Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease methods
Aim. Evidence-backed execution summary for Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease methods from Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Materials and Methods
reagent used in the protocol.
- Use
- Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine which of these antibodies displayed the most specific binding to human α-syn, of these antibodies, 9E4 displayed the most specificity and...
Materials and Methods
reagent used in the protocol.
- Use
- For studies of antibody trafficking into the CNS the mouse monoclonal antibody 9E4 was concentrated with a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) and linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) accord...
Preparation of antibodies for passive immunization
reagent used in the protocol.
- Use
- Previous active immunization experiments have suggested that the most effective antibodies were against CT-epitopes of α-syn, for this study a panel of antibodies were developed 1) clone 8A5 (α-syn epitope 125-140; IgG1); 2) clone 9E4 (α-syn epitope 118-126; IgG1) and 6H7 (α-syn epit...
ELISA analysis of brain and plasma antibody concentrations
reagent used in the protocol.
- Use
- Antibody levels in the brain and plasma of immunized mice were determined as previously described. Briefly, using 96-well microtiter plates coated with 0.4 µg per well of purified full-length α-syn. Samples were incubated overnight followed by goat anti-mouse IgG alkaline phosphatase-conjugated antibody (...
Immunoblot analysis
reagent used in the protocol.
- Use
- Briefly, as previously described, brains were homogenized and divided into cytosolic and membrane fractions,. For immunoblot analysis, 20 µg of total protein per lane was loaded into 4-12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes. For characterization of the antib...
Immunocytochemical and neuropathological analyses
reagent used in the protocol.
- Use
- For characterization of the antibodies used for immunotherapy, vibratome sections from untreated non-tg and α-syn tg mice were incubated the with monoclonal antibodies against CT and NT-α-syn (9E4, 6H7 and 8A5, ELAN Pharmaceuticals). Analysis of α-syn accumulation for the immunotherapy experiment was...
Neuronal cell cultures and treatments
reagent used in the protocol.
- Use
- The rat neuroblastoma cell line B103 was used for in vitro experiments. This model was selected because over expression of α-syn in these cells interferes with neuronal plasticity (reduced neurite outgrowth and adhesion) but does not result in overt cell death,. This model mimics the early pathogenic process...
An antibody against CT-α-syn ameliorates motor and learning deficits and synaptic pathology in α-syn tg mice
reagent used in the protocol.
- Use
- Following the initial screening and subsequent selection of the 9E4 antibody, α-syn tg and non-tg mice were passively immunized with either 9E4 or the control IgG1. Antibody titers in the passively immunized α-syn tg and non-tg mice were analyzed by ELISA. On average titer levels were comparable between pa...
Materials and Methods
Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine which of these antibodies displayed the most specific binding to human α-syn, of these antibodies, 9E4 displayed the most specificity and...
- Use
- Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine which of these antibodies displayed the most specific binding to human α-syn, of these antibodies, 9E4 displayed the most specificity and...
Materials and Methods
For studies of antibody trafficking into the CNS the mouse monoclonal antibody 9E4 was concentrated with a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) and linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) accord...
- Use
- For studies of antibody trafficking into the CNS the mouse monoclonal antibody 9E4 was concentrated with a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) and linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) accord...
Behavioral testing
In patients with LBD, α-syn accumulates not only in subcortical nuclei but also in the temporal cortex and limbic system and accounts for cognitive deficits in these patients. Similarly, in our PDGF-α-syn tg mice, protein accumulation occurs in the temporal cortex and hippocampus. In this context and as...
- Use
- In patients with LBD, α-syn accumulates not only in subcortical nuclei but also in the temporal cortex and limbic system and accounts for cognitive deficits in these patients. Similarly, in our PDGF-α-syn tg mice, protein accumulation occurs in the temporal cortex and hippocampus. In this context and as...
Rotarod
Mice were analyzed for 2 days in the Rotarod (San Diego Instruments, San Diego, CA), as previously described (Masliah et al., 2000). On the first day, mice were trained for five trials: the first one at 10 rpm, the second at 20 rpm, and the third to the fifth at 40 rpm. On the second day, mice were tested for seven...
- Use
- Mice were analyzed for 2 days in the Rotarod (San Diego Instruments, San Diego, CA), as previously described (Masliah et al., 2000). On the first day, mice were trained for five trials: the first one at 10 rpm, the second at 20 rpm, and the third to the fifth at 40 rpm. On the second day, mice were tested for seven...
Immunoblot analysis
Briefly, as previously described, brains were homogenized and divided into cytosolic and membrane fractions,. For immunoblot analysis, 20 µg of total protein per lane was loaded into 4-12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes. For characterization of the antib...
- Use
- Briefly, as previously described, brains were homogenized and divided into cytosolic and membrane fractions,. For immunoblot analysis, 20 µg of total protein per lane was loaded into 4-12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes. For characterization of the antib...
Immunocytochemical and neuropathological analyses
For characterization of the antibodies used for immunotherapy, vibratome sections from untreated non-tg and α-syn tg mice were incubated the with monoclonal antibodies against CT and NT-α-syn (9E4, 6H7 and 8A5, ELAN Pharmaceuticals). Analysis of α-syn accumulation for the immunotherapy experiment was...
- Use
- For characterization of the antibodies used for immunotherapy, vibratome sections from untreated non-tg and α-syn tg mice were incubated the with monoclonal antibodies against CT and NT-α-syn (9E4, 6H7 and 8A5, ELAN Pharmaceuticals). Analysis of α-syn accumulation for the immunotherapy experiment was...
Immunocytochemical and neuropathological analyses
Stereological analysis and quantification of neocortical and hippocampal intra-neuronal FL-αsyn and CC-αsyn immunoreactivity was conducted by the disector method using the Stereo-Investigator System (MBF Bioscience, Williston, VT) and the results were averaged and expressed as cell counts per 0.1 mm 3. Ne...
- Use
- Stereological analysis and quantification of neocortical and hippocampal intra-neuronal FL-αsyn and CC-αsyn immunoreactivity was conducted by the disector method using the Stereo-Investigator System (MBF Bioscience, Williston, VT) and the results were averaged and expressed as cell counts per 0.1 mm 3. Ne...
Double immunolabeling and fluorescence co-labeling
To determine the co-localization between α-syn and lysosomal and autophagy markers double-labeling experiments were performed, as previously described. For this purpose, vibratome sections were immunolabeled with the rabbit polyclonal antibodies against α-syn (Millipore, affinity purified polyclonal, 1...
- Use
- To determine the co-localization between α-syn and lysosomal and autophagy markers double-labeling experiments were performed, as previously described. For this purpose, vibratome sections were immunolabeled with the rabbit polyclonal antibodies against α-syn (Millipore, affinity purified polyclonal, 1...
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Materials and Methods
Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine which of these antibodies displayed the most specific binding to human α-syn, of these antibodies, 9E4 displayed the most specificity and was chosen for the immunization study. A total of 40 α-syn tg mice (6 m/o, n = 20 mice per group) received weekly intraperitoneal (IP) injections (10 mg/kg) for 6 months with the CT-α-syn antibody (9E4) and IgG1 control. An additional group of non-tg mice treated with the 9E4 antibody (n = 12) and the IgG1 control (n = 12) was included as control for behavioral and neuropathological studies. Mice were bled once a month and antibody titers monitored by enzyme-linked immunosorbent assay (ELISA). At the end of the studies, mice wer...
Materials and Methods
For studies of antibody trafficking into the CNS the mouse monoclonal antibody 9E4 was concentrated with a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) and linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. For this experiment non-tg (total n = 18) and α-syn tg mice (total n = 18) (6 m/o) were injected intravenously (IV) with the 9E4-FITC or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg. Mice were sacrificed 3, 14 and 30 days after injection (n = 3 per group). CSF from these mice was used to immunolabel cortical sections from antibody-naive animals.
Materials and Methods
As an additional control to monitor the passage of FITC-labeled antibodies across the blood-brain barrier, mice were injected with FITC tagged β -syn. The For this experiment non-tg (total n = 8) and α-syn tg mice (total n = 8) (6 m/o) were injected intravenously (IV) with the FITC-labeled β-syn or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg. Mice were sacrified at 14 days post-injection. CSF from these mice was also used to immunolabel slides from FITC tagged β -syn niave non-tg and α-syn tg mice.
Materials and Methods
Upon sacrifice, the right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was snap-frozen and stored at -70°C for subsequent protein analysis. All experiments described were approved by the animal subjects committee at the University of California at San Diego (UCSD) and were performed according to NIH recommendations for animal use. UCSD is an Institutional Animal Care and Use Committee accredited institution and the UCSD Animal Subjects Committee approved the experimental protocol ( S02221 ) followed in all studies according to the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines.
Behavioral testing
In patients with LBD, α-syn accumulates not only in subcortical nuclei but also in the temporal cortex and limbic system and accounts for cognitive deficits in these patients. Similarly, in our PDGF-α-syn tg mice, protein accumulation occurs in the temporal cortex and hippocampus. In this context and as previously described, in order to evaluate the functional effects of passive immunization treatment in mice, groups of non-tg and α-syn tg animals were tested in the water maze. For this purpose, a pool (diameter 180 cm) was filled with opaque water (24°C) and mice were first trained to locate a visible platform (days 1-3) and then a submerged hidden platform (days 4-7) in three daily trials 2-3 min apart. Mice that failed to find the hidden platform within 90 seconds were placed on it for 30 seconds. The same platform location was used for all...
Pole Test
For the pole test, animals were placed head upward on top of a vertical wooden pole 50 cm long and 1 cm in diameter. When placed on the pole, animals orient themselves downward and descend the length of the pole. Groups of mice received training that consisted of five trials for each session. For testing, animals received five trials and the time taken to descend (T-total) was measured.
Rotarod
Mice were analyzed for 2 days in the Rotarod (San Diego Instruments, San Diego, CA), as previously described (Masliah et al., 2000). On the first day, mice were trained for five trials: the first one at 10 rpm, the second at 20 rpm, and the third to the fifth at 40 rpm. On the second day, mice were tested for seven trials at 40 rpm each. Mice were placed individually on the cylinder and the speed of rotation increased from 0 to 40 rpm over a period of 240 s. The length of time mice remained on the rod (fall latency) was recorded and used as a measure of motor function.
Immunocytochemical and neuropathological analyses
Stereological analysis and quantification of neocortical and hippocampal intra-neuronal FL-αsyn and CC-αsyn immunoreactivity was conducted by the disector method using the Stereo-Investigator System (MBF Bioscience, Williston, VT) and the results were averaged and expressed as cell counts per 0.1 mm 3. Neocortical and hippocampal FL-αsyn and CC-αsyn immunoreactive neuropil was assessed in digital images analyzed with the Image Quant software by selecting and area to exclude cell bodies, setting the threshold levels and expressing the data as pixel intensity (arbitary units).
Measurement outputs
What raw and processed outputs should exist?
Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine whi...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Upon sacrifice, the right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was sn...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
In patients with LBD, α-syn accumulates not only in subcortical nuclei but also in the temporal cortex and limbic system and accounts for cognitive deficits in these patien...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Mice were analyzed for 2 days in the Rotarod (San Diego Instruments, San Diego, CA), as previously described (Masliah et al., 2000). On the first day, mice were trained for five...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
All experiments were done blind-coded and in triplicate.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine whi...; Upon sacrifice, the right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was sn...; In patients with LBD, α-syn accumulates not only in subcortical nuclei but also in the temporal cortex and limbic system and accounts for cognitive deficits in these patien...; Mice were analyzed for 2 days in the Rotarod (San Diego Instruments, San Diego, CA), as previously described (Masliah et al., 2000). On the first day, mice were trained for five....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
All experiments were done blind-coded and in triplicate. Values in the figures are expressed as means ± SEM. To determine the statistical significance, values were compared...; (A) Antibody titers determined by ELISA in non-tg or α-syn tg mice immunized with the C-terminal antibody (9E4) or IgG1 controls. Horizontal lines represent the mean of the...; The effects of passive immunization on motor behavior in the α-syn tg mice was assessed using the rotarod and pole test. Results from the pole test demonstrated a motor imp...; Statistical analysis of the Rotarod results using repeated-measures two-way ANOVA demonstrated that IgG1-treated α-syn tg mice spent significantly less time on the rotating...
from paperReporting output
Report representative outputs alongside summary comparisons for Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine whi..., Upon sacrifice, the right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was sn..., In patients with LBD, α-syn accumulates not only in subcortical nuclei but also in the temporal cortex and limbic system and accounts for cognitive deficits in these patien..., Mice were analyzed for 2 days in the Rotarod (San Diego Instruments, San Diego, CA), as previously described (Masliah et al., 2000). On the first day, mice were trained for five....
inferred from protocolStructured statistical methods
All experiments were done blind-coded and in triplicate. Values in the figures are expressed as means ± SEM. To determine the statistical significance, values were compared...; (A) Antibody titers determined by ELISA in non-tg or α-syn tg mice immunized with the C-terminal antibody (9E4) or IgG1 controls. Horizontal lines represent the mean of the...; The effects of passive immunization on motor behavior in the α-syn tg mice was assessed using the rotarod and pole test. Results from the pole test demonstrated a motor imp...; Statistical analysis of the Rotarod results using repeated-measures two-way ANOVA demonstrated that IgG1-treated α-syn tg mice spent significantly less time on the rotating...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine which of these antibodies displayed the most specific binding to human α-syn, of these antibodies, 9E4 displayed the most specificity and was chosen for the immunization study. A total of 40 α-syn tg mice (6 m/o, n = 20 mice per group) received weekly intraperitoneal (IP) injections (10 mg/kg) for 6 months with the CT-α-syn antibody (9E4) and IgG1 control. An additional group of non-tg mice treated with the 9E4 antibody (n = 12) and the IgG1 control (n = 12) was included as control for behavioral and neuropathological studies. Mice were bled once a month and antibody titers monitored by enzyme-linked immunosorbent assay (ELISA). At the end of the studies, mice were tested for functional effects in the water maze. Brains and peripheral tissues were removed and divided sagittally.
For studies of antibody trafficking into the CNS the mouse monoclonal antibody 9E4 was concentrated with a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) and linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. For this experiment non-tg (total n = 18) and α-syn tg mice (total n = 18) (6 m/o) were injected intravenously (IV) with the 9E4-FITC or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg. Mice were sacrificed 3, 14 and 30 days after injection (n = 3 per group). CSF from these mice was used to immunolabel cortical sections from antibody-naive animals.
As an additional control to monitor the passage of FITC-labeled antibodies across the blood-brain barrier, mice were injected with FITC tagged β -syn. The For this experiment non-tg (total n = 8) and α-syn tg mice (total n = 8) (6 m/o) were injected intravenously (IV) with the FITC-labeled β-syn or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg. Mice were sacrified at 14 days post-injection. CSF from these mice was also used to immunolabel slides from FITC tagged β -syn niave non-tg and α-syn tg mice.
Upon sacrifice, the right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was snap-frozen and stored at -70°C for subsequent protein analysis. All experiments described were approved by the animal subjects committee at the University of California at San Diego (UCSD) and were performed according to NIH recommendations for animal use. UCSD is an Institutional Animal Care and Use Committee accredited institution and the UCSD Animal Subjects Committee approved the experimental protocol ( S02221 ) followed in all studies according to the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines.
In patients with LBD, α-syn accumulates not only in subcortical nuclei but also in the temporal cortex and limbic system and accounts for cognitive deficits in these patients. Similarly, in our PDGF-α-syn tg mice, protein accumulation occurs in the temporal cortex and hippocampus. In this context and as previously described, in order to evaluate the functional effects of passive immunization treatment in mice, groups of non-tg and α-syn tg animals were tested in the water maze. For this purpose, a pool (diameter 180 cm) was filled with opaque water (24°C) and mice were first trained to locate a visible platform (days 1-3) and then a submerged hidden platform (days 4-7) in three daily trials 2-3 min apart. Mice that failed to find the hidden platform within 90 seconds were placed on it for 30 seconds. The same platform location was used for all sessions and all mice. The starting point at which each mouse was placed into the water was changed randomly between two alternative entry points located at a similar distance from the platform. In addition, on the final day of testing the platform was removed and the time spent by mice in the corre...
For the pole test, animals were placed head upward on top of a vertical wooden pole 50 cm long and 1 cm in diameter. When placed on the pole, animals orient themselves downward and descend the length of the pole. Groups of mice received training that consisted of five trials for each session. For testing, animals received five trials and the time taken to descend (T-total) was measured.
Mice were analyzed for 2 days in the Rotarod (San Diego Instruments, San Diego, CA), as previously described (Masliah et al., 2000). On the first day, mice were trained for five trials: the first one at 10 rpm, the second at 20 rpm, and the third to the fifth at 40 rpm. On the second day, mice were tested for seven trials at 40 rpm each. Mice were placed individually on the cylinder and the speed of rotation increased from 0 to 40 rpm over a period of 240 s. The length of time mice remained on the rod (fall latency) was recorded and used as a measure of motor function.
Stereological analysis and quantification of neocortical and hippocampal intra-neuronal FL-αsyn and CC-αsyn immunoreactivity was conducted by the disector method using the Stereo-Investigator System (MBF Bioscience, Williston, VT) and the results were averaged and expressed as cell counts per 0.1 mm 3. Neocortical and hippocampal FL-αsyn and CC-αsyn immunoreactive neuropil was assessed in digital images analyzed with the Image Quant software by selecting and area to exclude cell bodies, setting the threshold levels and expressing the data as pixel intensity (arbitary units).
Machine-readable layer
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"text": "Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine which of these antibodies displayed the most specific binding to human α-syn, of these antibodies, 9E4 displayed the most specificity and was chosen for the immunization study. A total of 40 α-syn tg mice (6 m/o, n = 20 mice per group) received weekly intraperitoneal (IP) injections (10 mg/kg) for 6 months with the CT-α-syn antibody (9E4) and IgG1 control. An additional group of non-tg mice treated with the 9E4 antibody (n = 12) and the IgG1 control (n = 12) was included as control for behavioral and neuropathological studies. Mice were bled once a month and antibody titers monitored by enzyme-linked immunosorbent assay (ELISA). At the end of the studies, mice wer..."
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