Journal: bioRxiv

Article Title: LRRK2 is activated by phosphatidylinositol 3-phosphate in conjunction with CASM

doi: 10.64898/2025.12.16.694573

Figure Lengend Snippet: (A) Schematic diagram of the PI3P metabolic pathway, where class III PI3K is responsible for the generation of PI3P from PI and PIKfyve is for the generation of PI(3,5)P 2 from PI3P. Compounds that inhibit each kinase are also shown. (B) Immunostaining of intracellular vacuoles formed by treatment with apilimod (Api) or YM201636 (YM) in RAW264.7 cells. Vacuoles were stained with anti-LAMP1 antibody (red) and nuclei were stained with DAPI (cyan). Scale bar = 10 μm. (C-F) Immunoblot analysis of Thr73-phosphorylated Rab10 (pRab10) and other indicated proteins in RAW264.7 cells treated with PIKfyve inhibitors as well as VPS34-IN1 (C, D) or Compound19 (Cmpd19) (E, F). Bar graphs in D and F show quantification of pRab10 relative to total Rab10. Data represent mean ± SEM, n = 6 (D) or 3 (F), and the difference was analyzed using two-way ANOVA with Tukey’s test. (G, H) Immunoblot analysis of pRab10 and other proteins in A549 cells treated with PIKfyve inhibitors as well as VPS34-IN1. A bar graph in H shows quantification of pRab10 relative to total Rab10. Data represent mean ± SEM, n = 4, two-way ANOVA with Tukey’s test. (I-J) Immunoblot analysis of pRab10 in HEK293 cells that overexpress GFP, LRRK2-wild type (WT) or pathogenic mutants (R1441C, Y1699C) and are treated with PIKfyve inhibitors. A bar graph in J shows quantification of pRab10/total Rab10. Data represent mean ± SEM, n = 4, two-way ANOVA with Tukey’s test. (K) Schematic illustration of the activation induction mechanism of PI3K by Tat-D11. (L-N) Immunoblot analysis of pRab10 and LC3B upon Tat-D11 and/or MLi-2 treatment in RAW264.7 cells. Bar graphs show quantifications of pRab10 relative to total Rab10 (M) and LC3B-II relative to α-tubulin (N). Data represent mean ± SEM, n = 6 (K) or 5 (L), two-way ANOVA with Tukey’s test.

Article Snippet: RAW264.7 mouse macrophage cells (ECACC, RRID: CVCL_0493) and Lrrk2 knockout RAW264.7 cells (ATCC, RRID: CVCL_UL72) were routinely cultured on culture dishes for suspended cells (Sumitomo Bakelite Co.) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS).

Techniques: Immunostaining, Staining, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: LRRK2 is activated by phosphatidylinositol 3-phosphate in conjunction with CASM

doi: 10.64898/2025.12.16.694573

Figure Lengend Snippet: (A) Schematic diagram of the mechanism of the CASM-LRRK2 pathway induced by pH/ionic imbalances. LAP: LC3-associated phagocytosis. (B-C) Immunoblot analysis of pRab10 and other indicated proteins in RAW264.7 cells that were pretreated with siRNAs for Atg5 or Atg16l1 and then treated with apilimod or CQ. Knockdown efficiencies of Atg5 and Atg16l1 are also shown. A bar graph in C shows quantification of pRab10 relative to total Rab10, as shown in B. Data represent mean ± SEM, n = 3, two-way ANOVA with Tukey’s test. (D-E) Immunoblot analysis of pRab10 and other proteins in RAW264.7 cells treated with PIKfyve inhibitors as well as bafilomycin A 1 (BafA1). A bar graph in E shows quantification of pRab10/total Rab10, as shown in D. Data represent mean ± SEM, n = 6, two-way ANOVA with Tukey’s test. (F-G) Immunoblot analysis of Rab10 in WT or E230 bone marrow-derived macrophages (BMDMs) treated with or without apilimod. A bar graph in G shows quantification of pRab10/total Rab10, as shown in F. Data represent mean ± SEM, n = 3, two-way ANOVA with Tukey’s test. (H-K) Immunoblot analysis of pRab10 and LC3B in RAW264.7 cells treated with apilimod or zymosan (zym) in addition to DPI. Bar graphs show quantifications of pRab10/total Rab10 upon treatment with apilimod (I) or zymosan (J) and that of LC3B-II relative to α-tubulin upon treatment with zymosan (K). Data represent mean ± SEM, n = 3, two-way ANOVA with Tukey’s test.

Article Snippet: RAW264.7 mouse macrophage cells (ECACC, RRID: CVCL_0493) and Lrrk2 knockout RAW264.7 cells (ATCC, RRID: CVCL_UL72) were routinely cultured on culture dishes for suspended cells (Sumitomo Bakelite Co.) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS).

Techniques: Western Blot, Knockdown, Derivative Assay

Journal: bioRxiv

Article Title: LRRK2 is activated by phosphatidylinositol 3-phosphate in conjunction with CASM

doi: 10.64898/2025.12.16.694573

Figure Lengend Snippet: (A) Confocal microscopic analysis of the colocalization of endogenous LRRK2 (red) and p40PX-EGFP (indicating PI3P, green) in RAW264.7 cells treated with PIKfyve inhibitors or CQ. Nuclei were stained with DAPI (cyan). Closed filled arrowheads indicate LRRK2-PI3P double-positive structures, and closed blank arrowheads indicate PI3P-positive but LRRK2-negative structures. (B) Colocalization of Thr73-phosphorylated Rab10 (pRab10, red) and p40PX-EGFP (PI3P, green) in RAW264.7 cells treated with PIKfyve inhibitors or CQ. Nuclei were stained with DAPI (cyan). Closed filled arrowheads indicate pRab10-PI3P double-positive vesicular structures, and closed blank arrowheads indicate PI3P-positive but pRab10-negative structures. Scale bars in A and B = 10 μm. (C-D) Quantitative analysis of the colocalization of PI3P and LRRK2 (C) or PI3P and pRab10 (D), as shown in A and B respectively, using Pearson’s correlation coefficient. (E) Colocalization of endogenous LRRK2 (red), LAMP1 (magenta), and p40PX-EGFP (PI3P, green) in RAW264.7 cells treated with apilimod or CQ. Closed filled arrowheads indicate triple-positive vesicular structures. Scale bars = 10 μm.

Article Snippet: RAW264.7 mouse macrophage cells (ECACC, RRID: CVCL_0493) and Lrrk2 knockout RAW264.7 cells (ATCC, RRID: CVCL_UL72) were routinely cultured on culture dishes for suspended cells (Sumitomo Bakelite Co.) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS).

Techniques: Staining

Journal: bioRxiv

Article Title: LRRK2 is activated by phosphatidylinositol 3-phosphate in conjunction with CASM

doi: 10.64898/2025.12.16.694573

Figure Lengend Snippet: (A) Protein-lipid overlay assays for the detection of the interaction of full-length (left) or N-terminally truncated (970-2527aa, right) LRRK2 with various phosphoinositides. The names of phospholipids corresponding to each spot are shown on the right. Representative pictures from three independent experiments are shown. (B) Structure of the positively charged cluster in the N-terminal region of LRRK2, predicted to interact with PI3P. Side chains of candidate residues predicted to face the membrane are indicated by red arrows. (C) Analysis of apilimod-induced activation of mutant LRRK2 harboring alanine substitutions for the N-terminal positively charged amino acids in HEK293 cells. (D) Quantitative analysis of Thr73-phosphorylated Rab10 relative to total Rab10, as shown in C. Data represent mean ± SEM, n = 4, two-way ANOVA with Tukey’s test. (E) Protein-lipid overlay assays for the detection of interaction of wild-type LRRK2 (left) or PentaA LRRK2 (right) with phosphoinositides. The arrangement of phospholipids is the same as that shown in A. Representative pictures from three independent experiments are shown. (F) Model of LRRK2 activation via PI3P, multiple Rabs and CASM effector GABARAP on membranes. N-terminal region of LRRK2 functions as a hot spot of interaction with these molecules, mediating LRRK2 activation.

Article Snippet: RAW264.7 mouse macrophage cells (ECACC, RRID: CVCL_0493) and Lrrk2 knockout RAW264.7 cells (ATCC, RRID: CVCL_UL72) were routinely cultured on culture dishes for suspended cells (Sumitomo Bakelite Co.) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS).

Techniques: Membrane, Activation Assay, Mutagenesis

Journal: NPJ Parkinson's Disease

Article Title: LRRK2 p.G2385R and p.R1628P variants in a multi-ethnic Asian Parkinson’s Cohort: epidemiology and clinical insights

doi: 10.1038/s41531-025-01166-x

Figure Lengend Snippet: Frequency of LRRK2 p.G2385R and p.R1628P among PD patients acrossdiverse ancestral groups in Malaysia (n = 3058 index cases).

Article Snippet: Genotyping of LRRK2 p.G2385R ( NM_198578.4 ; c.7153 G > A) and p.R1628P ( NM_198578.4 ; c.4883 G > C) variants was performed using TaqMan allelic discrimination assays (C__63498855_10 and C__63497592_10 respectively) using the StepOnePlusTM Real-Time PCR (RTPCR) Systems platform following the manufacturer’s instructions, and genotype calling with the StepOneTM software.

Techniques: