Parkinson's disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration methods
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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.
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Gait recovery and locomotor function
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- Per-run gait capture with paw placement, timing, and stride features for each animal
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Protein expression by western blot
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Quantify the primary readouts for this experiment: Gait recovery and locomotor function; Protein expression by western blot.
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Report representative outputs alongside summary comparisons for Gait recovery and locomotor function, Protein expression by western blot.
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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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