Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release

Abstract

Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca(2+) homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca(2+) channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca(2+) release, measured using a genetically encoded Ca(2+) indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1's expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca(2+)-dependent lysosomal trafficking.

Figure 1. Activation of TRPML1 by small molecule synthetic agonists

(a) ML-SA1 induced increases in cytosolic [Ca²⁺], as measured by the Fura-2 ratio (F₃₄₀/F₃₈₀) in HEK293T cells expressing TRPML1-L¹⁵L/AA-L⁵⁷⁷L/AA (ML1-4A) at low pH (pH 4.6) external solution. SF-51 (10 μM) induced much smaller responses in the same cells. (b) SF-51 (30 μM) induced increases in cytosolic [Ca²⁺] in ML1-4A-expressing HEK293T cells at low pH external solution. (c) Chemical structures of SF-51 and ML-SA1. The difference between SF-51 and ML-SA1 is highlighted in red. (d) Activation of whole-cell currents by SF-51 and ML-SA1 in ML1-4A-expressing HEK293T cells. ML1-4A-mediated current (I_ML1-4A) was elicited by repeated voltage ramps (-140 to +140 mV; 400 ms) with a 4-s interval between ramps. Only a portion of the voltage protocol is shown; holding potential = 0 mV. (e) Activation of whole-endolysosome I_ML1 by ML-SA1 and PI(3,5)P₂. Enlarged vacuoles/endolysosomes were isolated from vacuolin-treated HEK293 cells stably-expressing ML1-4A. ML-SA1 and PI(3,5)P₂ were applied to the cytosolic sides of the isolated vacuoles. The pipette (luminal) solution was a standard external (Tyrode’s) solution adjusted to pH 4.6; the bath (internal/cytoplasmic) solution was a K⁺-based solution (140 mM K⁺-gluconate). Note that the inward current indicates cations flowing out of the endolysosome. (f) Synergistic activation of whole-endolysosome I_ML1 by ML-SA1 and PI(3,5)P₂ in human fibroblasts. I_ML1 was activated by PI(3,5)P₂ (1 μM) and ML-SA1 (10 μM); co-application of PI(3,5)P₂ and ML-SA1 further increased I_ML1. (g) ML-SA1 activated endogenous whole-endolysosome ML-like currents (I_ML-L) in CHO cells. (h) ML-SA1 activated whole-endolysosome I_ML-L in mouse primary macrophages. (i) ML-SA1 activated whole-endolysosome I_ML1 in WT (ML1^+/+), but not ML4 (ML1^−/−) human fibroblasts.

Figure 2. A lysosome-targeted genetically-encoded Ca²⁺ indicator

(a) GCaMP3-TRPML1 (GCaMP3-ML1) fusion strategy. GCaMP3 is fused to the N-terminus of TRPML1. (b) Co-localization of GCaMP3-ML1 fluorescence with Lamp-1 in HEK293T cells expressing both GCaMP3-ML1 and Lamp-1-mCherry. Scale bar = 5 μm. (c) Co-localization of GCaMP3-ML1 fluorescence with LysoTracker in GCaMP3-ML1-expressing HEK293T cells. Scale bar = 2 μm. (d) Preferential detection of Ca²⁺ release from lysosome stores by GCaMP3-ML1 in GCaMP3-ML1-transfected Cos-1 cells. ER and lysosome Ca²⁺ releases were triggered by thapsigargin (TG, 2 μM) and Glycyl-L-phenylalanine 2-naphthylamide (GPN, 200 μM), respectively, in low (nominally-free Ca²⁺ + 1 mM EGTA; free [Ca²⁺] estimated to be < 10 nM) external [Ca²⁺]. All cells responded to GPN; only 1 out 4 cells responded to TG.

Figure 3. ML-SA1 induces TRPML1-mediated Ca²⁺ release from lysosomes

(a) ML-SA1 (20 μM) induced rapid increases in GCaMP3 fluorescence (measured as change of GCaMP3 fluorescence ΔF over basal fluorescence F₀; ΔF/F₀) under low (< 10 nM) external Ca²⁺ in CHO cells transfected with GCaMP3-ML1. Subsequent application of GPN (200 μM) induced smaller responses. Maximal responses were induced by ionomycin (1 μM) application. (b) Representative micrographs from panel (a) to show the changes of GCaMP3 fluorescence upon bath application of ML-SA1 and ionomycin to GCaMP3-ML1-transfected CHO cells. Scale bar = 5 μm. (c) ML-SA1 failed to induce significant Ca²⁺ increases in CHO cells transfected with GCaMP3-ML1-KK (non-conducting pore mutation). (d) Basal GCaMP3 fluorescence (normalized to the maximal ionomycin-induced fluorescence) of GCaMP3-ML1-KK and GCaMP3-ML1. (e) GPN (200 μM) pretreatment abolished ML-SA1-induced responses in GCaMP3-ML1-expressing CHO cells. (f) Repetitive applications of ML-SA1 (20 μM) induced little or no responses in GCaMP3-ML1-expressing CHO cells. (g) Repetitive applications of GPN (200 μM) induced smaller GCaMP3 responses in GCaMP3-ML1-expressing CHO cells. (h) ML-SA1 failed to further increase GCaMP3 fluorescence in GCaMP3-ML1-expressing CHO cells in the presence of GPN (200 μM). (i) ML-SA1 induced small responses in GCaMP3-ML1-expressing CHO cells that had received an application of GPN. (j) Bafilomycin A1 (Baf-A1, 500 nM) pretreatment abolished ML-SA1-induced Ca²⁺ response in GCaMP3-ML1-expressing CHO cells. (k) ML-SA1-induced responses in CHO cells transfected with GCaMP3-ML1-KK (non-conducting pore mutation), and GCaMP3-ML1-transfected cells pretreated with Baf-A1, GPN, or TG. For panels d and k, the responses were averaged for 40-100 transfected cells from at least 3 independent experiments; data are presented as the mean ± SEM. Statistical comparisons were made using analysis of variance: * P <0.05.

Figure 4. TRPML1-mediated lysosomal Ca²⁺ release, but not lysosome Ca²⁺ store, is reduced in NPC cells

(a, b) ML-SA1-induced lysosomal Ca²⁺ release in GCaMP3-ML1-transfected WT (a) and NPC (b) CHO cells. (c) Co-localization of GCaMP3-ML1 fluorescence with Lamp-1 in NPC CHO cells doubly-transfected with GCaMP3-ML1 and Lamp-1-mCherry. Scale bar = 5 μm. (d) Comparable expression levels of GCaMP3-ML1 in WT and NPC CHO cells. Expression level of GCaMP3-ML1 was estimated by the maximal GCaMP3 fluorescence signal induced by ionomycin (1 μM). (e) Comparable GPN-induced GCaMP3 responses in WT, NPC, or U18666A-treated GCaMP3-ML1-transfected CHO cells. (f) NH₄Cl (10 mM) and GPN (200 μM) induced comparable levels of Ca²⁺ increases (measured with Fura-2 ratios) in WT, NPC, and U18666A-treated CHO cells. (g) NH₄Cl (10 mM) and GPN (200 μM) induced comparable levels of Fura-2 Ca²⁺ responses in WT and NPC human fibroblasts. (h) TRPML1-mediated, but not GPN-induced lysosomal Ca²⁺ release, was reduced in GCaMP3-ML1-transfected NPC human fibroblasts compared to WT cells. (i) WT CHO cells were treated with U18666A (2 μg/ml) for 16 h, and then subjected to filipin staining. Scale bar = 10 μm. (j) Sphingomyelins were stained with Lysenin (0.1 μg/ml) in WT CHO cells, WT CHO cells treated with U18666A (2 μg /ml) for 16 h, and NPC CHO cells. Scale bar = 10 μm. (k) Ca²⁺ responses in GCaMP3-ML1-transfected WT CHO cells treated with U18666A (2 μg /ml) for 16-20 h. (l) ML-SA1-induced peak GCaMP3 responses were reduced in GCaMP3-ML1-transfected NPC or U18666A-treated WT CHO cells. (m, n) Endogenous TRPML1-mediated lysosomal Ca²⁺ release (measured with Fura-2 ratios) in WT (m) and NPC (n) CHO cells. (o) Average endogenous ML-SA1-induced lysosomal Ca²⁺ responses in WT and NPC CHO cells, and WT, NPC1^−/−, and ML1^−/− mouse macrophages. For panels d, e, f, g, h, k, l, and o, the results were averaged for 40-100 cells from at least 3 independent experiments; data are presented as the mean ± SEM. Statistical comparisons were made using analysis of variance: * P <0.05.

Figure 5. TRPML-mediated currents are reduced in the lysosomes of NPC cells

(a, b) Concentration-dependent activation of whole-endolysosome I_ML-L in WT (a) and NPC (b) human fibroblasts. ML-SA1 (3, 10, 25 μM) was applied to the cytosolic sides of the vacuoles isolated from human fibroblasts. (c) Average current densities (pA/pF) of I_ML-L were reduced in NPC fibroblasts compared to WT cells. The current density was calculated from the current size (pA) normalized with the capacitance of the vacuole (pF). (d) RT-PCR analysis of TRPML1-3 mRNA expression in WT and NPC human fibroblasts, and NPC1 ^−/− mouse macrophages. Expression levels were normalized to that of GAPDH (human fibroblast) and L32 (mouse macrophage). For panels c and d, data are presented as the mean ± SEM; the n numbers are in parentheses. Statistical comparisons were made using analysis of variance: * P <0.05.

Figure 6. Regulation of TRPML1 by sphingomyelins and sphingomyelinases

(a) SF-51-activated whole-cell I_ML1-4A was inhibited by sphingomyelins (SMs; 20 μM) at low extracellular pH in ML1-4A-expressing HEK293T cells. (b) Insensitivity of whole-cell I_ML1-Va to SMs (60 μM). (c) Lack of effect of ceramide (Cer, 20 μM) on whole-cell I_ML1-4A. (d) Summary of the effects of various sphingolipids on whole-cell I_ML1-4A in ML1-4A-expressing HEK293T cells. PC: phosphocholine. (e) SF-51-activated whole-cell I_ML1-4A was potentiated by sphingosine (Sph; 5 μM) at low external pH in ML1-4A-expressing HEK293T cells. (f) The effects of SMase (50 ng/ml) treatment on I_ML1-4A at low external pH. I_ML1-4A was elicited by repeated voltage ramps (− 140 to + 140 mV; 400 ms) with a 4-s interval between ramps. Current amplitudes at -140 mV were used to plot the time dependence. (g) Representative traces of I_ML1-4A before (black) and after (dark yellow) SMase treatment at two different time points, as shown in f. (h, i) SMase (50 ng/ml) treatment alone had no (6 out of 7 cells; see h for an example) or small (1 out of 7 cells; i) activation effect on basal whole-cell I_ML1-4A in ML1-4A-expressing HEK293T cells. (j) The effects of SMase (50 ng/ml) treatment on SF-51-activated I_ML1-4A at neutral external pH (pH 7.4). (k) The potentiation effect of SMase was more dramatic at pH 4.6, but abolished by heat inactivation of the enzyme activity. HI-SMase: heat-inactivated SMase. For panels d and k, data are presented as the mean ± SEM; the n numbers are in parentheses. Statistical comparisons were made using analysis of variance: * P <0.05.

Figure 7. SM accumulation in the lysosome reduces TRPML1-mediated Ca²⁺ release

(a,b) ML-SA1-induced lysosomal Ca²⁺ release was reduced in GCaMP3-ML1-transfected NPA fibroblasts (b) compared to WT fibroblasts (a). (c) Average ML-SA1 (20 μM) and GPN (200 μM)-induced responses in GCaMP3-ML1-transfected WT and NPA fibroblasts. (d) Desipramine (25 μM) treatment for 2-4 hrs reduced TRPML1-mediated lysosomal Ca²⁺ release responses (20 μM ML-SA1) in GCaMP3-ML1-transfected CHO cells. (e) Pretreatment of a protease inhibitor Leupeptin (25 μM) abolished the effects of Desipramine in ML-SA1-induced GCaMP3 responses. (f) Summary of the effects of Desipramine (25 μM) and Fluoxetine (25 μM) treatment for 2-4 hrs on TRPML1-mediated lysosomal Ca²⁺ release responses (20 μM ML-SA1) in GCaMP3-ML1-transfected CHO cells. (g) Desipramine and fluoxetine treatment for 2-4 hrs reduced endogenous lysosomal Ca²⁺ release responses (measured with Fura-2 ratios; 100 μM ML-SA1) in CHO cells. For panels c, f, and g, the results were averaged for 40-100 cells from at least 3 independent experiments; data are presented as the mean ± SEM. Statistical comparisons were made using analysis of variance: * P <0.05.

Figure 8. ML-SA1 and TRPML1 overexpression rescue the trafficking defects and reduce cholesterol accumulation in NPC cells

(a) A fluorescent analogue of lactosylceramide, BODIPY-LacCer, was mainly localized in the Golgi-like structures of WT macrophages after a pulse-chase for 1 h, but accumulated in the LEL-like vesicles of NPC macrophages. ML-SA1 (10 μM) treatment of NPC1^−/− macrophages for 12 h resulted in a primary Golgi-like localization for LacCer. Scale bar = 10 μm. (b) ML-SA1 reduced the number of LacCer puncta in NPC1^−/− macrophages. (c, d) ML-SA1 reduced unesterified cholesterol (filipin) staining in ML1, but not ML1-KK-expressing U18666A-treated WT CHO cells. Differential interference contrast (DIC) images are shown for comparison. Scale bar = 10 μm. (e) Unesterified cholesterol (filipin) staining in NPC CHO cells was partially reduced in cells expressing DsRed-aSMase. (f) Average filipin staining (intensity normalized to non-transfected control cells) in DsRed-aSMase-transfected cells. (g) Unesterified cholesterol (filipin) staining in NPC CHO cells was partially reduced in cells expressing ML1-Va-EGFP (ML1^Va). (h) Average filipin staining (intensity normalized to non-transfected control cells) in ML1^Va-transfected cells. Scale bar = 10 μm for panels a, c, e, and g. For panels b, d, f, and h, the results were averaged for multiple randomly-taken micrographs from at least 3 independent experiments. Data are presented as the mean ± SEM. Statistical comparisons were made using analysis of variance: * P <0.05.