Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: Antibodies, chemicals, and plasmids used in this study.

Article Snippet: LAMTOR1 AAV9 shRNA Virus , VectorBuilder , Custom.

Techniques: Virus, Control, shRNA, Plasmid Preparation, Recombinant

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: LAMTOR1 regulates lysosomal positioning in dendrites of cultured hippocampal neurons. See also – . (A) Images of cultured hippocampal neurons stained for LAMP2 (red). Neurons were infected with an shRNA AAV directed against LAMTOR1 (shLT1) or a scrambled shRNA control (shSc) with GFP co-expression before being processed for immunofluorescence assay and imaging. Insets: enlarged dendrites. Scale bar: 20 μm, and 5 μm in insets. (B) Quantification of lysosome distribution along the dendrites, as shown in (A) . Results are Means ± SEM of 17–18 neurons from 6 independent experiments, * p < 0.05, *** p < 0.001, as compared with shSc, two-way ANOVA with Sidak’s post-test. (C) Images of cultured hippocampal neurons stained for cathepsin B (red). Neurons were infected as described in (A) before being processed for immunofluorescence assay and imaging. Insets: enlarged dendrites. Scale bar: 20 μm, and 5 μm in insets. (D) Quantification of lysosome distribution along the dendrites, as shown in (C) . Results are Means ± SEM of 14–17 neurons from 3 independent experiments, * p < 0.05, *** p < 0.001, as compared with shSc, two-way ANOVA with Sidak’s post-test.

Article Snippet: LAMTOR1 AAV9 shRNA Virus , VectorBuilder , Custom.

Techniques: Cell Culture, Staining, Infection, shRNA, Control, Expressing, Immunofluorescence, Imaging

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: The effects of LAMTOR1 KD on lysosomal positioning depend on TRMPL1-mediated Ca 2+ release. (A) The TRPML1 inhibitor, ML-SI1, blocked the effects of LAMTOR1 KD on dendritic lysosomal positioning. Cultured hippocampal neurons were infected with AAV expressing either a LAMTOR1 shRNA (shLT1) or a scrambled shRNA (shSc); they were then treated with vehicle control or ML-SI1 before being processed for LAMP2 staining. Results are Means ± SEM of 6–18 neurons from 3 to 6 independent experiments, ** p < 0.01, *** p < 0.001, as compared with shSc (green line compared to red line), ## p < 0.01, ### p < 0.001, as compared with shLAMTOR1 (purple line compared to green line), two-way ANOVA with Tukey’s post-test. (B) TRPML1 KD blocked the effects of LAMTOR1 KD on dendritic lysosomal positioning. Cultured hippocampal neurons were infected with shLAMTOR1 (shLT1) or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were then being processed for LAMP2 staining. Results are Means ± SEM of 8–9 neurons from 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with shSc/Accell siControl (green line compared to red line), # p < 0.05, ### p < 0.001, as compared with shLAMTOR1/Accell siControl (purple line compared to green line), two-way ANOVA with Tukey’s post-test. Note that the data for shSc and shLT1 in (A) are the same as those shown in . (C) Representative images of with shLT1 or shSc AAV and Accell TRPML1 siRNA or control siRNA and stained with LAMP2 and GFP. Scale bar: 20 μm.

Article Snippet: LAMTOR1 AAV9 shRNA Virus , VectorBuilder , Custom.

Techniques: Cell Culture, Infection, Expressing, shRNA, Control, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: TRPML1 activation by ML-SA1 or blockade of LAMTOR1/TRPML1 inhibitory interaction reproduced the effects of LAMTOR1 KD on lysosome positioning in a dynein-dependent manner. (A) The TRPML1 activator ML-SA1 induced lysosomal accumulation in proximal dendrites. Neurons were infected with shLAMTOR1 (shLT1) or shSc AAV and treated with either a vehicle control or ML-SA1 before being processed for LAMP2 staining. Results are Means ± SEM of 12–18 neurons from 3 to 6 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with shSc. (B) Disruption of LAMTOR1-TRPML1 interaction induced lysosomal accumulation in proximal dendrites. Neurons were infected with shLAMTOR1 (shLT1) or shSc AAV, treated with TAT or TAT-2031 (10 μM) for 3 h, before being processed for LAMP2 staining. Results are Means ± SEM of 14–17 neurons from 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with shSc+TAT. (C) Inhibition of dynein blocked LAMTOR KD induced lysosomal accumulation. Neurons were infected with shLAMTOR1 (shLT1) or shSc AAV, treated with vehicle control or Ciliobrevin D (CilioD), before being processed for LAMP2 staining. Results are Means ± SEM of 10–18 neurons from 3 independent experiments, ** p < 0.01, *** p < 0.001, as compared with shSc, ### p < 0.001 compared with shLAMTOR1. P -values were derived by two-way ANOVA with Tukey’s post-test. Note that the data for shSc and shLAMTOR1 in (A,C) are the same as those shown in . (D) Diagram showing that deletion of LAMTOR1 or disruption of LAMTOR1/TRPML1 interaction (red X) enhances dynein-mediated retrograde lysosomal trafficking and proximal dendrite positioning.

Article Snippet: LAMTOR1 AAV9 shRNA Virus , VectorBuilder , Custom.

Techniques: Activation Assay, Infection, Control, Staining, Disruption, Inhibition, Derivative Assay