Fig 2: Insulin signalling in Lrrk2 deficient animals. (a) Insulin-triggered phosphorylation of IRß and AS160 (Thr642) in fibroblasts from 22 months old Lrrk2 deficient rats at different time-points after stimulation and the corresponding quantification of P-IRß (b, n = 7, normalized to IR-ß) and P-AS160 Thr642 (c, n = 10, normalized to AS160) signal intensity (mean ± SEM). (d) Western blot analysis (fibroblasts from 22 months old rats as example) and quantification of total GLUT4, AS160 and Rab10 expression in fibroblasts from 6 months (e) and 22 months old (f) Lrrk2 deficient and wild-type rats (normalized to tubulin, mean ± SEM). # are numbers of animals/cell lines. The difference in GLUT4 and AS160 signal intensity between 6 months und 22 months old sample-groups results from differences in experimental procedure and does not reflect the absolute quantity of GLUT4 or rather AS160 in these age groups. (g) Investigation of Rab10 phosphorylation by Mn2+ Phos-tag SDS-PAGE in fibroblasts from 6 months old Lrrk2 deficient and wild-type rats at different time points (0-10-30-40 min) after insulin addition and (h) corresponding quantification of P-Rab10 signal intensity in wild-type cells at different time points after stimulation (normalized to Rab10, n = 7, mean and SEM).

Fig 3: Insulin signalling in human fibroblasts from Parkinson’s patients with G2019S mutated LRRK2. (a) Example: Western blot analysis of protein extracts from fibroblasts of one PD patient with G2019S mutation (top) and one healthy control individual (button) and (b) the corresponding quantification of P-Akt Thr308 (top) and P-Akt Ser473 (button) signal intensity (normalized to Akt, n = 7 independent experiments, fibroblasts from 3 different healthy controls and 3 PD patients with G2019S mutation in LRRK2; mean ± SEM). (c) GLUT4 immunostaining on the plasma membrane of human fibroblasts derived from PD patients with G2019S mutation and healthy control individuals (red: GLUT4 immunostaining; green: wheat germ agglutinin (=plasma membrane) staining; blue: DAPI) and (d) the corresponding quantification of GLUT4 signal intensity (mean ± SEM). (e) Quantification of total GLUT4, AS160 and Rab10 protein expression in human fibroblasts (normalized to tubulin) analysed by Western blot (mean and SEM).

Fig 4: GLUT4 translocation in Lrrk2 deficient fibroblasts. (a) GLUT4 immunostaining on the cell surface of fibroblasts from 6 months old Lrrk2 deficient and wild-type rats without (w/o) insulin and 10 and 30 min after insulin addition and (c) the corresponding quantification of the GLUT4 signal intensity at different time-points (0, 10 and 30 min) after stimulation (mean and SEM). (b) GLUT4 immunostaining on plasma membrane of fibroblasts from 22 months old Lrrk2 deficient and wild-type rats without insulin and 10 and 30 min after stimulation and (d) the corresponding quantification of the GLUT4 signal intensity (mean and SEM). Red: GLUT4 immunostaining; green: wheat germ agglutinin (=plasma membrane) staining; blue: DAPI.

Fig 5: Primary podocytes can be cultured from kidney organoid glomeruli. a Isolated organoid glomeruli show evidence of podocyte cell migration (OrgPods) displaying thin arborized projections (TAPs) (inset). Inverted image shown to provide maximum contrast, scale bar 100 µm. b TAPS from newly emerged podocytes are composed of F-actin shown by phallodin immunofluorescent staining. Inverted image, scale bar 50 µm. c Immunostaining at 36 h post-plating shows a strong positively stained 3D OrgGlom with a migrating 2D OrgPod population. Left panel 2D images, right panel 3D reconstruction of Z-stack. Scale bars 50 µm. d At 48 h post-plating OrgPods display a flattened, arborized morphology with processes connecting adjacent cells (arrow), scale bar 50 µm. e Immunostaining of ciPods for SYNAPTOPODIN showed expression is absent in undifferentiated cells (ciPod: Un), only becoming evident following 14 days induced differentiation at 37 °C (ciPod: Diff). OrgPods also display strong SYNAPTOPODIN protein expression, aligned with F-actin stress fibres. Scale bars 100 µm. f OrgPods express the neonatal Fc receptor (FcRN) and actively endocytose fluorescein isothiocyanate (FITC)-labelled albumin at 37 °C resulting in FITC-accumulation in endosomes on the cell surface. This is process halted when performed at 4 °C. Scale bars 50 µm. g OrgPods stimulated with insulin (10 mg/ml) for 10 min showed cortical reorganisation of their actin cytoskeleton with GLUT4 translocation from a vesicular to plasma membrane localisation. Scale bars 50 µm. All representative images reflect a minimum of three biological replicates. For immunofluorescence, images are shown in greyscale for single channels, and merged images in colour