Fig 2: LAMTOR1 KD-induced learning and memory impairment in mice is ameliorated by TRPML1 inhibition Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA-injected mice exhibited a significant deficit 24 h after training, which was reversed by ML-SI1 treatment (N = 7–18 mice).% freezing for different experimental groups in context memory (N = 7–19 mice).Model illustrating the proposed role of LAMTOR1-mediated inhibition of lysosomal Ca2+ release via TRPML1 in the regulation of lysosome motility in dendrites and synaptic plasticity. More lysosomes with lower pH move faster and longer distances in LAMTOR1 KD neurons, as compared to control neurons. LAMTOR1 interaction with TRPML1 inhibits its Ca2+ release and LAMTOR1 KD increased its Ca2+ release and dynein-dependent dendritic lysosome trafficking (?). LAMTOR1 KD-induced TRPML1 Ca2+ release also activates CaN, efficiently dephosphorylating GluA1 and targeting internalized AMPARs to lysosomes for degradation, thereby regulating synaptic plasticity (?). Figs 3B and 5A, and 8C were created with BioRender.com. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey’s post-test. *P < 0.05, ***P < 0.001, as compared to shSc; # P < 0.05, ### P < 0.001, as compared to shLAMTOR1. See also Appendix Fig S6G. Source data are available online for this figure.

Fig 3: Effects of LAMTOR1 KD and ML-SI1 treatment on GluA1 phosphorylation and levels in hippocampal neurons A–DLAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p-GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML-SI1 treatment. (A) Representative images of CA1 pyramidal neurons stained with anti-p-GluA1 S845 (magenta), anti-GluA1 (red), and anti-GFP (green) antibodies. Scale bar = 20 µm. (B, C, D) Quantitative analysis of the mean fluorescence intensity (MFI) of p-GluA1 S845 (B), GluA1 (C)-immunoreactivity, and the ratio of p-GluA1 to GluA1 (D) in hippocampal CA1 stratum radiatum (SR) 30 min after LFS. N = 4–6 slices from 4 to 6 mice.ERepresentative images of proximal dendrites of hippocampal neurons stained with anti-GluA1 (green), anti-LAMP2 (red), and anti-GFP (gray) antibodies. Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false-colored green and GFP false-colored gray to better show colocalization of GluA1 with LAMP2. Arrowheads indicate clearly colocalized puncta. Scale bar, 5 µm.FQuantitative analysis of the ratio of the number of GluA1/LAMP2 colocalized puncta to that of total lysosomes in (F) (n = 17–29 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. *P < 0.05, **P < 0.01 compared with shSc, # P < 0.05, ## P < 0.01 compared with shLAMTOR1; two-way ANOVA with Tukey’s post-test (B, C, D, F). A.U., Arbitrary unit. See also Appendix Fig S6. Source data are available online for this figure.