Pathogenic LRRK2 disrupts lysosomal morphology in a kinase-dependent manner. (A,B) Confocal images of Lysotracker® red fluorescence in live fibroblasts derived from a healthy control (A) and a Parkinson disease (PD) patient harbouring the LRRK2 G2019S mutation (B). Higher magnification images are shown in the right panels. Scale bars: 5 µm (and also apply to B–D,H,I). Fluorescence intensity was increased 1.4±0.07-fold (mean±s.e.m.) in Parkinson disease cells (n = 108 cells from three independent platings of two patient and paired control lines). (C,D) Confocal images of LAMP1 staining in fixed fibroblasts. Nuclei (stained with DAPI) are shown in blue. (E) Pooled data quantifying LAMP1 intensity (left) or the proportion of cells displaying perinuclear lysosome clustering (right). Data (mean±s.e.m.) are from 969 healthy control and 1181 LRRK2-PD cells from 21 independent platings of a single patient and paired control line. (F) Representative electron micrographs of endolysosomes from a healthy (left) and LRRK2-PD (right) fibroblast. L, lysosome. Scale bars: 200 nm. (G) Pooled data quantifying lysosome area (left) and density (right). Data (mean±s.e.m.) are from 100 lysosomes. (H,I) LAMP1 staining in LRRK2-PD fibroblasts treated for three days with the LRRK2 kinase inhibitors LRRK2-In1 (100 nM, H) or GSK2578215A (32 nM, I). (J) Pooled data quantifying LAMP1 intensity for the cells shown in H and I (mean±s.e.m., n = 136–335 cells from five independent platings of two patient and paired control lines). ***P<0.001.

TPC2 but not TPC1 mediates lysosomal morphology disturbances. (A–D) LAMP1 staining in fibroblasts from a healthy control (CTRL) (A) and a Parkinson disease patient (PD) (B–D) treated with either a control siRNA (Scr. siRNA) (A,B) or siRNA to TPC2 (C) or TPC1 (D). Scale bars: 5 µm. (E) Pooled data quantifying LAMP1 intensity for the cells shown in A–D (mean±s.e.m., n = 381–532 cells from six independent knockdowns from two patient and paired control lines). (F) Quantitative PCR analysis of TPC2 (left) and TPC1 (right) levels in cells treated with the indicated TPC siRNA. Data are from two patients and are normalised to TPC levels in cells treated with scrambled control siRNA. (G–I) Lysosomal morphology in control fibroblasts (G) or LRRK2-PD fibroblasts treated without (H) or with (I) the Rab7 GTPase inhibitor CID 1067700 (1 µM, 3 days). (J,K) Pooled data quantifying LAMP1 intensity (J) or the proportion of cells displaying perinuclear lysosome clustering (K) for the cells shown in G–I (mean±s.e.m., n = 237–281 cells from five independent platings of three patient and paired control lines). ***P<0.001. (L) Concentration–effect relationship (mean±s.e.m., n = 130–281 cells from five independent platings of three patient lines) for the Rab7 GTPase inhibitor on lysosome clustering in LRRK2-PD fibroblasts.

Lysosomal defects are NAADP- and PtdIns(3,5)P₂-dependent. (A) Schematic of the TPC (yellow) showing proposed ion permeability (Ca²⁺ and Na⁺; grey arrows) and activating ligands (NAADP and PtdIns(3,5)P₂; green arrows). Drugs used are highlighted in italics and their loci of action is shown in red. (B–E) LAMP1 staining in fibroblasts from a healthy control (CTRL) (B) and a Parkinson disease (PD) patient (C–E) treated for 3 days with either DMSO (B,C) or the NAADP antagonists, Ned-19 (100 µM, D) and Ned-K (100 µM, E). Scale bars: 5 µm. (F) Pooled data quantifying LAMP1 intensity for the cells shown in B–E (mean±s.e.m., n = 92–322 cells from six independent platings of three patient and paired controls). (G) Concentration–effect relationships (mean±s.e.m., n = 40–206 cells from six independent platings of three patient lines) for Ned-19 (black circles) and Ned-K (white circles) on lysosomal morphology in LRRK2-PD fibroblasts. (H,I) LAMP1 staining in LRRK2-PD fibroblasts treated without (H) or with the PIKfyve inhibitor YM-201636 (1 µM, 2 h) (I). (J) Pooled data quantifying LAMP1 intensity for the cells shown in H and I (mean±s.e.m., n = 73–292 cells from two independent platings of two patient and paired control lines). ***P<0.001.

Pathogenic LRRK2 disrupts local and global Ca²⁺ signalling. (A–D) LAMP1 staining in fibroblasts from a healthy control (CTRL) (A) and a Parkinson disease patient (PD) (B–D) treated for 2 h with either DMSO (A,B) or 10 µM acetoxymethyl (AM) esters of the Ca²⁺ chelators BAPTA (C) or EGTA (D). Scale bars: 5 µm. (E) Pooled data quantifying LAMP1 intensity for the cells shown in A–D (mean±s.e.m., n = 150–307 cells from six independent platings of two patient and paired controls). ***P<0.001. (F,G) Cytosolic Ca²⁺ responses of individual Fura-2-loaded healthy (F) or Parkinson disease (G) fibroblasts microinjected with either vehicle (blue traces) or NAADP (20 µM pipette; black traces). (H) Pooled data (n = 6) quantifying the change in ratio upon microinjection of NAADP (left) or vehicle (right).