consisting of ketamine, xylazine, acepromazine, and intra-cardially perfused with
150-200 mL of saline followed by 300-400 mL of a modified Zamboni’s solution (4%
paraformaldehyde, 0.18% picric acid). Brains were removed and placed in the same
fixative solution for a minimum of 2 hours and then transferred into 30% sucrose in 0.1
M phosphate buffer at 4°C for 2 to 3 days for cryoprotection. Forty m thick sections
were collected on a freezing-stage microtome into a 24-well plate and stored in
cryoprotectant at –20°C until time of analysis.
For all IHC staining procedures, sections were pretreated with hydrogen peroxide in TBS.
For NTN IHC, brain sections were immersed in ethanol in Tris-buffered saline (TBS) for
10 minutes and then transferred to hydrogen peroxide. A 1-in-12 series of brain sections
was incubated overnight at 4°C with 400 ng/mL goat anti-human NTN polyclonal
antibody (R&D Systems) in antibody dilution buffer (AbDB, TBS containing 4% horse
serum (HS) and 0.25% Triton-X100). For GDNF IHC, a 1-in-12 series of brain sections
was incubated overnight at 4°C with 0.1 ng/mL goat anti-GDNF polyclonal antibody
(R&D Systems) in AbDB. For tyrosine hydroxylase (TH) IHC, a 1-in-6 series of sections
was incubated overnight at 4°C with 135 ng/mL of rabbit anti-TH polyclonal antibody
(Pel-Freez, Milwaukee, WI) in AbDB.
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Depending on the protein of interest, sections were then incubated with either 4.5 g/mL
biotinylated horse anti-goat or 2.2 g/mL biotinylated donkey anti-rabbit secondary
antibodies (Vector Laboratories, Burlingame, CA), and subsequently with avidin-biotinperoxidase complex (Vector Laboratories, Burlingame, CA). Staining was developed
with 3,3-diaminobenzadine (DAB) prepared according to the manufacturer’s instructions
(KPL, Gaithersburg, MD). Sections were placed onto glass slides, dried overnight,
mounted with DPX Mounting Medium (Electron Microscopy Sciences, Ft. Washington,
PA), and coverslipped.
Volumetric analysis of striatal NTN distribution
For volumetric analysis, images were captured from 1-in-12 series of brain sections
spanning the striatum using a microscope interfaced with a camera and SPOT Advanced
software (v3.4, Diagnostic Instruments, Sterling Heights, MI). The area of NTN
distribution was subsequently determined by measuring the area of positive staining in
mm2. The total volume was then calculated using Cavalieri’s formula: V=AxFxT where:
A is the total sum of areas calculated across all measured sections; F is the sampling
frequency (i.e., 12 for a 1-in-12 series); and T is the sections thickness.
TH positive nigral cell counts
The optical fractionator method [1] was used to estimate the total neuronal cell
population in the SNc using a computerized microscope with the Stereoinvestigator
software v5.0 (MicroBrightField Inc., Colchester, VT). The sections used for counting
the TH-positive neuronal cell population spanned the rostral tip of the substantia nigra
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pars compacta to the caudal end of the substantia nigra pars reticulata. Results are
presented as the percentage of TH-positive cells in the lesioned hemisphere relative to the
number of neurons present in the intact hemisphere.
Striatal Fiber Optical Densitometry
Optical densitometry (OD) was performed on TH-positive fibers in the striatum using the
NIH Image J software v1.3 (National Institute of Health). For each animal, the average
optical density of 4 separate sections through the striatum was determined. According to
the atlas of Paxinos and Watson [2], the approximate sections were as follows relative to
bregma: +0.22 mm, -0.26 mm, -0.74 mm, and -1.22 mm. OD from a region of cortex
immediately adjacent to the corpus callosum was used to correct striatal OD
measurements for non-specific background.
West, M. J., Slomianka, L., and Gundersen, H. J. (1991). Unbiased stereological
estimation of the total number of neurons in thesubdivisions of the rat hippocampus using
the optical fractionator. Anat Rec 231: 482-497.
Paxinos, G., and Watson, C. (2004). The rat brain in stereotaxic coordinates,
Elsevier, New York, NY.
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