Fig 1: Overexpression of TRPML1 alleviates NAD and cognitive impairment in mice with Alzheimer-like phenotypes. (a) A volcano plot of differential gene expression analysis of the AD-related GSE5281 dataset. The abscissa represents -log10 (p value), the ordinate represents the logFC value, the red dots represent highly expressed genes, and the green dots represent poorly expressed genes. (b) Venn diagram of AD-related genes from the GeneCards and DisGeNet databases as well as differentially underexpressed genes. (c) A heat map of candidate gene expression in the AD-related GSE5281 dataset. The color scale from green to red indicates that the gene expression value is from low to high. (d) Expression of TRPML1 in AD samples (n = 10) and control samples (n = 13) in the AD-related GSE5281 dataset. (e) The protein expression of TRPML1 in the hippocampal tissues of APP/PS1 mice, determined by Western blot analysis. (f) The protein expression of TRPML1 in the hippocampal tissues of APP/PS1 mice in the absence or presence of TRPML1+/+, determined by Western blot analysis. (g) Escape latency of APP/PS1 mice in the absence or presence of TRPML1+/+. (h) The travel length of APP/PS1 mice in the absence or presence of TRPML1+/+. (i) Time crossing the platform of APP/PS1 mice in the absence or presence of TRPML1+/+. (j) Recognition indexes of APP/PS1 mice in the absence or presence of TRPML1+/+. (k) Expression of TRPML1 in primary neurons in response to Aβ1-42 alone or combined with oe-TRPML1/sh-TRPML1, determined by Western blot analysis. (l) Axonal length of neurons observed under an inverted microscope. Measurement data were described as the mean ± standard deviation. An unpaired t-test was used for comparison between the two groups. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post hoc test. ANOVA of repeated measurements with Bonferroni's post hoc test was applied for multigroup comparisons at different time points. n = 12 for mice in each group. ∗p < 0.05 vs. C57BL/6J WT mice or untreated primary hippocampal neurons; #p < 0.05 vs. APP/PS1 mice; ^p < 0.05 vs. primary hippocampal neurons treated with sh-NC+Aβ1-42; &p < 0.05 vs. primary hippocampal neurons treated with oe-NC+Aβ1-42. The cell experiment was repeated 3 times independently.
Fig 2: TRPML1 facilitates autophagosome-lysosome fusion through p62-mediated recruitment of dynein. (a) Changes of autolysosome formation in neurons detected by immunofluorescence staining. (b) Nuclear expression of BDNF in mouse hippocampal neurons identified by immunofluorescence staining. (c) Venn diagram flagging the intersection of TRPML1-related genes and AD-related genes in the GeneCards database and DisGeNet database. (d) Interaction network diagrams of the candidate genes, wherein the circle color from blue to orange represents the gene degree from small to large. (e) The interaction between TRPML1, p62, and dynein, confirmed by Co-IP. Neurons were treated with sh-p62 and/or oe-TRPML1 in the presence of Aβ1-42. (f) The extent of dynein phosphorylation in neurons. (g) Fusion of lysosomes and autophagosomes in neurons detected by immunofluorescence staining. Measurement data were described as the mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post hoc test. The cell experiment was repeated 3 times independently. ∗p < 0.05 vs. untreated HT22 cells; #p < 0.05 vs. cells treated with oe-NC+Aβ1-42; ^p < 0.05 vs. cells treated with sh-NC+Aβ1-42 or oe-TRPML1+Aβ1-42; &p < 0.05 vs. cells treated with oe-TRPML1+Aβ1-42 or ciliobrevin D+Aβ1-42.
Fig 3: Overexpression of TRPML1 protects axons by interacting with p62 in APP/PS1 transgenic mice. (a) Protein expression of BDNF and the ratio of p-TrkB/TrkB and p-CREB/CREB in hippocampal tissues of mice evaluated by Western blot analysis. (b) Dendrites and dendritic spines in hippocampal tissues of mice evaluated by Golgi-Cox staining. (c) Cell apoptosis in hippocampal tissues of mice, tested by TUNEL assay. (d) Senile plaques in hippocampal tissues of mice, evaluated by Congo red staining. Measurement data were described as the mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post hoc test. n = 12 for mice in each group. ∗p < 0.05 vs. C57BL/6J WT mice; #p < 0.05 vs. APP/PS1 mice; &p < 0.05 vs. APP/PS1/TRPML1+/+ mice.
Fig 4: Overexpression of TRPML1 promotes autophagosome-lysosome fusion and stimulates BDNF nuclear transport to block NAD in AD. (a) Protein expression of BDNF and the ratio of p-TrkB/TrkB and p-CREB/CREB in neurons, evaluated by Western blot analysis. (b) Changes of autolysosome formation in neurons identified by immunofluorescence staining. (c) Changes of BDNF nuclear translocation detected by immunofluorescence staining. (d) Length of axons in HT22 cells observed under an inverted microscope. Measurement data were described as the mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post hoc test. The cell experiment was repeated 3 times independently. ∗p < 0.05 vs. untreated HT22 cells; #p < 0.05 vs. cells treated with Aβ1-42; &p < 0.05 vs. cells treated with oe-NC+Aβ1-42. ^p < 0.05 vs. cells treated with oe-TRPML1+Aβ1-42.
Fig 5: TRPML1/p62 mediates the BDNF/TrkB signaling pathway and axonal development in vitro. (a) Interaction between TRPML1 and p62 assessed by FRET. (b) Protein expression of BDNF and the ratio of p-TrkB/TrkB and p-CREB/CREB in neurons, evaluated by Western blot analysis. (c) BDNF nuclear translocation in neurons with alternative transfection detected by immunofluorescence staining. (d) Length of axons in neurons observed under an inverted microscope. Measurement data were described as the mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post hoc test. The cell experiment was repeated 3 times independently. ∗p < 0.05 vs. untreated HT22 cells; ^p < 0.05 vs. cells treated with sh-NC; #p < 0.05 vs. cells treated with oe-NC; &p < 0.05 vs. cells treated with oe-TRPML1.
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