VAMP associated proteins are required for autophagic and lysosomal degradation by promoting a PtdIns4P-mediated endosomal pathway

Abstract

Mutations in the ER-associated VAPB/ALS8 protein cause amyotrophic lateral sclerosis and spinal muscular atrophy. Previous studies have argued that ER stress may underlie the demise of neurons. We find that loss of VAP proteins (VAPs) leads to an accumulation of aberrant lysosomes and impairs lysosomal degradation. VAPs mediate ER to Golgi tethering and their loss may affect phosphatidylinositol-4-phosphate (PtdIns4P) transfer between these organelles. We found that loss of VAPs elevates PtdIns4P levels in the Golgi, leading to an expansion of the endosomal pool derived from the Golgi. Fusion of these endosomes with lysosomes leads to an increase in lysosomes with aberrant acidity, contents, and shape. Importantly, reducing PtdIns4P levels with a PtdIns4-kinase (PtdIns4K) inhibitor, or removing a single copy of Rab7, suppress macroautophagic/autophagic degradation defects as well as behavioral defects observed in Drosophila Vap33 mutant larvae. We propose that a failure to tether the ER to the Golgi when VAPs are lost leads to an increase in Golgi PtdIns4P levels, and an expansion of endosomes resulting in an accumulation of dysfunctional lysosomes and a failure in proper autophagic lysosomal degradation. Abbreviations: ALS: amyotrophic lateral sclerosis; CSF: cerebrospinal fluid; CERT: ceramide transfer protein; FFAT: two phenylalanines in an acidic tract; MSP: major sperm proteins; OSBP: oxysterol binding protein; PH: pleckstrin homology; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns4K: phosphatidylinositol 4-kinase; UPR: unfolded protein response; VAMP: vesicle-associated membrane protein; VAPA/B: mammalian VAPA and VAPB proteins; VAPs: VAMP-associated proteins (referring to Drosophila Vap33, and human VAPA and VAPB).

Keywords: ALS8; PI4KB; VAPA/VAPB; autophagy.

Figure 1.

Loss of VAP proteins impairs autophagy. (a) Immunofluorescence staining and quantification of ref(2)P (green) in muscle tissue of the Vap33^∆31 mutant. Images showing the muscle 6 and 7 of the abdominal segments 3. GR, 20-kb genomic rescue construct that contains the Vap33 locus and serves as a WT control [5] (b) Crawling assay showing the distance that individual larvae traveled within 1 min (n > 10). (c) Immunofluorescence staining and quantification of ref(2)P (red) in Vap33^∆20 mutant clones (GFP negative) of Drosophila fat body and salivary gland. (d) Immunofluorescence staining and quantification of poly-ubiquitin (red) in Vap33^∆20 mutant clones (GFP negative) of Drosophila salivary glands. (e) Western Blots (WBs) with ref(2)P antibody in flies. (f) Western blots of HEK293T cells transfected with control or VAPA/B siRNA for 4 days and starved with DMEM or HBSS for 4 h prior to blotting (g) Western blots with SQSTM1 antibody 4 days after VAPA/B siRNA transfection. HEK293T cells are treated with DMSO or bafilomycin A₁ (0.1 mg/ml) for 4 h. Note the accumulation of SQSTM1. The quantification of SQSTM1 normalized to GAPDH is shown below each blot.

Figure 2.

Loss of VAPs leads to autophagic vesicle accumulation. (a) Western blots with Atg8a antibody of late 3rd instar larvae. (b) HEK293T cells were transfected with control or VAPA/B siRNA for 4 days, then treated with DMSO or bafilomycin A₁ (0.1 mg/ml) for 4 h. WBs show accumulation of LC3-I and LC3-II. Quantifications of LC3-II normalized to LC3-I and GAPDH are shown below each blot. LC3 long is a longer exposure of the LC3 blots. * indicates overexposure. (ci – iv) TEM images showing autophagic vesicles in salivary glands and fat body cells from Vap33^∆20 and Vap33^∆20;GR-Vap33^WT 3rd instar larvae. Arrowheads indicate autophagosomes (green), lysosomes (red) and autolysosomes/amphisomes (blue). (cii’ and iv’) Higher magnification of autolysosomes/amphisomes in Vap33^∆20 salivary gland and fat body. Scale bars: 600 nm. (d) Quantifications of the total area and number of autophagic vesicles in (c). Three animals were imaged from each genotype and 5 representative images were selected for each animal for quantification. Error bars represent s.e.m; *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3.

Loss of VAP proteins impairs autophagic degradation. (a) Left: mCherry-Atg8a (red) in Vap33^∆20 mutant clones (GFP negative) of Drosophila fat body. Middle: Zooms of wild-type and mutant cells. Right: Quantification of the size and intensity of mCherry-Atg8a puncta. (b) Left: HEK293T cells were transfected with control or VAPA/B siRNA for 4 days and serum starved for 4 h prior to immunofluorescence staining of endogenous LC3 and LAMP1. Right: Quantification of the signal colocalization of LC3 and LAMP1 using Coloc 2 (ImageJ). (c,d) HeLa cells were transfected with control or VAPA/B siRNA for 3 days and then transfected with the RFP-GFP-LC3 or SQSTM1-RFP-GFP expression vector. Sixteen h after transfection, cells are starved with HBSS for 5 h and imaged live. (e) Quantification of the red and yellow puncta of RFP-GFP-LC3 or SQSTM1-RFP-GFP transfected cells.

Figure 4.

Loss of Vap33 in Drosophila leads to expansion of the lysosomal pool. (a) LysoTracker Red staining (red) and quantification in Vap33^∆20 mutant clones (GFP negative) of Drosophila salivary glands. Wild-type cells are green. (b) Magic Red™ CtsB1 substrate staining and quantification in Vap33^∆20 mutant clones of Drosophila salivary glands. Wild-type cells are green. (c,d) LAMP1-GFP (green) and quantification in Vap33^∆20 mutant clones of Drosophila fat body and salivary gland. Wild-type nuclei are labeled with RFP. (e) Immunostaining of LAMP1-GFP (green) with GFP antibody (red) in Vap33^∆20 mutant clones of Drosophila fat body. Acidic lysosomes should only be labeled in red. Wild-type cells are blue. Right panel, representative images showing lysosomes in wild-type and Vap33 mutant cells.

Figure 5.

Loss of VAPs affects lysosomal degradation. (a) LAMP2 (green) staining in HeLa cells transfected with control or VAPA/B siRNA. (b) LysoTracker Red staining (red) in HeLa cells transfected with control or VAPA/B siRNA. (c) Quantification of LAMP2 and intensity from (a,b). (d) WBs of lysosomal hydrolases in control or VAPA/B siRNA-transfected HEK293T cells before or after HBSS starvation (8 h). (e) Western blots of Cp1/cathepsin L in Drosophila fat body and salivary glands. (f) HEK293T cells transfected with control or VAPA/B siRNA and treated with or without bafilomycin A₁ for 6 h and probed for TFEB, p-RPS6KB/p70S6K and total RPS6KB/p70S6K. (g) HeLa cells transfected with control or VAPA/B siRNA and starved or treated with or without Torin for 2 h. The cytoplasm and nucleus are separated and then probed for TFEB. Starvation: Sixteen h serum starvation followed by 4 h serum and amino acid starvation.

Figure 6.

Loss of VAPs affects PtdIns4P and the endosome pathway. (a) Immunofluorescence staining of PtdIns4P (red) in Vap33 mutant clones (non-green) of Drosophila fat body and salivary gland cells. (b) Immunofluorescence staining of PtdIns4P in control or VAPA/B siRNA-transfected HEK293T cells. (c) Immunofluorescence staining of Rab5 and Rab7 (red) in Vap33 mutant clones (non-green) of Drosophila salivary glands (d) Immunofluorescence staining of Rab7 (red) in Drosophila muscles of control and Vap33 mutant animals (e) Immunofluorescence staining of RAB5 in control or VAPA/B siRNA-transfected HEK293T cells. (f) Sgs3-GFP (green, a marker for secretory granules) in Vap33 mutant clones of Drosophila salivary gland. Wild-type nuclei are labeled with RFP.

Figure 7.

Reducing PtdIns4P levels rescues the endo-lysosomal defects. (a) Left: Immunofluorescence staining of Rab7 in muscles of control and Vap33 mutant larvae. Vap33 mutant larvae are treated with or without PI4KIIIbeta-IN-10 (5 nM, a PtdIns4K inhibitor). Right: Quantification of the Rab7 intensity. (b) HEK293T cells were transfected with control or VAPA/B siRNA and treated with DMSO or PI4KIIIbeta-IN-10 (25 nM) for 4 h prior to immunofluorescence staining of endogenous PtdIns4P and LAMP2. (c) Quantification of PtdIns4P intensity and the lysosomes in (b).

Figure 8.

Reducing endosome levels rescues the lysosomal and autophagic defects. (a) Control or VAPA/B siRNA-transfected HEK293T cells were treated with DMSO or PI4KIIIbeta-IN-10 for 4 h. WB probed for TFEB and CTSB. (b) Control or VAPA/B siRNA-transfected HEK293T cells are treated with DMSO or PI4KIIIbeta-IN-10 for 18 h and treated with bafilomycin A₁ for 4 h. WBs probed with LC3. (c) Immunofluorescence staining of ref(2)P/SQSTM1 in muscles of Vap33^∆20 mutant and Vap33^∆20; Rab5^KO/+ larvae. Bottom: Quantification of the ref(2)P intensity. (d) Immunofluorescence staining of Rab7 and ref(2)P in muscles of Vap33^∆20 mutant and Vap33^∆20; Rab7^KO/+ larvae. Bottom: Quantification of the Rab7 intensity and number of ref(2)P puncta. (e) Crawling assay of control, Vap33^∆20 mutant, Vap33^∆20 mutant treated with PI4KIIIbeta-IN-10 (5 nM) and Vap33^∆20; Rab7^KO/+ larvae. Ten to 20 animals are tested for each genotype/treatment.

Figure 9.

Model. (a) In WT cells, OSBP is localized to the Golgi through the interaction of its PH domain with PtdIns4P present on the Golgi. OSBP also interacts with the VAP proteins through a FFAT motif thereby tethering the ER to the Golgi. This membrane tethering facilitates the PtdIns4P transfer from the Golgi to the ER for hydrolysis by SACM1L. Golgi PtdIns4P mediates vesicle trafficking from the Golgi to various compartments, including the plasma membrane and endosomes. Endosomes mature to lysosomes, which play a key role in the degradation of autophagic compartments. (b) Upon loss of VAPs, OSBP is dissociated from the ER, and ER-Golgi tethering is disrupted. Therefore, hydrolysis of PtdIns4P in the ER is impaired and PtdIns4P accumulates on the Golgi. Elevated PtdIns4P levels promote endosome formation from the Golgi, resulting in an accumulation of endosomes. Expansion of the endosomal pool disrupts the degradation properties of lysosomes upon fusion by affecting lysosomal enzyme composition. This in turn impairs autophagy.