Parkinson's disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration methods
Aim. Evidence-backed execution summary for Parkinson's disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration methods from Parkinson's disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Retromer Subunit Assembly and Levels in Brains of D620N VPS35 KI Mice.
reagent used in the protocol.
- Use
- Western blot analyses of ventral midbrain extracts from VPS35 D620N/D620N, VPS35 D620N/WT, and WT mice at 3 mo reveal normal levels of the cargo-selective complex subunits VPS35, VPS26, and VPS29 ( ). Therefore, the D620N mutation does not obviously influence overall retromer stability. To evaluate the interaction...
Retromer Subunit Assembly and Levels in Brains of D620N VPS35 KI Mice.
reagent used in the protocol.
- Use
- Normal retromer assembly in brain but reduced VPS35 and WASH complex in dopaminergic neurons of D620N VPS35 KI mice. ( A ) Hemibrain extracts from D620N VPS35 KI and WT mice at 3 mo ( n = 2 mice per genotype) were subjected to IP with anti-VPS35 antibody (or control anti-V5 IgG). Western blot analysis of IP and inpu...
The D620N Mutation Supports Survival and Is Inconsistent with a Full Loss-of-Function Mechanism.
reagent used in the protocol.
- Use
- Our data indicate that homozygous D620N VPS35 KI mice display a normal lifespan ( ), in contrast to homozygous KO mice (and VPS35 FLOX/FLOX mice; ) which are embryonic-lethal ( ), suggesting that the D620N mutation is not likely to act via a loss-of-function mechanism. To further address this mechanism, we crossed V...
The D620N Mutation Supports Survival and Is Inconsistent with a Full Loss-of-Function Mechanism.
reagent used in the protocol.
- Use
- A single D620N VPS35 allele is sufficient for normal survival, early dopaminergic neuronal health, and tau pathology. Heterozygous VPS35 D620N/WT mice were crossed with VPS35 FLOX/WT mice to create compound heterozygous VPS35 D620N/FLOX progeny and their littermate controls. ( A ) Western blot analysis of hemibrain...
Cell Culture and Transient Transfection.
reagent used in the protocol.
- Use
- HEK-293T cells were maintained and transfected using X-tremeGENE HP Reagent (Roche) as described ( ).
Co-IP, Western Blotting, and MS.
reagent used in the protocol.
- Use
- For co-IP assays, hemibrains from 3-mo-old mice or transfected HEK-293T cells were prepared in IP buffer [20 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 200 mM sorbitol, 2 mM EDTA, 0.1% Triton X-100, and 1 × Complete Mini protease inhibitor mixture (Roche)] and subjected to IP with antibodies to VPS35 or V5 (...
Immunohistochemistry and Immunofluorescence.
reagent used in the protocol.
- Use
- Mice were perfused with 4% paraformaldehyde (pH 7.4), cryoprotected in 30% sucrose, and dissected into 35-µm-thick coronal sections. Immunostaining by incubation with primary and biotinylated secondary antibodies (Vector Labs), ABC reagent (Vector Labs), and DAB (Vector Labs) has been described (, ). Images we...
Modest Motor Deficits in D620N VPS35 KI Mice.
To evaluate the potentially deleterious impact of D620N VPS35 expression on motor behavior, we conducted open-field, rotarod, and gait analyses on VPS35 D620N/WT and VPS35 D620N/D620N mice compared with WT littermate control mice at different time points as the mice aged. VPS35 D620N/WT and VPS35 D620N/D620N mice do...
- Use
- To evaluate the potentially deleterious impact of D620N VPS35 expression on motor behavior, we conducted open-field, rotarod, and gait analyses on VPS35 D620N/WT and VPS35 D620N/D620N mice compared with WT littermate control mice at different time points as the mice aged. VPS35 D620N/WT and VPS35 D620N/D620N mice do...
RNA Extraction and Northern Blot Analysis.
Total RNAs were extracted from hemibrains and subjected to Northern blot analysis using a 32 P-labeled DNA probe (nucleotides 163-984 of mVPS35). Signal was detected using a Typhoon phosphor-imaging system (Amersham).
- Use
- Total RNAs were extracted from hemibrains and subjected to Northern blot analysis using a 32 P-labeled DNA probe (nucleotides 163-984 of mVPS35). Signal was detected using a Typhoon phosphor-imaging system (Amersham).
Immunohistochemistry and Immunofluorescence.
Mice were perfused with 4% paraformaldehyde (pH 7.4), cryoprotected in 30% sucrose, and dissected into 35-µm-thick coronal sections. Immunostaining by incubation with primary and biotinylated secondary antibodies (Vector Labs), ABC reagent (Vector Labs), and DAB (Vector Labs) has been described (, ). Images we...
- Use
- Mice were perfused with 4% paraformaldehyde (pH 7.4), cryoprotected in 30% sucrose, and dissected into 35-µm-thick coronal sections. Immunostaining by incubation with primary and biotinylated secondary antibodies (Vector Labs), ABC reagent (Vector Labs), and DAB (Vector Labs) has been described (, ). Images we...
Stereological Analysis and Optical Densitometry.
Unbiased stereological quantitation of SNpc dopaminergic neurons was performed using the optical fractionator probe of StereoInvestigator software (MicroBrightField Biosciences), as previously described (, ). The optical density of TH-immunoreactive nerve terminals in the striatum was determined using the ScanScope...
- Use
- Unbiased stereological quantitation of SNpc dopaminergic neurons was performed using the optical fractionator probe of StereoInvestigator software (MicroBrightField Biosciences), as previously described (, ). The optical density of TH-immunoreactive nerve terminals in the striatum was determined using the ScanScope...
Behavioral Assessments.
Behavioral testing was performed on male and female mice at ages indicated for each test, as described ( ). Open-field testing using an Ethovision XT video tracking system (Noldus Information Technology), accelerating rotarod (5 to 50 rpm over 5 min), and digital Gait analysis using the automated CatWalk XT system (...
- Use
- Behavioral testing was performed on male and female mice at ages indicated for each test, as described ( ). Open-field testing using an Ethovision XT video tracking system (Noldus Information Technology), accelerating rotarod (5 to 50 rpm over 5 min), and digital Gait analysis using the automated CatWalk XT system (...
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Retromer Subunit Assembly and Levels in Brains of D620N VPS35 KI Mice.
Western blot analyses of ventral midbrain extracts from VPS35 D620N/D620N, VPS35 D620N/WT, and WT mice at 3 mo reveal normal levels of the cargo-selective complex subunits VPS35, VPS26, and VPS29 ( ). Therefore, the D620N mutation does not obviously influence overall retromer stability. To evaluate the interaction of VPS35 with retromer complex subunits in the brain of D620N KI mice, we conducted coimmunoprecipitation (co-IP) assays with a VPS35-specific antibody using soluble hemibrain extracts from VPS35 D620N/D620N and WT mice. WT and D620N VPS35 interact robustly and equivalently with VPS26 in brain ( ). However, we are unable to detect an interaction of WT or D620N VPS35 with the WASH complex subunits FAM21 and WASH1 ( ). We could further confirm the equivalent interaction with retromer subunits VPS26A, VPS26B, and VPS29 by mass spectrometric analysis of VPS35 IPs from WT or D6...
Retromer Subunit Assembly and Levels in Brains of D620N VPS35 KI Mice.
Normal retromer assembly in brain but reduced VPS35 and WASH complex in dopaminergic neurons of D620N VPS35 KI mice. ( A ) Hemibrain extracts from D620N VPS35 KI and WT mice at 3 mo ( n = 2 mice per genotype) were subjected to IP with anti-VPS35 antibody (or control anti-V5 IgG). Western blot analysis of IP and input fractions reveal equivalent interactions of endogenous WT and D620N VPS35 with retromer subunit VPS26, whereas WASH complex subunits (FAM21 and WASH1) are not detected. ( B ) LC-MS/MS analysis of anti-VPS35 IP (or anti-V5 IP) fractions from A reveal equivalent interactions of WT and D620N VPS35 with retromer subunits VPS26A, VPS26B, and VPS29 in mouse brain. WASH complex subunits are not detected. MS/MS spectral counts for each protein are shown. ( C ) HEK-293T cells expressing V5-tagged human VPS35 (WT or D620N) were subjected to IP with anti-V5 antibody, and IP and inpu...
Co-IP, Western Blotting, and MS.
For co-IP assays, hemibrains from 3-mo-old mice or transfected HEK-293T cells were prepared in IP buffer [20 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 200 mM sorbitol, 2 mM EDTA, 0.1% Triton X-100, and 1 × Complete Mini protease inhibitor mixture (Roche)] and subjected to IP with antibodies to VPS35 or V5 (5 µg) as described ( ). IPs and input lysates were subjected to Western blotting, detection, and quantitation as described ( ). For MS, anti-VPS35 IPs from WT or VPS35 KI mouse brain were subjected to analysis by nano liquid chromatography/tandem MS (LC-MS/MS) (ThermoFisher Q Exactive) at MS Bioworks as previously described ( ).
Stereotactic Delivery of Preformed Fibrils.
Mouse α-synuclein was expressed in Escherichia coli and purified as previously described ( ). To generate fibrils, 5 mg/mL of purified α-synuclein in 1 × PBS, pH 7.4, was shaken at 1,000 rpm for 7 d, sonicated with 60 pulses at 10% power (30 s total time, 0.5 s on, 0.5 s off), aliquoted, and stored at -80 °C. Sedimentation assays and thioflavin T assays were performed as described (, ) to ensure fibril quality. Male adult littermate mice ( n = 3 mice per genotype) were subjected to stereotactic surgery for the unilateral delivery of PFF-αSyn into the dorsal striatum. The following coordinates were used relative to bregma: anterior-posterior, +0.2 mm; medial-lateral, +2.0 mm; and dorsal-ventral, -2.6 mm. Mice received PFF-αSyn (5 µg) in 1 µL at a flow rate of 0.1 µL/min. Animals were euthanized at 30 d postinjec...
Behavioral Assessments.
Behavioral testing was performed on male and female mice at ages indicated for each test, as described ( ). Open-field testing using an Ethovision XT video tracking system (Noldus Information Technology), accelerating rotarod (5 to 50 rpm over 5 min), and digital Gait analysis using the automated CatWalk XT system (Noldus) were conducted.
Measurement outputs
What raw and processed outputs should exist?
To evaluate the potentially deleterious impact of D620N VPS35 expression on motor behavior, we conducted open-field, rotarod, and gait analyses on VPS35 D620N/WT and VPS35 D620N...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
To determine whether the expression of D620N VPS35 in mice influences the integrity of the nigrostriatal dopaminergic pathway with age, cohorts of KI mice were aged up to 13 mo....
- 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
Progressive loss of nigrostriatal pathway dopaminergic neurons in D620N VPS35 KI mice. Unbiased stereological analysis of TH+ and Nissl+ neurons in the SNpc reveal dopaminergic...
- 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
Since the neuropathological spectrum associated with familial VPS35 mutations in PD is not yet clear, we next evaluated the development of PD-related neuropathology using a numb...
- 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
To determine whether the expression of D620N VPS35 in mice influences the integrity of the nigrostriatal dopaminergic pathway with age, cohorts of KI mice were aged up to 13 mo.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To evaluate the potentially deleterious impact of D620N VPS35 expression on motor behavior, we conducted open-field, rotarod, and gait analyses on VPS35 D620N/WT and VPS35 D620N...; To determine whether the expression of D620N VPS35 in mice influences the integrity of the nigrostriatal dopaminergic pathway with age, cohorts of KI mice were aged up to 13 mo....; Progressive loss of nigrostriatal pathway dopaminergic neurons in D620N VPS35 KI mice. Unbiased stereological analysis of TH+ and Nissl+ neurons in the SNpc reveal dopaminergic...; Since the neuropathological spectrum associated with familial VPS35 mutations in PD is not yet clear, we next evaluated the development of PD-related neuropathology using a numb....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
To determine whether the expression of D620N VPS35 in mice influences the integrity of the nigrostriatal dopaminergic pathway with age, cohorts of KI mice were aged up to 13 mo....; Progressive loss of nigrostriatal pathway dopaminergic neurons in D620N VPS35 KI mice. Unbiased stereological analysis of TH+ and Nissl+ neurons in the SNpc reveal dopaminergic...; Since the neuropathological spectrum associated with familial VPS35 mutations in PD is not yet clear, we next evaluated the development of PD-related neuropathology using a numb...; Axonal damage in D620N VPS35 KI mice. Representative photomicrographs of striatum and substantia nigra from 13-mo-old VPS35 D620N/WT, VPS35 D620N/D620N, and control VPS35 WT/W...
from paperReporting output
Report representative outputs alongside summary comparisons for To evaluate the potentially deleterious impact of D620N VPS35 expression on motor behavior, we conducted open-field, rotarod, and gait analyses on VPS35 D620N/WT and VPS35 D620N..., To determine whether the expression of D620N VPS35 in mice influences the integrity of the nigrostriatal dopaminergic pathway with age, cohorts of KI mice were aged up to 13 mo...., Progressive loss of nigrostriatal pathway dopaminergic neurons in D620N VPS35 KI mice. Unbiased stereological analysis of TH+ and Nissl+ neurons in the SNpc reveal dopaminergic..., Since the neuropathological spectrum associated with familial VPS35 mutations in PD is not yet clear, we next evaluated the development of PD-related neuropathology using a numb....
inferred from protocolStructured statistical methods
To determine whether the expression of D620N VPS35 in mice influences the integrity of the nigrostriatal dopaminergic pathway with age, cohorts of KI mice were aged up to 13 mo....; Progressive loss of nigrostriatal pathway dopaminergic neurons in D620N VPS35 KI mice. Unbiased stereological analysis of TH+ and Nissl+ neurons in the SNpc reveal dopaminergic...; Since the neuropathological spectrum associated with familial VPS35 mutations in PD is not yet clear, we next evaluated the development of PD-related neuropathology using a numb...; Axonal damage in D620N VPS35 KI mice. Representative photomicrographs of striatum and substantia nigra from 13-mo-old VPS35 D620N/WT, VPS35 D620N/D620N, and control VPS35 WT/W...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
Western blot analyses of ventral midbrain extracts from VPS35 D620N/D620N, VPS35 D620N/WT, and WT mice at 3 mo reveal normal levels of the cargo-selective complex subunits VPS35, VPS26, and VPS29 ( ). Therefore, the D620N mutation does not obviously influence overall retromer stability. To evaluate the interaction of VPS35 with retromer complex subunits in the brain of D620N KI mice, we conducted coimmunoprecipitation (co-IP) assays with a VPS35-specific antibody using soluble hemibrain extracts from VPS35 D620N/D620N and WT mice. WT and D620N VPS35 interact robustly and equivalently with VPS26 in brain ( ). However, we are unable to detect an interaction of WT or D620N VPS35 with the WASH complex subunits FAM21 and WASH1 ( ). We could further confirm the equivalent interaction with retromer subunits VPS26A, VPS26B, and VPS29 by mass spectrometric analysis of VPS35 IPs from WT or D620N KI brains, but we are unable to detect any WASH complex subunits ( ). Using similar co-IP assays, we can detect the robust interaction of V5-tagged WT VPS35 with endogenous WASH1 and FAM21 in HEK-293T cells and the marked impairment of both interactions with D620N VPS35 ( ), as previously report...
Normal retromer assembly in brain but reduced VPS35 and WASH complex in dopaminergic neurons of D620N VPS35 KI mice. ( A ) Hemibrain extracts from D620N VPS35 KI and WT mice at 3 mo ( n = 2 mice per genotype) were subjected to IP with anti-VPS35 antibody (or control anti-V5 IgG). Western blot analysis of IP and input fractions reveal equivalent interactions of endogenous WT and D620N VPS35 with retromer subunit VPS26, whereas WASH complex subunits (FAM21 and WASH1) are not detected. ( B ) LC-MS/MS analysis of anti-VPS35 IP (or anti-V5 IP) fractions from A reveal equivalent interactions of WT and D620N VPS35 with retromer subunits VPS26A, VPS26B, and VPS29 in mouse brain. WASH complex subunits are not detected. MS/MS spectral counts for each protein are shown. ( C ) HEK-293T cells expressing V5-tagged human VPS35 (WT or D620N) were subjected to IP with anti-V5 antibody, and IP and input fractions were probed for VPS26, FAM21, and WASH1. WT and D620N VPS35 interact equivalently with endogenous VPS26, whereas D620N VPS35 displays an impaired interaction with endogenous FAM21 and WASH1. ( D ) Immunofluorescent confocal colocalization analysis reveals markedly reduced levels of VPS35...
For co-IP assays, hemibrains from 3-mo-old mice or transfected HEK-293T cells were prepared in IP buffer [20 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 200 mM sorbitol, 2 mM EDTA, 0.1% Triton X-100, and 1 × Complete Mini protease inhibitor mixture (Roche)] and subjected to IP with antibodies to VPS35 or V5 (5 µg) as described ( ). IPs and input lysates were subjected to Western blotting, detection, and quantitation as described ( ). For MS, anti-VPS35 IPs from WT or VPS35 KI mouse brain were subjected to analysis by nano liquid chromatography/tandem MS (LC-MS/MS) (ThermoFisher Q Exactive) at MS Bioworks as previously described ( ).
Mouse α-synuclein was expressed in Escherichia coli and purified as previously described ( ). To generate fibrils, 5 mg/mL of purified α-synuclein in 1 × PBS, pH 7.4, was shaken at 1,000 rpm for 7 d, sonicated with 60 pulses at 10% power (30 s total time, 0.5 s on, 0.5 s off), aliquoted, and stored at -80 °C. Sedimentation assays and thioflavin T assays were performed as described (, ) to ensure fibril quality. Male adult littermate mice ( n = 3 mice per genotype) were subjected to stereotactic surgery for the unilateral delivery of PFF-αSyn into the dorsal striatum. The following coordinates were used relative to bregma: anterior-posterior, +0.2 mm; medial-lateral, +2.0 mm; and dorsal-ventral, -2.6 mm. Mice received PFF-αSyn (5 µg) in 1 µL at a flow rate of 0.1 µL/min. Animals were euthanized at 30 d postinjection.
Behavioral testing was performed on male and female mice at ages indicated for each test, as described ( ). Open-field testing using an Ethovision XT video tracking system (Noldus Information Technology), accelerating rotarod (5 to 50 rpm over 5 min), and digital Gait analysis using the automated CatWalk XT system (Noldus) were conducted.
Machine-readable layer
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"name": "Parkinson's disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration methods",
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"name": "Retromer Subunit Assembly and Levels in Brains of D620N VPS35 KI Mice.",
"text": "Western blot analyses of ventral midbrain extracts from VPS35 D620N/D620N, VPS35 D620N/WT, and WT mice at 3 mo reveal normal levels of the cargo-selective complex subunits VPS35, VPS26, and VPS29 ( ). Therefore, the D620N mutation does not obviously influence overall retromer stability. To evaluate the interaction of VPS35 with retromer complex subunits in the brain of D620N KI mice, we conducted coimmunoprecipitation (co-IP) assays with a VPS35-specific antibody using soluble hemibrain extracts from VPS35 D620N/D620N and WT mice. WT and D620N VPS35 interact robustly and equivalently with VPS26 in brain ( ). However, we are unable to detect an interaction of WT or D620N VPS35 with the WASH complex subunits FAM21 and WASH1 ( ). We could further confirm the equivalent interaction with retromer subunits VPS26A, VPS26B, and VPS29 by mass spectrometric analysis of VPS35 IPs from WT or D6..."
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"text": "Normal retromer assembly in brain but reduced VPS35 and WASH complex in dopaminergic neurons of D620N VPS35 KI mice. ( A ) Hemibrain extracts from D620N VPS35 KI and WT mice at 3 mo ( n = 2 mice per genotype) were subjected to IP with anti-VPS35 antibody (or control anti-V5 IgG). Western blot analysis of IP and input fractions reveal equivalent interactions of endogenous WT and D620N VPS35 with retromer subunit VPS26, whereas WASH complex subunits (FAM21 and WASH1) are not detected. ( B ) LC-MS/MS analysis of anti-VPS35 IP (or anti-V5 IP) fractions from A reveal equivalent interactions of WT and D620N VPS35 with retromer subunits VPS26A, VPS26B, and VPS29 in mouse brain. WASH complex subunits are not detected. MS/MS spectral counts for each protein are shown. ( C ) HEK-293T cells expressing V5-tagged human VPS35 (WT or D620N) were subjected to IP with anti-V5 antibody, and IP and inpu..."
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"text": "For co-IP assays, hemibrains from 3-mo-old mice or transfected HEK-293T cells were prepared in IP buffer [20 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 200 mM sorbitol, 2 mM EDTA, 0.1% Triton X-100, and 1 × Complete Mini protease inhibitor mixture (Roche)] and subjected to IP with antibodies to VPS35 or V5 (5 µg) as described ( ). IPs and input lysates were subjected to Western blotting, detection, and quantitation as described ( ). For MS, anti-VPS35 IPs from WT or VPS35 KI mouse brain were subjected to analysis by nano liquid chromatography/tandem MS (LC-MS/MS) (ThermoFisher Q Exactive) at MS Bioworks as previously described ( )."
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"name": "Stereotactic Delivery of Preformed Fibrils.",
"text": "Mouse α-synuclein was expressed in Escherichia coli and purified as previously described ( ). To generate fibrils, 5 mg/mL of purified α-synuclein in 1 × PBS, pH 7.4, was shaken at 1,000 rpm for 7 d, sonicated with 60 pulses at 10% power (30 s total time, 0.5 s on, 0.5 s off), aliquoted, and stored at -80 °C. Sedimentation assays and thioflavin T assays were performed as described (, ) to ensure fibril quality. Male adult littermate mice ( n = 3 mice per genotype) were subjected to stereotactic surgery for the unilateral delivery of PFF-αSyn into the dorsal striatum. The following coordinates were used relative to bregma: anterior-posterior, +0.2 mm; medial-lateral, +2.0 mm; and dorsal-ventral, -2.6 mm. Mice received PFF-αSyn (5 µg) in 1 µL at a flow rate of 0.1 µL/min. Animals were euthanized at 30 d postinjec..."
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"headline": "Parkinson's disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration",
"datePublished": "2019",
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