Fig 2: Generation of Vps35 p.D620N knock-in mice (VKI). a Schematic of the targeting design showing the murine Vps35 genomic sequence (Ensembl reference ENSMUSG00000031696), 5' and 3' homology arms (arrowed), exons 14–17 (blue), engineered loxP site in intron 14, the g.85,263,520G>A (p.D620N) mutation in exon 15 (encoding p.D620N) and endogenous stop codon in exon 17 (TAA). FlpE-recombinase deletion between FRT sites removed the PKG-neo-pA cassette to yield the cVKI allele (not shown). Subsequent Cre-recombinase deletion between loxP sites created the VKI allele. b cDNA sequencing in wild-type mice (top), heterozygous VKI (middle), and homozygous VKI (bottom), between g.8:85263511–85263528 (GRCm38) highlights the g.85,263,520G>A nucleotide mutation in exon 15 that encodes the p.D620N amino acid substitution. c Relative Vps35 mRNA (fold change) expression analysis in cerebellar tissue using quantitative RT-PCR. Data normalized to WT (1-way ANOVA F2,24 = 0.05, p = 0.95). d Total Vps35 protein levels were quantified by western blotting, showing no difference in Het and Homo mice compared to their WT littermates (1-way ANOVA F2,33 = 0.05, p = 0.94)

Fig 3: Accumulation of VPS35- FAM21 co-clusters and Rab 11 clusters in VKI dendrites. A–D Cultured cortical neurons were immunostained for MAP2 (blue), VPS35 (cyan), and VPS26, FAM21, NEEP21, or Rab11 (magenta) A VPS35 and VPS26 (i); There was no genotype effect on dendritic cluster density of VPS35 (ii, Kruskal–Wallis p = 0.30). VPS26 cluster density was significantly reduced in both mutants (iii, Kruskal–Wallis p < 0.04; Uncorrected Dunn’s Het *p < 0.04 & Ho *p < 0.02), yet there was no genotype effect on co-cluster density or Pearson’s coefficient (iv & v, Kruskal–Wallis p = 0.45 & 0.10, respectively). B VPS35 and FAM21 (i); there was no genotype effect on FAM21 cluster density (ii, Kruskal–Wallis p = 0.24) or Pearson’s coefficients (iv, 1-way ANOVA p = 0.24). There was a significant genotype effect on co-cluster density due to increases in homozygous cells (iii, Kruskal–Wallis p < 0.03; Uncorrected Dunn’s Ho *p < 0.02). C NEEP21 and VPS35 (i); there was no genotype effect on NEEP21 cluster (ii, Kruskal–Wallis p = 0.37) or co-cluster density (iii, Kruskal–Wallis p = 0.38), but a significant genotype effect on Pearson’s coefficient due to a significant reduction in homozygous VKI dendrites (iv, Welch’s ANOVA p < 0.03; Unpaired t with Welch’s correction Ho *p < 0.02). D VPS35 and Rab11 (i); Rab11 cluster density was increased in both genotypes (ii, 1-way ANOVA p < 0.03; Uncorrected Fisher’s LSD *p < 0.02 & *p < 0.03). There was no genotype effect on co-cluster density (iii, 1-way ANOVA p = 0.92), but a significant genotype effect in the Pearson’s coefficient due to reduced correlation in heterozygous dendrites (iv, Welch’s ANOVA p < 0.009; Unpaired t with Welch’s correction Het **p < 0.003)

Fig 4: Altered FAM21 binding and LRRK2 kinase inhibitor-reversible Rab-GTPase phosphorylation in VKI mouse brain. A Co-immunoprecipitations of VPS26 and FAM21 with VPS35 from VKI whole-brain lysate run on a WES capillary-based western blot system (i) uncovered no effect on VPS26 pulled by VPS35 (ii, 1-way ANOVA p = 0.44) but a significant reduction in FAM21 pulled by mutant VPS35 (iii, 1-way ANOVA p < 0.003; Uncorrected Fisher’s LSD Het **p < 0.01; Ho **p < 0.01). B Western blot of Rab10, Rab10 pT73, and ß-actin in VKI whole brain lysate (i) revealed no significant genotype effect on Rab10 levels (ii, Kruskal–Wallis p = 0.16), but a significant increase in Rab10 pT73 in VKI (iii, Kruskal–Wallis p < 0.0001; Uncorrected Dunn’s Het **p < 0.003, Ho **p < 0.003). C Western blot of LRRK2, LRRK2 pS935, GAPDH, Rab10, and Rab10 pT73 in VKI whole brain lysate (i) revealed that MLi2 treatment significantly reduced LRRK2 pS935 in all genotypes (ii, 2-way ANOVA treatment p < 0.0001; Uncorrected Fisher’s LSD WT-WTMLi2 ****p < 0.0001; Het-HetMLi2 ***p < 0.0002; Ho-HoMLi2 ****p < 0.0001) and had significant genotype and treatment effects on Rab10 pT73 due to significant reductions in homozygous cells (iii, 2-way ANOVA genotype p = 0.04; treatment p < 0.009; Uncorrected Fisher’s LSD WT-WTMLi2 p = 0.82; Het-HetMLi2 p = 0.10; Ho-HoMLi2 **p = 0.007). D Western blot of LRRK2, LRRK2 pS935, GAPDH, Rab10, and Rab10 pT73 in whole brain lysate revealed no significant effect of the solubilizing agent Captisol on LRRK2 pS935 (ii, Mann–Whitney p = 0.89) or Rab10 pT73 (iii, Mann–Whitney p = 0.69). For C.ii–iii, WTCap n = 5, WTMLi2 n = 6, HetCap n = 6, HetMLi2 n = 6, HoCap n = 5, HoMLi2 n = 5