(a) Golgi-Cox staining of SPNs from P15 littermate LRRK2^+/+ and LRRK2^−/− pups. The bottom panels are enlargement of boxed areas of the upper panels. Scale bar: 5μm. (b) The density of dendritic spines and filopodia (n=3 three pups per genotype). Data represent mean ± SEM. Mann-Whitney Test (spine: z=1.964, filopodia: z=−1.964). ^*P<0.05. The inset shows schematic representation of filopodium and different types of dendritic spines. (c) The distribution of different types of dendritic spines in LRRK2^+/+ and LRRK2^−/− SPNs (n_LRRK2^+/+=839 spines, n_LRRK2^−/− =835 spines from six neurons of three pups per genotype, Chi-Squared test (χ²₍₂₎=50.13), p<0.0001. (d,e) Cumulative frequencies of spine head diameter (d, KS-test, p<0.001) and spine length (e, KS-test, p<0.001) in the spines of SPNs described above. Scatter blots show average head diameter and length. Data represent mean ± SEM. Unpaired t-test, t₍₄₎=1.839 and 3.853, respectively. ^*P<0.05. (f) Traces of mEPSCs recorded from P15 LRRK2^+/+ and LRRK2^−/− SPNs. (g, h) Mean values and cumulative distribution for amplitude (g, Unpaired t-test, t₍₈₎=2.446; KS-test, *p<0.01) and frequency (h, Unpaired t-test, t₍₈₎=2.667; KS-test, p<0.001) of mEPSCs from P15 pups (n_LRRK2^+/+=11 neurons, n_LRRK2^−/− =13 neurons of five pups per genotype). Data represent mean ± SEM. ^*P<0.05. (i) Paired-pulse facilitation measured at a 50ms inter-pulse interval and summary data for n_LRRK2^+/+=5 neurons, n_LRRK2^−/−=7 neurons (t₍₁₂₎=0.6323).

(a) Western blot analysis of forebrain homogenates from LRRK2^+/+ and LRRK2^−/− mice. The full-length images are in shown in . (b) The pS3 cofilin levels normalized against total cofilin levels (n=3 per genotype). Data represent mean±SEM, Unpaired t-test, t₍₄₎=0.8906, 0.1047, 11.11, and 4.117, respectively. ^*p<0.05, ^***p<0.001. (c) Representative images of spines and their parental dendrites of cultured LRRK2^+/+ and LRRK2^−/− hippocampal neurons transfected with mCherry (red) and stained with pS3 cofilin (green). Scale bar: 10μm. (d) Staining of F-actin (green), PSD95 (red), and βIII-tubulin (blue) in dendritic spines and corresponding dendrites of LRRK2^+/+ and LRRK2^−/− hippocampal neurons at 15DIV. Scale bar: 5μm. (e) F-actin area staining in the dendritic spine of LRRK2^+/+ and LRRK2^−/− hippocampal neurons (n=6–8 neurons per genotype; n=85–90 spines per genotype). Data represent mean ± SEM. Unpaired t-test, ^***p<0.001. (f, j) Spines and dendritic regions of LRRK2^−/− (f) and LRRK2^+/+ (j) hippocampal neurons transfected with GFP, GFP-S3A, and GFP-S3D cofilin plasmids, along with mCherry at 15DIV. Scale bar: 5μm. (g, k) Density of dendritic spines and filopodia was quantified from LRRK2^−/− and LRRK2^+/+ neurons. (h–i, l–m) Bar graphs showing average head spine diameter and dendritic spine length of transfected LRRK2^−/− (h, i) and LRRK2^+/+ neurons (l, m) (LRRK2^−/−, n_GFP=73 spines, n_S3A-GFP=80 spines; LRRK2^+/+ n_GFP=101 spines, n_S3D-GFP=79 spines; Four to seven neurons per genotype). Data represent mean ± SEM. Unpaired t-test, ^*p<0.05, ^**p<0.01, ^***p<0.001

(a, b) Western Blot analysis of cultured LRRK2^+/+ and LRRK2^−/− cortical neurons treated with DMSO or FSK (50μM for 1 hr) and (c, d) DMSO or PKI (5μM for 24 hrs) for the expression of phosphorylated and total cofilin. Three or more independent cultures were analyzed per genotype and per treatment. Data represent mean ± SEM. Unpaired t-test, t₍₄₎=6.019, 3.820, and 0.0764, respectively in (b). t₍₄₎=3.441, 2.851, and 3.278, respectively in (d). *P<0.05, ^**p<0.01. The full-length images in Fig. 3a and 3c are shown in .

(a) Co-IP of transgenic human wild-type LRRK2 and endogenous mouse PKARIIβ from the forebrain homogenate of P15 CaMKII-tTA/tetO-LRRK2 double and littermate CaMKII-tTA single transgenic mice. The N-terminal of transgenic LRRK2 is HA-tagged. (b,c) Co-IP of GST-tagged recombinant human LRRK2 proteins with maltose-binding protein (MBP)-tagged recombinant mouse PKARIIβ proteins (b) and GST-tagged recombinant mouse PKACα proteins (c). (d) Co-IP of endogenous mouse LRRK2 and PKARIIβ from the light membrane fractions of P15 mouse striatal tissues. (e) Co-IP of endogenous mouse LRRK2, PKACα and PKARIIβ from the light membrane fractions of P15 mouse striatal tissues using a PKARIIβ antibody. (f) Co-staining of endogenous LRRK2 (green) and PKARIIβ (red) in cultured mouse hippocampal neurons at 15DIV. The middle two panels show the boxed areas in the top panel. The bottom panel shows LRRK2 (green) and PKARIIβ (red) staining in 15DIV LRRK2^−/− hippocampal neurons (arrowheads point to dendritic spines). Scale bars: 20μm (top panel), 5μm (lower panels). (g) Representative image shows subcellular localization of endogenous PKARIIβ (green) in the dendrite of mCherry-transfected 15DIV LRRK2^+/+ hippocampal neurons. Arrowheads indicate the localization of PKARIIβ at the base of dendritic spines. Scale bars: 5μm. (h) Co-IP of GFP-PKARIIβ with myc-tagged human wild-type full-length LRRK2, as well as LRRK2 kinase, RCK (Roc-COR-Kinase), ROC, COR, and WD40 domains from transfected HEK293 cells. (i) Co-IP of myc-tagged human wild-type full-length LRRK2 with GFP, GFP-PKARIIβ, and GFP-PKARIIβΔ2–5 from transfected HEK293 cells. DM: dimerization domain, AI: auto-inhibitory domain, CB: cAMP binding domain. The full-length images in Fig. 4a–e, 4h, and 4j are shown in .

(a) Fluorescent images of GFP-PKARIIβ and mCherry in dendrites of transfected LRRK2^+/+ and LRRK2^−/− hippocampal neurons at 15DIV. Scale bars: 5μm (lower magnification), 1μm (higher magnification). The RGB signal profile shows mCherry (red) and GFP-PKARIIβ (green) expression across the spine and adjacent dendritic shaft section depicted by the discontinuous white line on LRRK2^+/+ and LRRK2^−/− hippocampal neurons. (b,c) The relative ratio of GFP-PKARIIβ (b) and GFP-PKACα (c) in the dendritic spine versus adjacent dendritic shaft of LRRK2^+/+ and LRRK2^−/− hippocampal neurons at 15DIV (n=52–95 spines of seven neurons per genotype). Data represent mean ± SEM. Unpaired t-test, t₍₁₁₄₎=7.013 in (c). ^***p<0.001, ^****p<0.0001. (d) Western blot analysis of endogenous PKARIIβ, PKACα, PSD95, and LRRK2 expression in the total, pelleted nuclear and other insoluble (P1′), cytosol, light membrane (LM), synaptosome (synap) and PSD fractions of P15 LRRK2^+/+ and LRRK2^−/− mouse brain homogenates. The full-length images are shown in . (e,f) Quantified data of PKARIIβ (e) and PKACα (f) protein levels in the PSD fraction of P15 LRRK2^+/+ and LRRK2^−/− mouse brain homogenates (n=4 per genotype). Data represent mean ± SEM. Unpaired t-test, t₍₆₎=9.929 and 3.935, respectively. ^**P<0.01, ^****p<0.0001.

(a) Western blot analysis of pS845 GluR1, total GluR1 and LRRK2 expression in the forebrain homogenates of P2, P5, P15, and P30 LRRK2^+/+ and LRRK2^−/− mice. (b) Quantification analysis of pS845 GluR1 levels normalized to total GluR1 levels derived of three independent experiments. Data represent mean ± SEM. Unpaired t-test, t₍₄₎=0.5791, 6.282, 3.576, and 1.061, respectively. ^*p<0.05, ^**p<0.01. (c) Western blot analysis of GluR1 and PSD95 in the S1 and PSD fractions of P15 LRRK2^+/+ and LRRK2^−/− mouse brains. (d,e) Quantification analysis of GluR1 levels in the PSD versus S1 fractions of brain extracts (d), as well as GluR1 levels versus PSD95 levels in the PSD fraction of brain extracts (e). n=3 per genotype. Data represent mean ± SEM. Unpaired t-test, t(4)=4.218 (d) and 4.206 (e). ^*p<0.05. (f) Western blot analysis of pS845 and total GluR1 in the striatum of P21 LRRK2^+/+ and LRRK2^−/− mice after treated with saline (0) or Drd1 agonist SKF81297 at 2 and 10mg per kg bodyweight. Actin was used as the loading control. (g,h) Quantification analysis of pS845 GluR1 ratio in the striatal tissues of P21 (g) and 18-month-old mice (h) treated with SKF81297. n=4 per genotype per treatment. Data represent mean ± SEM. Unpaired t-test. t₍₈₎=2.501, 2.177, and 4.605 in (g). t₍₆₎=3.703 and 4.401 in (h). ^*p<0.05, ^**p<0.01. The full-length images of Fig. 6a,c,f are shown in .

(a) Co-IP of GFP-PKARIIβ with wild-type, R1441C, G2019S, and G2835 LRRK2 from transfected cells (a). (b) The relative binding affinity of GFP-PKARIIβ with LRRK2 proteins (n=3). t₍₄₎=3.070, 0.2735, and 2.016. (c) Co-IP of GST-LRRK2 and GST-R1441C with MBP-PKARIIβ recombinant proteins. (d) GFP-PKARIIβ and mCherry signals in the spines and dendrites of 15DIV cultured hippocampal neurons. Scale bar: 5μm. (e,f) Quantification of GFP-PKARIIβ signal in the spines (f, n=70–75 spines from six neurons per genotype), and the spine size (g, n=60 spines from four neurons per genotype)..t=2.552 and 2,145, respectively. (h–k) The pS3 versus total cofilin (h,i) and pS845 versus total GluR1 (j,k) in the forebrain homogenate of P15 LRRK2^+/+, LRRK2^+/R1441C and LRRK2^{R1441C/R1441C} mice. n=3 per genotype. t₍₄₎=1.935 and 3.182 in (h), 3.918 and 5.191 in (j). (l–n) The pS845 versus total GluR1 in the striatum of P21 LRRK2^+/+ (n=8), LRRK2^+/R1441C (n=4), and LRRK2^{R1441C/R1441C} (n=4) mice (l,m), and 18-month-old LRRK2^+/+ (n=4) and LRRK2^RC/RC (n=4) mice (n). t₍₁₀₎=0.3237, 0.7222, 2.865, and 3.283 in (m). t₍₆₎=3.303 and 4.083 in (m). (o–q) The ambulatory (o), fine (p), and rearing movement (q) of P21 LRRK2^+/+, LRRK2^−/−, and LRRK2^+/RC mice after treated with saline (n=16, 37, and 10) or SKF81297 at 2 (n=21, 28, and 11) and 10mg per kg bodyweight (n=27, 28, and 10). Data represent mean ± SEM. Unpaired test (b,e,f,h,j,l,m) and two-way ANOVA plus Bonferroni post-tests (m–q) were used.