Lysosomal TFEB-TRPML1 Axis in Astrocytes Modulates Depressive-like Behaviors

Abstract

Lysosomes are important cellular structures for human health as centers for recycling, signaling, metabolism and stress adaptation. However, the potential role of lysosomes in stress-related emotions has long been overlooked. Here, it is found that lysosomal morphology in astrocytes is altered in the medial prefrontal cortex (mPFC) of susceptible mice after chronic social defeat stress. A screen of lysosome-related genes revealed that the expression of the mucolipin 1 gene (Mcoln1; protein: mucolipin TRP channel 1) is decreased in susceptible mice and depressed patients. Astrocyte-specific knockout of mucolipin TRP channel 1 (TRPML1) induced depressive-like behaviors by inhibiting lysosomal exocytosis-mediated adenosine 5'-triphosphate (ATP) release. Furthermore, this stress response of astrocytic lysosomes is mediated by the transcription factor EB (TFEB), and overexpression of TRPML1 rescued depressive-like behaviors induced by astrocyte-specific knockout of TFEB. Collectively, these findings reveal a lysosomal stress-sensing signaling pathway contributing to the development of depression and identify the lysosome as a potential target organelle for antidepressants.

Keywords: ATP; astrocytes; depressive‐like behaviors; lysosomes; mucolipin TRP channel 1; transcription factor EB.

Figure 1

Lysosomal morphology and TRPML1 levels in mPFC astrocytes are altered following CSDS. A,B) Transmission electron microscopy images of lysosomes in mPFC astrocytes and neurons from Ctrl, Sus, and Res mice following the CSDS paradigm. The black arrows indicate lysosomes (red marks) in the cell. Scale bars, 2 µm (overview) and 500 nm (inset). C) Quantification of lysosomal size in the mPFC of Ctrl, Sus and Res mice (Astrocyte: n = 16 cells from 4 Ctrl mice, n = 22 cells from 6 Sus mice, n = 14 cells from 5 Res mice, p = 0.027; Neuron: n = 25 cells from 4 Ctrl mice, n = 10 cells from 6 Sus mice, n = 14 cells from 5 Res mice). D) Representative 3D‐reconstructed images of lysosomes immunostained with Lamp1 in mPFC astrocytes of Aldh1l1‐EGFP mice after the CSDS paradigm (EGFP, gray; Lamp1, red). Scale bars, 5 µm (overview) and 500 nm (inset). E) Quantification of lysosomal size in mPFC astrocytes from Aldh1l1‐EGFP mice after the CSDS paradigm (n = 11 cells from 3 Ctrl mice, n = 19 cells from 3 Sus mice, n = 7 cells from 3 Res mice, p = 0.0302). F) mRNA levels of lysosome‐related genes in the mPFC of Ctrl, Sus and Res mice (n = 9–10, each sample was analyzed in duplicate, Lgals3: p = 0.0143; Mcoln1: p = 0.0238; Atg16l2: p = 0.0206). G) MCOLN1 mRNA levels in the peripheral blood of MDD patients and healthy controls (n = 18–22, each sample was analyzed in duplicate, p = 0.0341). H) Mcoln1 mRNA levels in the peripheral blood of Ctrl, Sus and Res mice (n = 12–16, each sample was analyzed in duplicate, p = 0.015). I) Correlation between Mcoln1 mRNA levels and SI ratio. p = 0.0334. J,K) Protein levels of TRPML1 in the mPFC of Ctrl, Sus and Res mice (n = 5–7, p = 0.0112). L,M) Western blotting analysis of TRPML1 protein levels in cultured astrocytes and neurons treated with DXMS (1 µM) for 0, 4, 12, 24, or 48 h (n = 5–6, p = 0.023 and p = 0.0017). One way ANOVA followed by Dunnett's post‐hot test or Kruskal‐Wallis test followed by Dunn's multiple comparisons test C,E,F,H,K and M); Mann Whitney test G) and correlations evaluated with the Pearson correlation coefficient I). All data are presented as the mean ± SEM. n.s., not significant; *p <0.05, **p <0.01.

Figure 2

Selective deletion of astrocytic Mcoln1 induces depressive‐like behaviors in mice. A) Generation of astrocyte‐specific TRPML1 knockout mice (TRPML1 AcKO) and experimental timeline. B) Western blotting analysis of TRPML1 expression in the mPFC of TRPML1 AcKO and Ctrl mice (n = 3, p = 0.0425). C–F) Behavioral performances of TRPML1 AcKO and Ctrl mice in FST C) (n = 11–12, p = 0.0383), SI test D) (n = 8–11, p = 0.0009), EPM E) and OFT F) (n = 11–12). G) Generation of neuron‐specific TRPML1 knockout mice (TRPML1 NcKO) and experimental timeline. H) Western blotting analysis of TRPML1 expression in the mPFC of TRPML1 NcKO and Ctrl mice (n = 5–6, p = 0.043). I–L) Behavioral performances of TRPML1 NcKO and Ctrl mice in FST I) (n = 13), SI test J) (n = 12), EPM K), and OFT L) (n = 13). Two‐tailed unpaired Student's t test B,C,E,F,H,I,K and L); two‐way ANOVA followed by Sidak's multiple comparisons test D and J). All data are presented as the mean ± SEM. n.s., not significant; *p <0.05, ***p <0.001.

Figure 3

Astrocytic TRPML1 in the mPFC modulates depressive‐like behaviors. A) Schematic of the AAV vectors and design of the studies performed to analyze the behavioral performances of Mcoln1^flox/flox mice infected with AAV‐gfaABC1D‐EGFP‐iCre or AAV‐gfaABC1D‐EGFP virus. B) Representative confocal images of AAV‐gfaABC1D‐EGFP‐iCre expression in astrocytes of the mPFC. Scale bars = 500 µm (left), 50 µm (right). C) Western blotting images and quantitative analysis of TRPML1 expression in the mPFC of GFAP/PFC^△ML1 and EGFP mice (n = 5, p = 0.025). D–G) Behavioral performances of GFAP/PFC^△ML1 and EGFP mice in FST D) (n = 14–16, p = 0.0058), SI test E) (n = 13–15, p = 0.0105), EPM F) and OFT G) (n = 14–16). H) Schematic of the AAV vectors and design of the studies performed to analyze the behavioral performances of TRPML1 AcKO mice infected with AAV‐DIO‐Mcoln1‐3×Flag‐EGFP or AAV‐DIO‐EGFP virus. I) Representative confocal images of AAV‐DIO‐Mcoln1‐3×Flag‐EGFP expression in mPFC astrocytes. Scale bars = 500 µm (left), 50 µm (right). J) Western blotting images and quantitative analysis of TRPML1 expression in the mPFC of TRPML1 AcKO mice infected with AAV‐DIO‐Mcoln1‐3×Flag‐EGFP or AAV‐DIO‐EGFP virus (n = 4, p = 0.0286). K–N) Behavioral performances of TRPML1 AcKO mice infected with AAV‐DIO‐Mcoln1‐3×Flag‐EGFP or AAV‐DIO‐EGFP virus in FST K) (n = 9–10, p = 0.0013, p = 0.0283), SI test L) (n = 9–11, p = 0.0207), EPM M), and OFT N) (n = 9–11). Two‐tailed unpaired Student's t test C,D,F,G); Mann Whitney test J); one way ANOVA followed by Tukey's multiple comparisons test K,M and N); two‐way ANOVA followed by Sidak's multiple comparisons test E and L). All data are presented as the mean ± SEM. n.s., not significant; *p <0.05, **p <0.01.

Figure 4

Knockdown of astrocytic TRPML1 decreases lysosomal exocytosis‐mediated ATP release in the mPFC. A,B) TEM images A) and quantitative analysis B) of lysosomes in mPFC astrocytes from TRPML1 AcKO and Ctrl mice (n = 11 cells from 2 TRPML1 AcKO mice, n = 12 cells from 2 Ctrl mice, p = 0.0089 and p = 0.0391). The black arrows indicate lysosomes (red marks) in the cell. Scale bars, 2 µm (overview) and 500 nm (inset). C) Design of the procedure for knockdown of astrocytic TRPML1 in vitro. D) Western blotting analysis of TRPML1 expression in cultured Mcoln1^flox/flox astrocytes after infection with the Cre virus (n = 5, p = 0.0218). E) Lamp1 surface immunostaining in non‐permeabilized astrocytes. Scale bars, 10 µm. F‐H) Measurements of AP, β‐Gal activity and ATP levels in the medium of primary cultured astrocytes (n = 6). p = 0.0131 F), p = 0.0002 G) and p = 0.0002 H). I) Measurements of AP activity and ATP concentrations in the medium of primary cultured astrocytes treated with ML‐SA1 (50 µM, 1 h) and BAPTA‐AM (500 nM, 20 min) (n = 7–8). p = 0.0096 and p = 0.0119 (left), p = 0.0104 and p = 0.0199 (right). J) Measurements of ATP concentrations in the medium of primary cultured astrocytes treated with ML‐SA1 and Vacuolin‐1 (10 µM, 1 h) (n = 6, p = 0.0249 and p = 0.0161). K) Measurements of ATP concentrations in the medium of primary cultured astrocytes treated with ML‐SI3 (25 µM, 10 min) (n = 6, p <0.0001). L) ATP levels in the ACSF of isolated mPFC slices from TRPML1 AcKO mice (n = 6, p = 0.0313). M) Schematic of the AAV vectors engineered to express the ATP sensor and design of the fiber photometry experiment. N) Representative images of ATP sensor expression in mPFC astrocytes. Scale bars = 500 µm (left), 50 µm (right). O) Representative heat maps of z‐score changes over all trials in individual mice. P,Q) Time course of the average ATP transient z‐score event in the forced interaction test (FIT) (p) and quantification of the average peak z score during the FIT (Q) (n = 5–7, p <0.0001). R) Behavioral performances of C57BL/6J mice in the FST after infusion of ML‐SI3 into the mPFC (n = 12, p = 0.0281). S) Behavioral performances of TRPML1 AcKO mice treated with ATP in FST (n = 11–14, p = 0.0229 and p = 0.0363). Two‐tailed unpaired Student's t test B,D,F,G,H,K and R); Wilcoxon matched‐pairs signed rank test L); one way ANOVA followed by Tukey's multiple comparisons test I,J and S); two‐way ANOVA followed by Sidak's multiple comparisons test Q). All data are presented as the mean ± SEM. n.s., not significant; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.

Figure 5

Astrocytic TFEB mediates ATP release in a TRPML1‐dependent manner. A) TFEB protein levels in the mPFC of Ctrl, Sus and Res mice (n = 5–6, p = 0.0014). B) Western blotting analysis of TFEB protein levels in cultured astrocytes and neurons treated with DXMS (1 µM) for 0, 4, 12, 24, or 48 h (n = 5–6, p <0.0001, p = 0.0005 and p <0.0001). C) Western blotting analysis of astrocytic TFEB levels in the nuclear and cytosolic fractions. D) Representative immunofluorescence images and quantification of TFEB nuclear translocation in cultured astrocytes treated with DXMS (n = 26–34 cells, p = 0.0248, p = 0.0287, p = 0.0494 and p = 0.0077). TFEB, red; GFAP, green; DAPI, blue. Scale bars, 10 µm. E) Western blotting analysis of pTFEB (Ser 211) levels in cultured astrocytes treated with DXMS (n = 5, p = 0.0125, p = 0.0012, p = 0.0387 and p = 0.0131). F) ChIP‐PCR assay using primary cultured astrocytes (n = 4, p = 0.0286). G) Western blotting analysis of TFEB and TRPML1 protein levels in cultured Tfeb^flox/flox astrocytes infected with the Cre virus (n = 5, p = 0.0248). H) Representative images of Lysotracker staining. Scale bars, 5 µm. I) Quantification of lysosomal number in TFEB knockdown astrocytes (n = 44–48 cells, p <0.0001 and p = 0.0435). J) ATP levels in the medium of TFEB knockdown astrocytes treated with the TRPML1 agonist ML‐SA1 (50 µM) or vehicle for 1 h (n = 5–6, p = 0.0034 and P <0.0001). K,L) Western blotting analysis of TFEB and TRPML1 protein levels in 293T cells transfected with the TFEB plasmid (n = 6, p = 0.0028 and p = 0.0008). M) ATP levels in the medium of primary cultured astrocytes treated with the TFEB agonist C1 (1 µM, 12 h) and the TRPML1 antagonist ML‐SI3 (n = 6, p = 0.0013 and p = 0.0002). One way ANOVA followed by Dunnett's post‐hot test A,B and E); Kruskal‐Wallis test followed by Dunn's multiple comparisons test D); two‐tailed unpaired Student's t test G and L) and Mann Whitney test F and I); one way ANOVA followed by Tukey's multiple comparisons test J and M). All data are presented as the mean ± SEM. n.s., not significant; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.

Figure 6

Overexpression of TRPML1 in mPFC astrocytes rescues depressive‐like behaviors in TFEB cKO mice. A) Generation of astrocyte‐specific TFEB knockout mice (TFEB cKO) and experimental timeline. B) Analysis of astrocytes isolated by FACS from the mPFC. C) Simple Western blotting analysis of astrocytic TFEB expression in the mPFC of TFEB cKO and Ctrl mice (n = 4, p = 0.0286). D–G) Behavioral performances of TFEB cKO and Ctrl mice in FST D) (n = 7–9, p = 0.0124), SI test E) (n = 7–8, p = 0.011), OFT F), and EPM G) (n = 7‐9). H) Representative images of AAV‐gfaABC1D‐EGFP‐iCre expression in mPFC astrocytes. Scale bars, left, 500 µm; right, 50 µm. I) Western blotting analysis of TRPML1 expression in the mPFC of GFAP/PFC^△EB and EGFP mice (n = 4, p = 0.0085). J‐K) Behavioral performances of GFAP/PFC^△EB and EGFP mice in FST J) and SI test K) (n = 9–10). p = 0.0423 J) and p = 0.0004 K). L) Representative images of AAV‐CMV‐Mcoln1‐3×Flag‐EGFP expression in mPFC astrocytes. Scale bars, left, 500 µm; right, 50 µm. M) Western blotting analysis of TRPML1 expression in the mPFC of TFEB cKO mice injected with AAV‐CMV‐Mcoln1‐3×Flag‐EGFP or AAV‐DIO‐EGFP virus (n = 5–6, P = 0.0178). N,O) Behavioral performances of TFEB cKO mice infected with AAV‐CMV‐Mcoln1‐3×Flag‐EGFP or AAV‐DIO‐EGFP virus in FST N) (n = 8–10, p = 0.0392 and p = 0.0481) and SI test O) (n = 7–8, p = 0.0189). Mann Whitney test C) and two‐tailed unpaired Student's t test D,F,G,I,J and M); one way ANOVA followed by Tukey's multiple comparisons test N); two‐way ANOVA followed by Sidak's multiple comparisons test E,K and O). All data are presented as the mean ± SEM. n.s., not significant; *p <0.05, **p <0.01, ***p <0.001.