Figure 2. Abnormal phosphorylation of cofilin in LRRK2^−/− neurons.

(a) Western blot analysis of forebrain homogenates from LRRK2^+/+ and LRRK2^−/− mice. The full-length images are in shown in Supplementary Fig. 2e. (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