Additional file 1 of Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition
Creators: name: Kadgien, Chelsie A.
name: Kamesh, Anusha
name: Milnerwood, Austen J.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: 69999 Biological Sciences not elsewhere classified
subject: 110309 Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Fig. S1. Retromer protein levels not altered in VKI. A) Co-immunoprecipitation performed in VKI whole brain lysate pulling with FAM21 antibody and blotted on a WES capillary-based western blotting system shows VPS35 association with FAM21 in brain tissue. B) WES capillary-based western blot of VPS35, VPS26, FAM21, and β-tubulin in VKI whole-brain lysate (i) revealed no significant genotype effects on levels of VPS35 (ii, 1-way ANOVA p = 0.97), VPS26 (iii, Kruskal–Wallis p = 0.73), or WASH complex member FAM21 (iv, 1-way ANOVA p = 0.88). Fig. S2. Neuronal cargo and LRRK2 binding are not altered i n VKI. A) Western blot of striatal lysates and co-immunoprecipitates from 3-month-old VKI mice (pulling with VPS35 antibody) were probed for VPS35, GluN1, D2R, GluA1, LRRK2, and GAPDH (i). There were no genotype effects on VPS35 levels or pull by the antibody (ii–iii, Kruskal–Wallis p = 0.97; p = 0.13, respectively); GluN1 levels or coIP (iv–v, Kruskal–Wallis p = 0.51; p = 0.42, respectively); D2R levels or coIP (vi–vii Kruskal–Wallis p = 0.70; p = 0.45, respectively); GluA1 levels or coIP (viii–ix, Kruskal–Wallis p = 0.83; p = 0.44, respectively); or LRRK2 levels or coIP (x–xi, Kruskal–Wallis p &gt; 0.99; p = 0.40, respectively). Fig. S3. Phospho-LRRK2 is increased in VKI and MLi-2 does not alter protein levels. A) Western blot of LRRK2 pS935, LRRK2, and β-actin (i) revealed no genotype effect on LRRK2 expression levels (ii, Kruskal–Wallis p = 0.09). LRRK2 pS935 levels were significantly altered, due to an increase in phosphorylation in homozygous tissue (iii, 1-way ANOVA p 0.99), VPS35 (vi, 2-way ANOVA interaction p = 0.23; genotype p = 0.94; treatment p = 0.89), VGluT1 (vii, 2-way ANOVA interaction p = 0.55; genotype p = 0.39; treatment p = 0.69), or Rab10 (iv, 2-way ANOVA genotype x treatment p = 0.5258; genotype p = 0.4683; treatment p = 0.9659). For Bii-vi, WTCap n = 5, WTMLi2 n = 6, HetCap n = 6, HetMLi2 n = 6, HoCap n = 5, HoMLi2 n = 5. Fig. S4. Captisol does not affect protein levels. A) Further analysis of blots from Fig. 1. There were no significant genotype effects on the level of LRRK2 (i; Mann–Whitney p &gt; 0.99) or Rab10 (ii; Mann–Whitney p = 0.89). B) Western blots of WT brain lysate following acute treatment with Captisol or saline, probed for GluA1, VPS35, VGluT1, and β-actin (loading control). There were no significant effects of Captisol treatment on protein levels of VPS35, GluA1, or VGluT1 (ii–iv; Mann–Whitney p = 0.49; p &gt; 0.99; p = 0.49, respectively). Fig. S5. Cell density and dendritic morphology are not altered in VKI. A) Cortical cells were nucleofected with CAG-AAV-GFP plasmids on the day of plating and fixed at DIV21. GFP signal was amplified and imaged (top panel), then 2D in silico cell reconstruction performed in ImageJ (bottom panel). B) There was no effect of genotype on neuron density, indicating equivalent survival and no cell death (1-way ANOVA p = 0.47). C) Sholl analysis revealed no significant effect of genotype upon neurite complexity (2-way RM ANOVA radial distance x genotype interaction p = 0.87; genotype p = 0.78). D-F) There were also no genotype effects on total branch number, average branch length, or maximum branch length (Kruskal–Wallis p = 0.79, 0.34 &amp; 0.29, respectively). Fig. S6. VGluT1 cluster intensity reduced in VKI and channel kinetics not affected. A) Supplemental analysis from untreated cortical cell culture synapse staining presented in Fig. 3. PSD95 and VGluT1 densities were not altered by genotype (i-ii, Welch’s ANOVA p = 0.29 &amp; 0.42, respectively). There was a genotype effect on VGluT1 cluster intensity, due to significant reductions in homozygous cells only (iii, Kruskal–Wallis p