ERM phosphorylation is impaired in PD patients carrying LRRK2 G2019S mutation.

(A) Schematic of human frontal cortex regions analyzed for phospho-ERM staining. (B) Representative confocal images of GFAP (green) and phospho-ERM (purple) in the frontal cortex of human controls (n = 4; 3M, 1F) and LRRK2 G2019S mutation carriers (n = 3; 2M, 1F), aged >80 years. Scale bar, 200 μm. (C) Quantification of GFAP integrated density in (B). GFAP: t (5) = 2.822, p = 0.037; phospho-ERM: t (5) = 5.695, p = 0.0023. Grey dots: individual images; black dots: control averages; blue dots: mutation carrier averages. (D) Schematic of mouse anterior cingulate cortex (ACC) and primary motor cortex (MOp) analyzed for phospho-ERM staining. (E-F) Representative confocal images of phospho-ERM in the ACC and the MOp of WT or LRRK2 G2019Ski/ki Aldh1L1-eGFP mice at P21. Scale bar, 10 μm. (G-H) Representative confocal images of phospho-ERM in the ACC and MOp of WT (n = 4; 2M, 2F) or LRRK2 G2019Ski/ki Aldh1L1-eGFP mice (n = 4; 2M, 2F) at P21. Scale bar, 200 μm. (I) Quantification of phospho-ERM integrated density in (G-H). For the ACC, nested t-test, unpaired two-tailed t-test. t (6) = 4.247, p = 0.0054. For the MOp, nested t-test, unpaired two-tailed t-test. t (6) = 2.392, p = 0.0536. (J-K) Representative confocal images of phospho-ERM (purple) in the ACC and MOp of WT (n = 4; 2M, 2F) or LRRK2 G2019Ski/ki Aldh1L1-eGFP (n = 4; 2M, 2F) mice at P84. Scale bar, 200 μm. (L) Quantification of phospho-ERM integrated density in (J-K), For the ACC, nested t-test, unpaired Two-tailed t-test. t (6) = 4.383, p = 0.0047. For the MOp, nested t-test, unpaired Two-tailed t-test. t (6) = 3.898, p = 0.0080. Grey dots: individual images; black dots: WT averages; blue dots: mutant averages.

LRRK2 G2019S affects excitatory and inhibitory synapse densities in the ACC and MOp.

(A-B) Schematics of analyzed brain regions (ACC and MOp) and methods for quantifying VGluT1-PSD95 and VGAT-GEPHYRIN colocalized puncta. (C) Representative images of VGluT1-PSD95 staining in ventral ACC L1 of WT and LRRK2 G2019Ski/ki mice at P84. Scale bar, 10 μm. (D) Quantification of VGluT1-PSD95 puncta in ventral ACC L1 and L2-3, normalized to WT means. n = 5/group (3M, 2F). Nested One-way ANOVA: [F(3, 56) = 12.48, p < 0.0001]. Bonferroni’s test: L1 (p < 0.0001), L2-3 (p = 0.0002). (E) Representative images of VGluT1-PSD95 staining in MOp L1 at P84. Scale bar, 10 μm. (F) Quantification of VGluT1-PSD95 puncta in MOp L1 and L2-3. n = 5/group (3M, 2F). No significant differences (Bonferroni’s test: p > 0.05). (G) Representative images of VGAT-GEPHYRIN staining in ventral ACC L1. Scale bar, 10 μm. (H) Quantification of VGAT-GEPHYRIN puncta in ACC L1 and L2-3, normalized to WT means. n = 4/group (2M, 2F). No significant differences (Bonferroni’s test: p > 0.05). (I) Representative images of VGAT-GEPHYRIN staining in MOp L1. Scale bar, 10 μm. (J) Quantification of VGAT-GEPHYRIN puncta in MOp L1 and L2-3. n = 6/group (3M, 3F). Nested One-way ANOVA: [F(3, 68) = 25.88, p < 0.0001]. Bonferroni’s test: L1 (p < 0.0001), L2-3 (p < 0.0001). Grey dots: individual images; black dots: WT averages; blue dots: mutant averages.

LRRK2 G2019S affects excitatory and inhibitory synapse function in the ACC and MOp.

(A) Representative mEPSC traces from ventral ACC L2-3 pyramidal neurons in WT and LRRK2 G2019Ski/ki mice. (B) Cumulative probability and quantification of mEPSC frequency: n = 15 (WT), 13 (LRRK2 G2019Ski/ki) neurons, 4 mice/genotype. Kolmogorov-Smirnov test: D = 0.504, p < 0.001. Mean frequency: WT (2.878 ± 0.6355), LRRK2 G2019Ski/ki (0.9673 ± 0.2328). Unpaired t-test: t (6) = 2.823, p = 0.0302. (C) Cumulative probability and quantification of mEPSC amplitude: n = 14 (WT), 12 (LRRK2 G2019Ski/ki) neurons. Kolmogorov-Smirnov test: D = 0.194, p < 0.0001. Mean amplitude: WT (15.5 ± 0.6325), LRRK2 G2019Ski/ki (13.9 ± 0.9017). Unpaired t-test: t (6) = 1.451, p = 0.197. (D) Representative mIPSC traces from MOp L2-3 pyramidal neurons in WT and LRRK2 G2019Ski/ki mice. (E) Cumulative probability and quantification of mIPSC frequency: n = 12 (WT), 11 (LRRK2 G2019Ski/ki) neurons, 4 WT, 3 LRRK2 G2019Ski/ki mice. Kolmogorov-Smirnov test: D = 0.424, p < 0.0001. Mean frequency: WT (0.7788 ± 0.1012), LRRK2 G2019Ski/ki (1.301 ± 0.07291). Unpaired t-test: t (5) = 3.883, p = 0.0116. (F) Cumulative probability and quantification of mIPSC amplitude: n = 12 (WT), 11 (LRRK2 G2019Ski/ki) neurons. Kolmogorov-Smirnov test: D = 0.382, p < 0.0001. Mean amplitude: WT (8.633 ± 0.4883), LRRK2 G2019Ski/ki (9.834 ± 0.5558). Unpaired t-test: t (5) = 1.62, p = 0.1662. Data are presented as mean ± s.e.m.

Overexpression of phospho-dead Ezrin in adult LRRK2 G2019Ski/ki astrocytes restores excitatory synapse number and function in the ventral ACC.

(A) Representative ventral ACC images of WT and LRRK2 G2019Ski/ki mice injected with AAV-HA-WT or Phospho-dead EZRIN, stained for HA and phospho-ERM (P84). Scale bar, 200 μm. (B) Quantification of phospho-ERM intensity (n = 6/group, 3 males, 3 females). One-way ANOVA [F(3, 20) = 31.62, p < 0.0001], Bonferroni’s test: significant differences between WT + WT EZRIN vs. LRRK2 + WT EZRIN (p < 0.0001), and LRRK2 + WT EZRIN vs. LRRK2 + Phospho-dead EZRIN (p < 0.0001). No significant differences for other comparisons (p > 0.05). (C-D) Representative ACC L1 and L2-3 images of VGluT1/PSD95 staining (P84). Scale bar, 10 μm. Quantification of co-localized puncta normalized to WT + WT EZRIN (n = 5–6/group). L1: One-way ANOVA [F(3, 19) = 5.882, p = 0.0051], Bonferroni’s test: significant differences among WT + WT EZRIN, LRRK2 + WT EZRIN, and their Phospho-dead counterparts (p < 0.05). L2-3: One-way ANOVA [F(3, 18) = 10.45, p = 0.0003], Bonferroni’s test: significant group differences (p < 0.05). (E) Representative mEPSC traces from L2-3 pyramidal neurons. (F) Synaptic event frequency quantification (n = 11–15 neurons from 4 mice/group). Kruskal-Wallis test [H(3) = 36.83, p < 0.0001], Dunn’s posthoc: significant differences between WT + WT EZRIN vs. all other groups (p < 0.0001), except LRRK2 + Phospho-dead EZRIN (p > 0.9999). (G) Synaptic event amplitude (n = 11–15 neurons from 4 mice/group). Kruskal-Wallis test [H(3) = 7.051, p = 0.0703]; no significant differences across groups (p > 0.05). Data are mean ± s.e.m. Data are mean ± s.e.m.

Astrocytic LRRK2 controls astrocyte morphology by balancing ERM phosphorylation levels in vivo.

(A) Representative images of ACC and MOp L2-3 astrocytes in WT and LRRK2 G2019Ski/ki (P21) expressing PB-mCherry-CAAX ± Phospho-dead EZRIN. Astrocyte territory in cyan. Scale bar, 10 μm. (B) Astrocyte territory volume (n = 16–18 astrocytes, 6 mice/group). One-way ANOVA [F(3, 20) = 7.987, p = 0.0011] with Bonferroni’s test showed significant differences: WT vs. LRRK2 G2019Ski/ki (p = 0.0178), WT vs. WT + Phospho-dead EZRIN O/E (p = 0.0037), and WT + Phospho-dead EZRIN O/E vs. LRRK2 G2019Ski/ki + Phospho-mimetic EZRIN O/E (p = 0.0165); other comparisons were not significant (p > 0.05). (C) Astrocyte branching complexity. N = 16–18 astrocytes (6 mice per group). Two-way ANOVA showed main effects of condition [F(2.505, 3305) = 158.4, p < 0.0001], radius [F(88, 1602) = 218.5, p < 0.0001], and their interaction [F(264, 3958) = 10.62, p < 0.0001]. Bonferroni’s test identified significant differences between groups (all p < 0.0001), except WT vs. LRRK2 G2019Ski/ki + Phospho-dead EZRIN O/E (p = 0.5469). (D) Proposed model of Ezrin conformational transition and LRRK2 regulation in astrocyte morphogenesis. (E) Images of ACC and MOp L2-3 astrocytes expressing shControl-mCherry-CAAX or shLRRK2-mCherry-CAAX ± Phospho-mimetic EZRIN. Scale bar, 10 μm. (F) Astrocyte territory volume. n = 18–22 astrocytes (5–6 mice/group). One-way ANOVA [F(3, 19) = 5.47, p = 0.0070] with Bonferroni’s test showed significant differences between shControl and shLRRK2 (p = 0.0132), and shLRRK2 vs. shLRRK2 + Phospho-mimetic EZRIN O/E (p = 0.0152); no other comparisons were significant (p > 0.05). (G) Astrocyte branching complexity. n = 16–22 astrocytes. Two-way ANOVA revealed significant main effects of condition [F(2.435, 7008) = 225.0, p < 0.0001], radius [F(88, 8633) = 325.4, p < 0.0001], and their interaction [F(264, 8633) = 5.908, p < 0.0001]. Bonferroni’s test identified significant differences between most groups (all p < 0.0001) except shControl vs. shControl + Phospho-mimetic EZRIN O/E (p > 0.9999). Data are mean ± s.e.m.

LRRK2 G2019S alters the interactome of astrocytic Ezrin in vivo.

(A) astrocyte-specific AAVs (serotype PHP.eB) were used to express Ezrin or nonspecific cytosolic control. NES, nuclear export sequence; ITR, inverted terminal repeats; GfaABC1D truncated GFAP promoter; HA, hemagglutinin tag; pA, polyadenylation. (B) outline of the experimental paradigm. n = 3 biological replicates/construct/genotype (1 replicate = 2 animals per pooled sample). (C) Volcano plot showing the differential abundance of proteins detected by Astro-EZRIN-BioID in WT and LRRK2 G2019Ski/ki cortices. (D-E) Bars show the top 10 most significant Gene Ontology (GO) terms, ordered by lowest adjusted p-value, for the proteins differentially detected by Astro-EZRIN-BioID in WT compared to LRRK2 G2019Ski/ki (D) Molecular function (E) Biological Process. (F) The interaction network depicts 58 high-confidence proteins that gained or lost proximity to Ezrin in LRRK2 G2019Ski/ki compared to WT mice. (G-H) heatmaps depict fold-change in abundance (Astro-EZRIN-BioID / Asto-Cyto-BioID) of proteins with high confidence changed proximity to Ezrin in LRRK2 G2019Ski/ki astrocytes.

Interaction Between Atg7 and Ezrin Depends on Ezrin’s Phosphorylation State.

(A) Schematic of Atg7 domains. (B) Predicted Atg7 homodimer model. (C) Schematic of Ezrin domains. (D) Predicted Ezrin structures in closed and open conformations. (E) Predicted Atg7-Ezrin interaction. (F) Structural changes in Ezrin and Atg7 upon binding. (G) Co-immunoprecipitation of Ezrin-HA by Atg7-Myc in HEK293T cells expressing WT, phospho-dead, or phospho-mimetic Ezrin. Ezrin was detected with anti-HA and Atg7 was detected with anti-Myc. (H) Quantification of (G). One-way ANOVA [F(2,6) = 9.932, p = 0.0125] with Tukey’s test: WT vs. phospho-mimetic Ezrin (p = 0.0151), phospho-dead vs. phospho-mimetic Ezrin (p = 0.0272), WT vs. phospho-dead Ezrin (p = 0.8658). n = 3 experiments. (I) Images of ACC and MOp L2-3 astrocytes at P21 expressing shControl or shAtg7-PB-mCherry-CAAX. Scale bar, 10 μm. (J) Quantification of astrocyte territories. Nested ANOVA [F(3,28) = 9.484, p = 0.0002] with Bonferroni tests: WT shControl vs. LRRK2 G2019Ski/ki shControl (p = 0.0036), WT shControl vs. WT shAtg7 (p = 0.0003), WT shAtg7 vs. LRRK2 G2019Ski/ki shAtg7 (p = 0.0109). n = 20–25 cells from 4–5 mice/group. (K) Astrocyte branching complexity. Two-way ANOVA revealed significant condition [F(2.324, 472.6) = 43.1, p < 0.05], radius [F(9, 250) = 301.8, p < 0.05], and interaction effects [F(27, 610) = 4.539, p < 0.05]. Bonferroni tests showed significant differences between groups, including WT shControl vs. LRRK2 G2019Ski/ki shControl (p < 0.0001) and WT shControl vs. WT shAtg7 (p < 0.0001).

Human phospho-ERM is significantly increased and colocalizes with S100β and MAP2. Related to Figure 1.

(A-B) Representative confocal images of phospho-ERM (purple) and S100β (green) or MAP2 (green) in the frontal cortex of human control subjects or human PD patients carrying LRRK2 G2019S mutation carriers at age >80 years old. Scale bar, 50 μm. (C) Quantification of phospho-ERM integrated density in (A-B), n = 4 (Human control, 3 males and 1 female), 3 (LRRK2 G2019S mutation carriers, 2 males and 1 female) subjects, nested t-test, unpaired two-tailed t-test. For colocalized phospho-ERM with S100β, t (5) = 6.541, p = 0.0013. For colocalized phospho-ERM with MAP2, t (5) = 3.625, p = 0.0151. Grey dots are the data acquired from each image. Black dots are the averaged data acquired from each control subject. Blue dots are the averaged data acquired from each LRRK2 G2019S mutation carrier.

Phospho-ERM is upregulated in LRRK2 G2019Ski/ki astrocytes in ACC and MOp but not SSp and DMS. Related to Figure 1.

(A) Schematic representation of analyzed mouse brain regions for phospho-ERM staining, the ACC, and the MOp. (B-C) Representative confocal images of ERM phosphorylation (purple) in the ACC and MOp of WT or LRRK2 G2019Ski/ki Aldh1L1-eGFP mice at P84. Scale bar, 20 μm. (D) Schematic representation of analyzed mouse brain regions for phospho-ERM staining, the somatosensory cortex (SSp), and the dorsal medial striatum (DMS). (E-H) Representative confocal images of ERM phosphorylation (purple) in the SSp and DMS of WT or LRRK2 G2019Ski/ki Aldh1L1-eGFP mice at P21 (E-F) and at P84 (G-H). Scale bar, 200 μm. (I) Quantification of phospho-ERM integrated density in (E-H). For phospho-ERM integrated density at P21, nested t-test, unpaired two-tailed t-test. t (6) = 0.5005, p = 0.6346. n = 4 (WT, 2 males and 2 females), 4 (LRRK2 G2019Ski/ki, 2 males and 2 females) mice. For phospho-ERM integrated density at P84, nested t-test, unpaired two-tailed t-test. t (6) = 0.8383, p = 0.1123. n = 4 (WT, 2 males and 2 females), 4 (LRRK2 G2019Ski/ki, 2 males and 2 females) mice.

mouse phospho-ERM is significantly increased and colocalizes with S100β and MAP2. Related to Figure 1.

(A-B) Representative confocal images of phospho-ERM (purple) and S100β (green), MAP2 (green), or Iba1 (green) from the ACC and MOp of WT or LRRK2 G2019Ski/ki mice at P21. Scale bar, 20 μm. (C) Quantification of colocalized phospho-ERM integrated density with S100β, MAP2, and Iba1 in (D-E), n = 3 (WT, 1 male and 2 females), 3 (LRRK2 G2019Ski/ki, 1 male and 2 females) mice, nested t-test, unpaired two-tailed t-test. For colocalized phospho-ERM with S100β, t (4) = 12.37, p = 0.0002. For colocalized phospho-ERM with MAP2, t (4) = 3.939, p = 0.017. For colocalized phospho-ERM with Iba1, t (4) = 1.08, p = 0.3408. Grey dots are the data acquired from each image. Black dots are the averaged data acquired from each WT mouse. Blue dots are the averaged data acquired from each LRRK2 G2019Ski/ki mouse.

phospho-ERM is eliminated after Lambda protein phosphatase (λ-PPase) treatment. Related to Figure 1.

(A-B) Representative confocal images of S100β (green) and phospho-ERM (purple) in the ACC of WT or LRRK2 G2019Ski/ki mice at P21 with or without λ-PPase treatment. Scale bar, 200 μm. (C) Quantification of phospho-ERM integrated density in (A-B), n = 3 (WT, 1 male and 2 females), 3 (LRRK2 G2019Ski/ki, 1 male and 2 females) mice, Nested One-way ANOVA [F (3, 56) = 25.19, p < 0.0001], Bonferroni’s multiple comparisons test revealed a significant difference between WT mice and LRRK2 G2019Ski/ki mice without λ-PPase treatment (p < 0.0001, 95% C.I. = [-8107799, -3924949]), and between LRRK2 G2019Ski/ki mice with and without λ-PPase treatment (p < 0.0001, 95% C.I. = [2605448, 6788298]). alpha = 0.05. Grey dots are the data acquired from each image. Black dots are the averaged data acquired from each WT mouse. Blue dots are the averaged data acquired from each LRRK2 G2019Ski/ki mouse. (D-E) Representative confocal images of S100β (green) and phospho-ERM (purple) in the frontal cortex of human control subjects or human PD patients carrying LRRK2 G2019S mutation carriers at age >80 years old with or without λ-PPase treatment. Scale bar, 200 μm. (F) Quantification of phospho-ERM integrated density in (D-E), n = 4 (Human control, 3 males and 1 female), 3 (LRRK2 G2019S mutation carriers, 2 males and 1 female) subjects, Nested One-way ANOVA [F (3, 38) = 9.296, p < 0.0001], Bonferroni’s multiple comparisons test revealed a significant difference between human control subjects and human PD patients carrying LRRK2 G2019S mutation carriers without λ-PPase treatment (p = 0.0006, 95% C.I. = [-7928018, - 1867234]), and between human PD patients carrying LRRK2 G2019S mutation carriers with and without λ-PPase treatment (p < 0.0001, 95% C.I. = [2768186, 9247437]). alpha = 0.05. Grey dots are the data acquired from each image. Black dots are the averaged data acquired from each control subject. Blue dots are the averaged data acquired from each LRRK2 G2019S mutation carrier.

GFAP level is not altered in LRRK2 G2019Ski/ki ACC and MOp. Related to Figure 1.

(A-B) Representative confocal images of GFAP (purple) in the ACC and MOp of WT or LRRK2 G2019Ski/ki Aldh1l1-eGFP mice at P21. Scale bar, 200 μm. (C) Quantification of GFAP integrated density in (A-B), n = 3 (WT, 2 males and 1 female), 3 (LRRK2 G2019Ski/ki, 2 males and 1 female) mice, For GFAP quantification in the ACC, nested t-test, unpaired two-tailed t-test. t (4) = 1.023, p = 0.364. For GFAP quantification in the MOp, nested t-test, unpaired two-tailed t-test. t (4) = 0.375, p = 0.7267. Grey dots are the data acquired from each image. Black dots are the averaged data acquired from each WT mouse. Blue dots are the averaged data acquired from each LRRK2 G2019Ski/ki mouse.

LRRK2 G2019S does not change excitatory synapse numbers in the primary motor cortex. Related to Figure 2.

(A) Representative images from the ventral ACC of WT and LRRK2 G2019Ski/ki mice that were stained with VGluT1 and PSD95 antibodies at P21. Scale bar, 10 μm. (B) Quantification of VGluT1-PSD95 co-localized puncta, normalized using the means of WT values in the ventral ACC. n = 4 (WT, 2 males and 2 females), 4 (LRRK2 G2019Ski/ki, 2 males and 2 females) mice. Nested One-way ANOVA [F (3, 44) = 12.98, p < 0.0001], Bonferroni’s multiple comparisons test revealed a significant difference between WT ACC L1 and LRRK2 G2019Ski/ki ACC L1 (p = 0.0002, 95% C.I. = [13.71, 47.33]), and between WT ACC L2-3 and LRRK2 G2019Ski/ki ACC L2-3 (p < 0.0001, 95% C.I. = [16.48, 50.10]). alpha = 0.05. (C) Representative images from the MOp of WT and LRRK2 G2019Ski/ki mice that were stained with VGluT1 and PSD95 antibodies at P21. Scale bar, 10 μm. (D) Quantification of VGluT1-PSD95 co-localized puncta, normalized using the means of WT values in the MOp L1 and L2-3. n = 5 (WT), 5 (LRRK2 G2019Ski/ki) mice, 3 males and 2 females. Nested One-way ANOVA [F (3, 56) = 0.1837, p = 0.9070], Bonferroni’s multiple comparisons test revealed a significant difference between WT MOp L1 and LRRK2 G2019Ski/ki MOp L1 (p > 0.9999, 95% C.I. = [-14.69, 25.97]), and between WT MOp L2-3 and LRRK2 G2019Ski/ki MOp L2-3 (p > 0.9999, 95% C.I. = [-19.06, 21.61]). alpha = 0.05. Grey dots are the data acquired from each image. Black dots are the averaged data acquired from each WT mouse. Blue dots are the averaged data acquired from each LRRK2 G2019Ski/ki mouse.

Overexpression of AAV-HA-tagged-WT Ezrin and AAV-HA-tagged-Phospho-dead Ezrin in WT and LRRK2 G2019Ski/ki astrocytes. Related to Figure 4.

(A-B) Bar plots of Ezr, Rdx, and Msn gene expression in various brain cell types in mice (yellow) or humans (red). These plots are generated using publicly available data at https://www.brainrnaseq.org (C) Schematic of domains within Ezrin. (D) Experiment workflow of AAV-HA-tagged-WT Ezrin and AAV-HA-tagged-Phospho-dead Ezrin in WT and LRRK2 G2019Ski/ki astrocytes.

Overexpression of AAV-HA-tagged-WT Ezrin and AAV-HA-tagged-Phospho-dead Ezrin in WT and LRRK2 G2019Ski/ki astrocytes. Related to Figure 4.

(A) Representative images from the ventral ACC of WT and LRRK2 G2019Ski/ki mice injected with AAV-HA-tagged WT Ezrin or Phospho-dead Ezrin, stained with HA and phospho-Rab10 antibodies at P84. Scale bar, 20 μm. (B) Quantification of phospho-Rab10 integrated density. n = 4 per group (2 males and 2 females). One-way ANOVA [F(3,12) = 38.34, p < 0.0001] with Bonferroni’s post hoc test revealed significant differences between groups, except WT + WT Ezrin O/E vs. WT + Phospho-dead Ezrin O/E (p > 0.9999) and LRRK2 G2019Ski/ki groups (p = 0.3303).

WT Ezrin overexpression does not change excitatory synapse numbers in the ACC of WT mice. Related to Figure 4.

(A) Representative images from the ventral ACC of WT mice injected with AAV-HA-tagged BirA2 (cytoplasmic control) or WT Ezrin that were stained with VGLUT1 and PSD95 antibodies at P84. Scale bar, 10 μm. (B) Quantification of VGLUT1 puncta, normalized using the means of BirA2 values in the ventral ACC. n = 5 mice per group. For ACC L1, nested t-test, unpaired two-tailed t-test. t (8) = 0.5035, p = 0.6282. For ACC L2/3, nested t-test, unpaired two-tailed t-test. t (8) = 0.6465, p = 0.5361. (C) Quantification of PSD95 puncta, normalized using the means of BirA2 values in the ventral ACC. n = 5 mice per group. For ACC L1, nested t-test, unpaired two-tailed t-test. t (8) = 0.02872, p = 0.9778. For ACC L2/3, nested t-test, unpaired two-tailed t-test. t (8) = 0.4235, p = 0.6831. (D) Quantification of VGLUT1-PSD95 puncta, normalized using the means of BirA2 values in the ventral ACC. n = 5 mice per group. For ACC L1, nested t-test, unpaired two-tailed t-test. t (8) = 0.5400, p = 0.6039. For ACC L2/3, nested t-test, unpaired two-tailed t-test. t (8) = 0.6013, p = 0.5643.

Overexpression of AAV-HA-tagged-WT Ezrin and AAV-HA-tagged-Phospho-dead Ezrin in WT and LRRK2 G2019Ski/ki astrocytes. Related to Figure 4.

(A) Representative images from the MOp L1 of the same groups stained with VGAT and GEPHYRIN antibodies. Scale bar, 10 μm. Quantification of VGAT-GEPHYRIN co-localized puncta, normalized to WT + WT Ezrin O/E. n = 3–6 per group. One-way ANOVA [F(3,55) = 19.7, p < 0.0001] with Bonferroni’s post hoc test revealed significant differences between WT + WT Ezrin O/E and LRRK2 G2019Ski/ki + WT Ezrin O/E, and between WT + Phospho-dead Ezrin O/E and LRRK2 G2019Ski/ki + Phospho-dead Ezrin O/E (p < 0.0001). No differences were found between WT or LRRK2 G2019Ski/ki groups within Ezrin conditions (p > 0.05). (B) Representative images from the MOp L2-3 of WT and LRRK2 G2019Ski/ki mice injected with AAV-HA-tagged WT Ezrin or Phospho-dead Ezrin, stained with VGAT and GEPHYRIN antibodies at P84. Scale bar, 10 μm. Quantification of VGAT-GEPHYRIN co-localized puncta normalized to WT + WT Ezrin O/E. n = 3–6 per group. One-way ANOVA [F(3,54) = 15.10, p < 0.0001] with Bonferroni’s post hoc test showed significant differences between WT + WT Ezrin O/E and LRRK2 G2019Ski/ki + WT Ezrin O/E (p = 0.0107) and between WT + Phospho-dead Ezrin O/E and LRRK2 G2019Ski/ki + Phospho-dead Ezrin O/E (p < 0.0001). No differences were observed between WT groups (p > 0.9999) or between LRRK2 G2019Ski/ki groups (p = 0.0610).

LRRK2 G2019S does not change ALDH1L1+/SOX9+ cell numbers in the ACC and MOp. Related to Figure 5.

(A) Overview of postnatal astrocyte labeling by electroporation (PALE) with PiggyBac plasmids. (B-C) Representative confocal images of SOX9 (purple) in the ACC and MOp of WT or LRRK2 G2019Ski/ki Aldh1l1-eGFP mice at P21. Scale bar, 200 μm. (D) Quantification of ALDH1L1+/SOX9+ cell numbers in (B-C), n = 3 (WT, 2 males and 1 female), 3 (LRRK2 G2019Ski/ki, 2 males and 1 female) mice, For ALDH1L1+/SOX9+ cell counting in the ACC, nested t-test, unpaired Two-tailed t-test. t (4) = 0.9619, p = 0.3906. For ALDH1L1+/SOX9+ cell counting in the MOp, nested t-test, unpaired two-tailed t-test. t (4) = 1.684, p = 0.1675. Grey dots are the data acquired from each image. Black dots are the averaged data acquired from each WT mouse. Blue dots are the averaged data acquired from each LRRK2 G2019Ski/ki mouse.

Expression of LRRK2 in astrocytes. Related to Figure 5.

(A) Cultured WT, shControl, and shLrrk2 transfected rat cortical astrocytes were lysed, and cytoplasmic proteins were subjected to Western blotting using LRRK2 and GAPDH antibodies. Results are representative of 3 independent experiments. (B) Densitometric analysis of LRRK2 levels in (E). Signals corresponding to LRRK2 were first normalized to that for β-actin. Relative LRRK2 levels were then normalized to the LRRK2 signals in WT rat cortical astrocytes. Statistical significance was determined by One-way ANOVA [F (2, 6) = 92.25, p < 0.0001], Bonferroni multiple comparisons revealed a significant difference between WT and shLrrk2 (p < 0.0001, 95% C.I. = [0.6743, 1.183]) and between shLrrk2 and shControl (p < 0.0001, 95%C.I. = [-1.145, - 0.6363]) and no differences between WT and shControl (p > 0.9999, 95%C.I. = [-0.2165, 0.2923]), alpha = 0.05.

BioID constructs expression and biotinylation in vivo and in vitro. Related to Figure 6.

(A-B) Representative images of in vivo expression in the cortex of different BioID constructs labeled with HA and the biotinylating activity labeled with streptavidin. Merged images show the colocalization of HA and biotin signals in astrocytes. Scale bar, 500 μm (A). Scale bar, 20 μm (B). (C) Western blot analysis of BioID constructs expression (HA) and biotinylation activity (Streptavidin) in vitro in cortical lysates and subsequent immunoprecipitation.

LRRK2 G2019S changes the composition of cytoplasmic proteins in astrocytes in vivo. Related to Figure 6.

(A) Principle component analyses (PCA) of Cyto-BioID (control) and Ezrin-BioID (bait) samples. (B) The plot shows that Radixin and Moesin, known as Ezrin interacting proteins, are significantly enriched in the Ezrin-BioID sample compared to Cyto-BioID. (C-D) Interaction networks depict Ezrin interactions detected by BioID and known protein-protein interactions identified in the publicly available stringDB database for proteins with gained (C) or lost (D) proximity to Ezrin in LRRK2 G2019Ski/ki astrocytes compared to WT astrocytes. (E) Volcano plot showing the differential abundance of cytosolic proteins in WT and LRRK2 G2019Ski/ki cortices. (F-G) Bars show the top 10 most significant Gene Ontology (GO) terms, ordered by highest gene count and lowest adjusted p-value, for the proteins differentially detected by Astro-cyto-BioID in WT compared to LRRK2 G2019Ski/ki (F) Molecular function (G) Biological Process.

Astrocytic Atg7 is essential astrocyte morphological complexity in vitro. Related to Figure 7.

(A) Comparable expression of Atg7 mRNA transcripts by RT-PCR in N2A cells transfected with scrambled shRNA (shControl-GFP) or shRNA targeting Atg7 (shAtg7-GFP). n = 3 independent cultures. unpaired Two-tailed t-test. t (4) = 8.258, p = 0.0012. (B) Schematic of astrocyte-neuron co-culture assay. (C) (Upper) Rat cortical astrocytes transfected with scrambled shRNA (shControl-GFP) or shRNA targeting Atg7 (shAtg7-GFP) co-cultured with wild-type cortical neurons. Scale bar, 10 μm. (Lower) Quantification of astrocyte branching complexity. n = 30 (shControl-GFP), 30 (shAtg7-GFP) astrocytes co-cultured with WT cortical neurons compiled from two independent experiments. We fitted a linear mixed model with a number of intersections as the outcome variable, condition as a predictor, and a number of cells and cell radius entered as random effects. Within this model, shAtg7 (beta = 2.91, t(58) = 7.14) led to a significant decrease in the number of intersections compared to shControl astrocytes (beta = 7.89, t(58) = 19.35). Tukey multiple comparisons test revealed a significant difference between shControl and shAtg7 astrocytes co-cultured on WT neurons (p < 0.0001).