Phosphatidylinositol-5-Phosphate 4-Kinases Regulate Cellular Lipid Metabolism By Facilitating Autophagy

Abstract

While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P₂) in mammalian cells is generated by the conversion of phosphatidylinositol-4-phosphate (PI-4-P) to PI-4, 5-P₂, a small fraction can be made by phosphorylating phosphatidylinositol-5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2a^-/-Pip4k2b^-/- mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P₂ synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.

Keywords: PI5P4K; TFEB; autophagy; lipid kinase; lysosome; mTORC1; metabolism; phospholipid.

Figure 1. Autophagy defects in PI5P4K deficient mouse livers

(A) Experimental design of Pip4k2a^flx/flx Pip4k2b^−/− and Pip4k2a^−/−Pip4k2b^−/− livers. Pip4k2a^flx/flx Pip4k2b^−/− mice (~14-16 weeks of age) were retroorbitally injected with adenovirus, empty or Cre. Two weeks post injection, mice were fasted (18 hours) and subsequently livers were harvested. (B) Liver specific loss of Pip4k2a with Adeno-Cre virus. α-tubulin as loading control. (C) TEM of livers. Yellow arrows indicate LDs and the red asterisk indicates collagen. Scale bars, 1 μm. (D) Indicated livers from fed or fasted mice stained with oil red O. Scale bars, 20 μm. (E) Triglyceride measurements of indicated livers from fed or fasted mice. **p < 0.005, Student’s t-test. (F) Indicated livers from fed or fasted mice and stained for LC3B or LAMP1. Scale bars, 10 μm. (G) and (H) Quantification of results in (F). The number of LC3B and LAMP1 puncta was quantified in the cytoplasm and normalized to nuclei. Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to Pip4k2a^flx/flx Pip4k2b^−/− livers from fed mice, (n ≥ 50). (I) Western blot of p62 in indicated livers from fed or fasted mice. β-actin as loading control (n = 4). (J) Quantification of the western blot in (I). **p < 0.005, *p < 0.05, Student’s t-test (n = 4).

Figure 2. Loss of PI5P4Ks in mouse cells causes autophagy defects

(A) TEM of indicated MEFs. Red arrows indicate autophagic vacuoles (AVs). Scale bars, 1 μm. (B) Quantification of results in (A). The number of AVs was counted per cell. ***p< 0.0005, Student’s t-test, (n ≥ 30). (C) LDs (red) visualized in MEFs. Nuclei in blue. Scale bars, 10 μm. (D) Quantification of results in (C). *p< 0.05, Student’s t-test, (n ≥ 25). (E) MEFs were cultured overnight with either 10% or 0.3% serum and stained for LC3B (red) or LAMP1 (green). Nuclei in blue. Scale bars, 10 μm. (F) Quantification of LC3B puncta per cell in (E). Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to control MEFs cultured in 10% serum, (n ≥ 25). (G) Quantification of LAMP1 puncta per cell in (E). Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to control MEFs cultured in 10% serum, (n ≥ 25). (H) Colocalization of LC3B and LAMP1 puncta in (E). A Pearson correlation was calculated between the puncta for the LC3B and LAMP1 immunofluorescence signal. Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to control cultured in 10% serum, (n ≥ 25). (I) MEFs were cultured overnight with 10% serum and stained for LC3B (red) or LAMP1 (green) and visualized at 100× magnification. Autophagosomes marked by yellow and blue arrows. Nuclei in blue. Scale bars, 5 μm.

Figure 3. Autophagic flux perturbed in Pip4k2a^−/−Pip4k2b^−/− shTrp53 MEFs

(A) MEFs were transfected with the tandem fluorescent-tagged LC3 reporter and treated +/− CQ (20 M) 12 hours before fixation. Nuclei in blue. Scale bars, 15 μm. (B) Quantification of LC3B puncta per cell in (A). Statistical significance determined by ANOVA (*p< 0.05, ***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to control MEFs – CQ. (n ≥ 25). (C) Quantification of puncta color percentage in (A). Total number of autophagosomes (yellow) and autolysosomes (red) were quantified and compared to total puncta per cell (n=30 cells). Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. (D) Western blot of LC3B levels compared to β-actin in MEFs treated +/- 20uM CQ for 12 hours (n=4). (F) Western blot comparison of endogenous PI5P4Kα and p62 to αtod p62enin MEFs (n =3). (G) MEFs were infected with a lentivirus expressing GFP or GFP-PIP4K2A and stained with lysotracker (red). Nuclei in blue. Scale bars, 10 μm. (H) Increased magnification of the box in (G). Scale bars, 4 μm. (I) Quantification of the lysosomes in (G). Statistical significance determined by ANOVA (*p < 0.05) with Dunnett multiple comparison post-test.

Figure 4. Loss of PI5P4Ks induces autophagy-lysosome gene program and metabolic deficiencies

(A) Pathway enrichment for genes significantly differentially expressed (47 Genes, False Discovery Rate (FDR) <0.2) and overlapping Autophagy Signature (Perera et al., 2015), between control and double knockout MEFs. Ranked by −log10 (p-value). (B) Normalized enrichment score (NES) for genes identified as part of the KEGG Lysosomal and Hallmark mTORC1 gene sets. The upper part shows the NES, which is calculated by ranked ordered gene set, increasing the score when a gene is in the set and decreasing it when it is not. Each blue line represents a hit from the gene set. The lower portion shows the rank ordered genes for the double knockout MEFs when compared to the control MEFs, with highly expressed to the far left and down-regulated genes to the right. Enrichment score (ES) for KEGG Lysosome=0.62 and Hallmark mTORC1 = −0.49, FDR < 0.2 for both. (C) Heat map of lysosomal genes and mTORC1 genes from (B). P-values were adjusted for multiple testing using the Benjamini-Hochburg method with p < 0.05 and q < 0.1, (n = 3).

Figure 5. TFEB nuclear localization and downstream gene activation is increased with PI5P4K loss

(A) Heat map of TFEB-dependent genes in the double knockout MEFs compared to the control MEFs. *p < 0.05, (n = 3). (B) MEFs were cultured in 10% or 0.3% FBS for 18 hours and stained for TFEB (green). Nuclei stained in blue. Scale bars, 10 μm. (C) Quantification of results in (B). The intensity of TFEB immunofluorescence was quantified in the nucleus and the cytoplasm, and used to calculate the ratio. Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to control MEFs grown in 10% serum, (n ≥ 30). (D) MEFs were serum starved for 16 hours in 0.3% serum and subsequently harvested for qPCR of TFEB targets. Fold change is calculated by comparison to MEFs grown in complete media. *p < 0.05, Student’s t-test, (n ≥ 8). (E) Pip4k2a^−/−Pip4k2b^−/− shTrp53 MEFs infected with a lentivirus expressing mCherry or mCherry-PIP4K2A were serum starved for 16 hours in 0.3% serum and subsequently harvested for qPCR of TFEB targets. Fold change is calculated by comparison to MEFs grown in complete media. *p < 0.05, Student’s t-test, (n ≥ 8). (F) Lysates from indicated MEFs were assayed for the levels and phosphorylation states of the indicated mTORC1 targets (n =3).

Figure 6. Acetyl-CoA replenishment modulates autophagy in cells lacking PI5P4K

(A) Lysates from indicated MEFs were assayed by metabolomics for levels of Acetyl-CoA. *p < 0.05, Student’s t-test, (n ≥ 7). (B) Pip4k2a^−/−Pip4k2b^−/− shTrp53 MEFs were cultured overnight in 10% serum +/- 5 mM DMKG and stained for LAMP1 (green) and LC3B (red). Nuclei in blue. Scale bars, 10 μm. (C) Quantification of LC3B puncta per cell in (B). In addition to DMKG, MEFs were treated with acetate overnight. Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to vehicle treated MEFs, (n ≥ 25). (D) Quantification of LAMP1 puncta per cell in (B). Conditions same as (C). Statistical significance determined by ANOVA (***p < 0.0005) with Dunnett multiple comparison post-test. Each group was compared to vehicle treated MEFs, (n ≥ 25). (E) MEFs were cultured overnight in media containing 10% serum, 0.3% serum or 0.3% serum with 5 mM DMKG. The cells were then harvested for qPCR of TFEB targets. *p < 0.05, Student’s t-test, (n ≥6).

Figure 7. Conservation of PI5P4K pathway in C. elegans

(A) Lifespan assay of wild-type and ppk-2 mutant animals. (B and C) Oxidative stress resistance assay of indicated worm strains on specified conditions. Synchronized post fertile animals were transferred to plates supplemented with 4 mM paraquat and surviving animals were scored daily. (D) Oil red O staining in wild-type and ppk-2 mutant day 1 adult animals. (E) Quantification of the oil red O staining intensity. Data are expressed as means ±SEM. Statistical analyses for all data were performed by Student’s t-test (*p<0.05, **p<0.01). (F) Model of PI5P4K regulation of autophagy. In WT cells, during energy stress, autophagy is activated as a cellular recycling mechanism. Cells will capture cytoplasm and organelles and consume them in the lysosome following fusion of the autophagosome to the lysosome and the PI5P4Ks are required for proper fusion. In PI5P4K deficient cells, lysosomes fail to fuse with autophagosomes at the rate needed to clear the autophagosomes when cells are under energy stress. After multiple cell divisions following the loss of PI5P4Ks, a build-up of autophagic vesicles and undigested LDs accumulate leading to a drop in cellular metabolites, in particular amino acids and Acetyl-CoA. The decrease in metabolites impairs mTORC1 activation, thereby turning on TFEB dependent gene expression.