Source Paper
Eliezer Masliah, Edward Rockenstein, Michael Mante, Leslie Crews, Brian Spencer et al.
PLoS ONE • 2011
Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB.
Objective: To evaluate the effects of passive immunization with 9E4 antibody targeting C-terminal α-synuclein on behavioral deficits and neuropathological changes in α-synuclein transgenic mice, and to assess antibody trafficking into the central nervous system
This is a Passive Immunization Study protocol using mouse as the model organism. The procedure involves 13 procedural steps, 2 equipment items, 7 materials. Extracted from a 2011 paper published in PLoS ONE.
Model and subjects
mouse • α-synuclein transgenic (Line D) and non-transgenic • unknown • 6 months old • not specified • 82
Study window
~4.3 week study window | ~96 hours hands-on
Core workflow
Antibody selection and characterization • Passive immunization - main study initiation • Monthly blood collection and antibody titer monitoring
Primary readouts
Key equipment and reagents
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Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (6H7) and C-terminus of α-synuclein (8A5, 9E4) to determine which antibodies displayed the most specific binding to human α-synuclein. 9E4 was selected for its superior specificity.
Note: 9E4 antibody displayed the most specificity and was chosen for the immunization study
“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”
A total of 40 α-syn transgenic mice (6 months old, n=20 mice per group) received weekly intraperitoneal injections of 9E4 antibody or IgG1 control at 10 mg/kg dose. An additional group of non-transgenic mice (n=12 per group) treated with 9E4 antibody and IgG1 control was included as control.
Note: Injections were administered weekly via intraperitoneal route
“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)”
Mice were bled once a month throughout the 6-month immunization period. Antibody titers were monitored by enzyme-linked immunosorbent assay (ELISA).
Note: ELISA used to quantify antibody titers in serum
“Mice were bled once a month and antibody titers monitored by enzyme-linked immunosorbent assay (ELISA)”
At the end of the 6-month immunization study, mice were tested for functional effects in the water maze to assess cognitive and motor performance.
Note: Conducted at end of 6-month treatment period
“At the end of the studies, mice were tested for functional effects in the water maze”
Following behavioral testing, brains and peripheral tissues were removed and divided sagittally for neuropathological and protein analysis.
Note: Right hemibrain and left hemibrain processed separately
“Brains and peripheral tissues were removed and divided sagittally”
The mouse monoclonal antibody 9E4 was concentrated using a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) in preparation for FITC conjugation.
Note: Concentration step performed before fluorescent labeling
“the mouse monoclonal antibody 9E4 was concentrated with a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA)”
The concentrated 9E4 antibody was linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions.
Note: Performed according to kit manufacturer's instructions
“linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions”
Non-transgenic (n=18) and α-synuclein transgenic mice (n=18), 6 months old, were injected intravenously with 9E4-FITC or non-immune control FITC-tagged IgG1 at a concentration of 1 mg/kg.
Note: Intravenous route used for this trafficking study
“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 from the 9E4-FITC injection study were sacrificed at 3, 14, and 30 days after injection (n=3 per timepoint group). Cerebrospinal fluid (CSF) was collected from these mice.
Note: Three separate timepoint groups with n=3 per group
“Mice were sacrificed 3, 14 and 30 days after injection (n=3 per group). CSF from these mice was used to immunolabel cortical sections”
As an additional control to monitor the passage of FITC-labeled antibodies across the blood-brain barrier, non-transgenic (n=8) and α-synuclein transgenic mice (n=8), 6 months old, were injected intravenously with FITC-labeled β-synuclein or non-immune control FITC-tagged IgG1 at a concentration of 1 mg/kg.
Note: β-synuclein used as control protein to assess BBB permeability
“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 from the β-synuclein injection study were sacrificed at 14 days post-injection. Cerebrospinal fluid (CSF) was collected and used to immunolabel slides from FITC-tagged β-synuclein naive non-transgenic and α-synuclein transgenic mice.
Note: Single timepoint collection at 14 days
“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”
The right hemibrain was post-fixed in phosphate-buffered 4% paraformaldehyde (PFA) at pH 7.4 at 4°C for 48 hours for neuropathological analysis.
Note: pH 7.4 phosphate buffer used
“the right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis”
The left hemibrain was snap-frozen and stored at -70°C for subsequent protein analysis.
Note: Snap-freezing preserves protein integrity for biochemical analysis
“the left hemibrain was snap-frozen and stored at −70°C for subsequent protein analysis”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
To evaluate the effects of passive immunization with 9E4 antibody targeting C-terminal α-synuclein on behavioral deficits and neuropathological changes in α-synuclein transgenic mice, and to assess antibody trafficking into the central nervous system
Objective
To evaluate the effects of passive immunization with 9E4 antibody targeting C-terminal α-synuclein on behavioral deficits and neuropathological changes in α-synuclein transgenic mice, and to assess antibody trafficking into the central nervous system
Subjects
From papermouse • α-synuclein transgenic (Line D) and non-transgenic • unknown • 6 months old • not specified
Sample count
From paper82
Cohort notes
From paperα-syn tg mice (n=20 per group for main study, n=18 for trafficking study, n=8 for β-syn control); non-tg mice (n=12 per group for main study, n=18 for trafficking study, n=8 for β-syn control)
Antibody selection and characterization (not specified)
Passive immunization - main study initiation (6 months)
Monthly blood collection and antibody titer monitoring (Monthly for 6 months)
Water maze behavioral testing (not specified)
Behavioral deficits assessed via water maze performance
From paperNot explicitly specified in methods section
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Neuropathological changes in brain tissue
From paperNot explicitly specified in methods section
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Antibody titers in blood (via ELISA)
From paperNot explicitly specified in methods section
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Antibody trafficking into central nervous system (via FITC fluorescence)
From paperNot explicitly specified in methods section
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Behavioral deficits assessed via water maze performance
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Neuropathological changes in brain tissue
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Antibody titers in blood (via ELISA)
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Antibody trafficking into central nervous system (via FITC fluorescence)
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Not explicitly specified in methods section
Scoring or quantification
Quantify the primary readouts for this experiment: Behavioral deficits assessed via water maze performance; Neuropathological changes in brain tissue; Antibody titers in blood (via ELISA); Antibody trafficking into central nervous system (via FITC fluorescence).
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Behavioral deficits assessed via water maze performance, Neuropathological changes in brain tissue, Antibody titers in blood (via ELISA), Antibody trafficking into central nervous system (via FITC fluorescence).
Source links and direct wording from the methods section for validation and deeper review.
Citation
Eliezer Masliah et al. (2011). Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease. PLoS ONE
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