Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease methods
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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).
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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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