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: Test for functional cognitive effects in transgenic alpha-synuclein mice following passive immunization treatment with monoclonal antibody 9E4
This is a Water Maze Behavioral Testing protocol using mouse as the model organism. The procedure involves 11 procedural steps, 1 equipment items, 8 materials. Extracted from a 2011 paper published in PLoS ONE.
Model and subjects
mouse • Transgenic mice over-expressing alpha-synuclein under PDGF-beta promoter (Line D) and non-transgenic controls • unknown • 6 months old • Not specified • 64
Study window
~4.3 week study window | ~96 hours hands-on
Core workflow
Antibody selection and characterization • Passive immunization treatment initiation • Non-transgenic control group treatment
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 (8A5, 9E4) of alpha-synuclein to determine which antibody displayed the most specific binding to human alpha-synuclein. The 9E4 antibody was selected based on superior specificity.
Note: 9E4 antibody targets C-terminus of alpha-synuclein and displayed the most specificity
“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 alpha-synuclein transgenic mice (6 months old, n=20 per group) received weekly intraperitoneal injections of either the CT-alpha-synuclein antibody (9E4) or IgG1 control at a dose of 10 mg/kg.
Note: Treatment administered via intraperitoneal injection 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-transgenic mice was included as control, with n=12 treated with the 9E4 antibody and n=12 treated with IgG1 control for behavioral and neuropathological studies.
Note: Non-tg controls matched to tg treatment groups
“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 were monitored by enzyme-linked immunosorbent assay (ELISA) throughout the 6-month treatment period.
Note: ELISA used to quantify circulating antibody levels
“Mice were bled once a month and antibody titers monitored by enzyme-linked immunosorbent assay (ELISA)”
At the end of the 6-month treatment period, mice were tested for functional cognitive effects in the water maze.
Note: Testing performed after completion of immunization treatment
“At the end of the studies, mice were tested for functional effects in the water maze”
The mouse monoclonal antibody 9E4 was concentrated using a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) and then linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) according to manufacturer instructions.
Note: Preparation for blood-brain barrier trafficking studies
“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”
Non-transgenic (n=18) and alpha-synuclein transgenic mice (n=18) at 6 months of age were injected intravenously with either 9E4-FITC or non-immune control FITC-tagged IgG1 at a concentration of 1 mg/kg.
Note: Intravenous route used for blood-brain barrier trafficking studies
“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 at 3, 14, and 30 days after injection (n=3 per group). Cerebrospinal fluid (CSF) was collected from these mice and used to immunolabel cortical sections from antibody-naive animals.
Note: Multiple timepoints used to assess antibody trafficking kinetics
“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 passage of FITC-labeled antibodies across the blood-brain barrier, non-transgenic (n=8) and alpha-synuclein transgenic mice (n=8) at 6 months of age were injected intravenously with FITC-labeled beta-synuclein or non-immune control FITC-tagged IgG1 at a concentration of 1 mg/kg.
Note: Beta-synuclein used as control protein to assess blood-brain barrier 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 were sacrificed at 14 days post-injection. Cerebrospinal fluid (CSF) was collected and used to immunolabel slides from FITC-tagged beta-synuclein naive non-transgenic and alpha-synuclein transgenic mice.
Note: Single timepoint used for beta-synuclein control study
“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, brains were removed and divided sagittally. 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.
Note: Sagittal division allows for parallel neuropathological and biochemical analyses
“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”
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.
Test for functional cognitive effects in transgenic alpha-synuclein mice following passive immunization treatment with monoclonal antibody 9E4
Objective
Test for functional cognitive effects in transgenic alpha-synuclein mice following passive immunization treatment with monoclonal antibody 9E4
Subjects
From papermouse • Transgenic mice over-expressing alpha-synuclein under PDGF-beta promoter (Line D) and non-transgenic controls • unknown • 6 months old • Not specified
Sample count
From paper64
Cohort notes
From paper40 alpha-syn tg mice (n=20 per group), 12 non-tg mice treated with 9E4 antibody, 12 non-tg mice treated with IgG1 control
Antibody selection and characterization (Not specified)
Passive immunization treatment initiation (6 months)
Non-transgenic control group treatment (6 months)
Monthly antibody titer monitoring (Monthly for 6 months)
Cognitive function assessed by water maze performance
From paperNot specified in the provided methods section
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Antibody titers measured by ELISA
From paperNot specified in the provided methods section
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Blood-brain barrier penetration of FITC-labeled antibodies
From paperNot specified in the provided 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 specified in the provided methods section
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Cognitive function assessed by 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
Antibody titers measured by 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
Blood-brain barrier penetration of FITC-labeled antibodies
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
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Not specified in the provided methods section
Scoring or quantification
Quantify the primary readouts for this experiment: Cognitive function assessed by water maze performance; Antibody titers measured by ELISA; Blood-brain barrier penetration of FITC-labeled antibodies; Neuropathological changes in brain tissue.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Cognitive function assessed by water maze performance, Antibody titers measured by ELISA, Blood-brain barrier penetration of FITC-labeled antibodies, Neuropathological changes in brain tissue.
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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