Increased expression or activation of TRPML1 reduces hepatic storage of toxic Z alpha-1 antitrypsin

Abstract

Mutant Z alpha-1 antitrypsin (ATZ) accumulates in globules in the liver and is the prototype of proteotoxic hepatic disease. Therapeutic strategies aiming at clearance of polymeric ATZ are needed. Transient receptor potential mucolipin-1 (TRPML1) is a lysosomal Ca²⁺ channel that maintains lysosomal homeostasis. In this study, we show that by increasing lysosomal exocytosis, TRPML1 gene transfer or small-molecule-mediated activation of TRPML1 reduces hepatic ATZ globules and fibrosis in PiZ transgenic mice that express the human ATZ. ATZ globule clearance induced by TRPML1 occurred without increase in autophagy or nuclear translocation of TFEB. Our results show that targeting TRPML1 and lysosomal exocytosis is a novel approach for treatment of the liver disease due to ATZ and potentially other diseases due to proteotoxic liver storage.

Keywords: TRPML1; alpha-1 antitrypsin; alpha-1 antitrypsin deficiency; lysosomal exocytosis; mucolipin-1.

Graphical abstract

Figure 1

TRPML1 gene transfer in PiZ livers reduces ATZ accumulation (A) Schematic representation of AAV8 vectors injected in PiZ mice. (B) RT-PCR analysis for TRPML1 expression in livers of wild-type mice injected with AAV-GFP or AAV-TRPML1 (AAV-GFP, n = 7; AAV-TRPML1, n = 9). Human TRPML1 expression was normalized on murine Trpml1 mRNA. (C) Representative immunofluorescence staining for GFP, TRPML1, and TFEB in livers of PiZ mice injected with AAV vectors and harvested at 12 weeks post injection (n = 9 per group). Representative PAS-D (D) and Sirius Red (E) staining of livers of PiZ mice harvested 12 weeks after AAV injections and their corresponding quantifications (n = 9 per group). (F) Representative immunofluorescence staining for ATZ polymers in livers of PiZ mice injected with AAV vectors and harvested at 12 weeks post-injection. (G) Immunoblots for soluble and insoluble fractions on livers of PiZ mice injected with AAV-GFP or AAV-TRPML1 and their corresponding quantifications (AAV-GFP, n = 8; AAV-TRPML1, n = 9). GAPDH and H3 were used for normalization. AAV-GFP and AAV-TRPML1 are on the same gel but not contiguous. (H) Changes in percentages of serum total AAT (n = 9 per group) or polymeric ATZ (AAV-GFP, n = 6; AAV-TRPML1, n = 5; AAV-TFEB, n = 6) in PiZ mice after AAV injections. Data are shown as mean ± standard error. Student’s t test: ∗p value < 0.05; ∗∗p value < 0.01; ∗∗∗p value < 0.001. A.U., arbitrary units; p.i., post-injection.

Figure 2

Increased lysosomal exocytosis in PiZ livers after TRPML1 gene transfer (A) Representative images of immunofluorescence for LAMP1 and TRPML1-Myc in livers of PiZ mice harvested 4 weeks after AAV injections. (B) Representative images of immunofluorescence for LAMP1 and polymeric ATZ in livers of PiZ mice harvested 4 weeks after AAV injections. The dashed lines are superimposed to the margins of the plasma membranes to highlight single cells. (C) Representative electron microscopy images of PiZ livers injected with AAV-GFP or AAV-TRPML1 and harvested either 4 or 12 weeks post injection showing lysosomes (white arrows) close to the plasma membrane shown in red. The relative quantifications of lysosomes close to the plasma membrane is shown (n = 3 per group). (D) Triple immunostaining for the lysosome marker LAMP1, the plasma membrane marker laminin 2, and the basolateral membrane marker Na-K ATPase on livers of PiZ mice injected with AAV-GFP or AAV-TRPML1 and harvested at 4 weeks post injection. Quantification of LAMP1/laminin 2 co-localization is shown (n = 3 per group; n = 4 fields/sample). (E) Serum IDS and GALNS activities in AAV-GFP- and AAV-TRPML1-injected PiZ mice at 2 weeks post injection (IDS, n = 6 per group; GALNS, n = 4 for AAV-GFP; and n = 6 per AAV-TRPML1). Data are shown as mean ± standard error. Student’s t test: ∗p value < 0.05; ∗∗p value < 0.01. Abbreviations are as follows: BC, bile canaliculi; lyso, lysosomes; PM, plasma membrane.

Figure 3

TFEB nuclear translocation is not induced in PiZ livers by TRPML1 gene transfer (A) Immunohistochemistry for TFEB in livers of wild-type or PiZ mice injected with AAV-GFP or AAV-TRPML1 and harvested at 4 weeks post injection. Quantification of TFEB-positive nuclei in livers is shown (n = 3 per group). (B) Immunoblots for LAMP1, SQSTM1/p62, and LC3 in livers of wild-type and PiZ mice injected with AAV-TRPML1 or AAV-GFP controls and relative quantifications (n = 3–4 per group). Data are shown as mean ± standard error. Student’s t test: ∗p value < 0.05; ∗∗p value < 0.01. WT, wild-type.

Figure 4

Pharmacological activation of TRPML1 reduces ATZ accumulation in PiZ mouse livers (A) Experimental plan for ML-SA5 treatment in PiZ mice; each arrow indicates an ML-SA5 administration and blood drops indicates timing of blood collections. (B) Liver PAS-D staining of vehicle- and ML-SA5-treated PiZ mice and relative quantifications (n = 7 per group). (C) Immunofluorescence for ATZ polymers on livers of vehicle- and ML-SA5-treated PiZ mice (n = 7 per group). (D) Immunoblot for soluble and insoluble fractions of livers from vehicle- or ML-SA5-treated PiZ mice and their quantifications (n = 3 per group). GAPDH and H3 were used as loading controls. (E) Changes in percentages of serum total AAT (n = 8 per group) and ATZ polymers (vehicle, n = 7; ML-SA5, n = 8) in PiZ mice. (F) Immunohistochemistry for TFEB in livers from wild-type or PiZ mice treated with vehicle or ML-SA5 (n = 3 per group). (G) Immunoblots for autophagy markers SQSTM1/p62 and LC3 in livers of wild-type (n = 3 per group) and PiZ mice (n = 5 per group) treated with vehicle or ML-SA5 and their relative quantifications. Vehicle and MLSA5 samples are on the same gel but are not contiguous. Data are shown as mean ± standard error. Student’s t test: ∗p value < 0.05; ∗∗p value < 0.01. A.U., arbitrary units; p.i., post-injection; WT, wild-type.

Figure 5

Genetic or pharmacological activation of TRPML1 ameliorates liver disease of PiZ mice Graphic representation of therapeutic efficacy of TRPML1-mediated lysosomal exocytosis in PiZ mice. Delivery of ATZ polymers to lysosome for exocytosis may rely on ER-to-lysosome-associated degradation pathway (ERLAD) (1) or autophagosome-lysosome fusion (2).