Cooperative lipolytic control of neuronal triacylglycerol by spastic paraplegia-associated enzyme DDHD2 and ATGL

Figures

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  Fig. 1. Enzymatic characterization of DDHD2. A, D, and F: Western blotting analysis of COS-7 cell extracts expressing His6-tagged recombinant proteins. Equal amounts of cellular protein were resolved by electrophoresis and proteins were detected using an Anti-His6 antibody. B, C, E, G, H, I, and J: Lipid hydrolase activities of COS-7 cell extracts expressing His6-tagged recombinant proteins. Cell extracts were incubated with lipid substrates as indicated and the release of fatty acids was quantified. Statistical significance was assessed by ANOVA (n = 3; #, P < 0.05). ATGL, adipose triglyceride lipase; DAG, diacylglycerol; DDHD1, DDHD domain-containing 1; DDHD2, DDHD domain-containing 2; HSL, hormone-sensitive lipase; MAG, monoacylglycerol; PC, phosphatidylcholine; Sec23ip, Sec23 interacting protein; TAG, triacylglycerol.

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  Fig. 2. Contribution of cytosolic lipases to acylglycerol hydrolase activities of Neuro-2a cells. A: Inhibition of recombinant lipase activities by small molecules. COS-7 cell extracts expressing His6-tagged recombinant enzymes were incubated with TAG substrate in the absence or presence of Atglistatin (40 μM), 76-0079 (1 μM), or KLH45 (100 nM), and the release of fatty acids was quantified. B: Inhibition of endogenous lipase activities in Neuro-2a cells. Cellular extracts were incubated with lipid substrates and small molecule inhibitors as indicated, and the release of fatty acids was quantified. C: Western blotting analysis of lipase expression in Neuro-2a cells. Cells were transduced with lentivirus-expressing scrambled shRNA or DDHD2 shRNA, and protein expression was analyzed using specific antibodies. GAPDH was detected as a loading control. D: Lipid hydrolase activities of Neuro-2a cells after stable silencing of DDHD2 expression. Cellular extracts were incubated with lipid substrates as indicated and the release of fatty acids was quantified. Statistical significance was assessed by ANOVA (n = 3; #, P < 0.05). ATGL, adipose triglyceride lipase; DAG, diacylglycerol; DDHD2, DDHD…

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  Fig. 3. Contribution of cytosolic lipases to LD and TAG homeostasis in Neuro-2a cells. A and B: LD size and abundance in Neuro-2a cells. Cells were incubated for 24 h in the presence and absence of inhibitors and LDs were imaged by confocal fluorescence microscopy. The left panel shows a representative Z-stack. Scale bars represent 10 μm. Each datapoint represents a mean of 17–25 cells. C and D: TAG species levels in Neuro-2a cells. Cells were treated with small molecule inhibitors and TAG species were assessed by targeted MS. E and F: TAG levels in Neuro-2a cells incubated with exogenous FAs. Cells were incubated with 400 μM 14C-oleic acid in the absence or presence of lipase inhibitors and radioactivity in TAG was determined. G: TAG catabolism and (H) FA release in Neuro-2a cells. Cellular lipids were labeled with 14C-oleic acid. Cells were incubated in unlabeled medium in the absence or presence of lipase inhibitors for 4 h, and radioactivity in TAG and secreted FA was determined. The dotted line indicates TAG levels before medium switch. Statistical significance was assessed by ANOVA (n = 3–4; #, P < 0.05). TAG, triacylglycerol.

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  Fig. 4. Impact of cytosolic lipases on DAG pools and other lipids in Neuro-2a cells. A, B, and D: Levels of DAG regiomers in Neuro-2a cells. Cellular lipids were labeled with 14C-oleic acid in the presence and absence of inhibitors and radioactivity in DAG regiomers was visualized by autoradiography and quantified thereafter. Cells in (D) were transduced with lentivirus-expressing scrambled or DDHD2 shRNA. (C) Levels of DAG 36:2 regiomers in Neuro-2a cells. Cells were incubated in the absence or presence of inhibitors, and DAG 36:2 regiomers were quantified by targeted MS. E, F, and G: Phospholipid levels in Neuro-2a cells. Cells were incubated in the absence or presence of lipase inhibitors, and cellular PC, PE and PS species were quantified by targeted MS analysis. Statistical significance was assessed by ANOVA (n = 3–5; #, P < 0.05). DDHD2, DDHD domain-containing 2; DAG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.

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  Fig. 5. Impact of cytosolic lipases on TAG hydrolysis of murine brain and primary cortical neurons. A: Western blotting analysis of cytosolic lipase expression in murine tissues. Tissue homogenates of male C57BL/6J were separated by gel electrophoresis, and protein expression of DDHD2, HSL, and ATGL was detected by immunoblotting. B: Inhibition of endogenous lipase activities in murine brain homogenates. Brain extracts were incubated with TAG substrates and small molecule inhibitors as indicated, and the release of fatty acids was quantified. C: Inhibition of DDHD2 activity in murine tissue homogenates. Tissue extracts were incubated with TAG substrates and KLH45 or DMSO as indicated, and the release of fatty acids was quantified. D: Levels of sn-1,3-DAG and (E) TAG in primary cortical neurons after lipase inhibition. Primary cortical neurons were incubated with 14C-oleic acid and lipase inhibitors as indicated, and radioactivity in sn-1,3-DAG and TAG, respectively, was quantified. Data are expressed as means +SD. Statistical significance was assessed by (C) multiple t-tests or (B, D, E) ANOVA (n = 4–6; #, P < 0.05). TAG, triacylglycerol; ATGL, adipose triglyceride lipase; BAT, brown…

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  Fig. 6. Subcellular localization of cytosolic lipases in Neuro-2a cells and primary cortical neurons. Cells were incubated with oleic acid to induce lipid droplet (LD) formation. A: Neuro-2a perinuclear supernatants (PNS) were separated into cytosol (Cyt), membrane (Mem), and LD fractions by ultracentrifugation, and protein distribution was assessed by Western blotting analysis. B: subcellular localization of recombinant lipases in living Neuro-2a cells. Cells transiently expressing ATGL-ECFP or DDHD2-ECFP were incubated with oleic acid to induce LD formation and imaged by confocal fluorescence microscopy. LDs were detected using LipidTOX. Scale bars full size: 10 μm; scale bars insets: 1 μm. C, D: subcellular localization of endogenous DDHD2 in (C) Neuro-2a cells and (D) primary mouse neurons. Cells were incubated in the presence of oleic acid and in the absence or presence of KLH45 as indicated. Cells were fixed, and DDHD2 was detected by immunocytochemistry. LDs and cis-Golgi were detected using LipidTOX and anti-GM130 antibody, respectively. Scale bars full size: 10 μm; scale bars insets: 1 μm. ATGL, adipose triglyceride lipase; DDHD2, DDHD domain-containing 2; GAPDH,…