SGPL1 (sphingosine phosphate lyase 1) modulates neuronal autophagy via phosphatidylethanolamine production

Abstract

Macroautophagy/autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. The roles of the bioactive signaling lipid sphingosine-1-phosphate (S1P) and its catabolic enzyme SGPL1/SPL (sphingosine phosphate lyase 1) in autophagy are increasingly recognized. Here we provide in vitro and in vivo evidence for a previously unidentified route through which SGPL1 modulates autophagy in neurons. SGPL1 cleaves S1P into ethanolamine phosphate, which is directed toward the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SGPL1^fl/fl/Nes mice with developmental neural specific SGPL1 ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased BECN1/Beclin-1 and SQSTM1/p62 levels were apparent. Alterations were also noticed in downstream events of the autophagic-lysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as APP (amyloid β [A4] precursor protein) and SNCA/α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SGPL1 in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of SQSTM1, APP and SNCA. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SGPL1^fl/fl/Nes brains. Experiments using EGFP-mRFP-LC3 provided further support for blockage of the autophagic flux at initiation stages upon SGPL1 deficiency due to PE paucity. These results emphasize a formerly overlooked direct role of SGPL1 in neuronal autophagy and assume significance in the context that autophagy modulators hold an enormous therapeutic potential in the treatment of neurodegenerative diseases.

Keywords: amyloid precursor protein; autophagy; lysosomes; neuropathology; phosphatidylethanolamine; sphingosine-1-phosphate lyase; α-synuclein/SNCA.

Figure 1.

PE and EAP content is significantly decreased in SGPL1-deficient brains. Mean ± SEM of PE (n ≥ 3; unpaired Student t test, P_h3m = 0.041, P_h9m = 0.0458, P_h12m = 0.0326, P_c3m = 0.0284, P_c9m = 0.0474, P_c12m = 0.0471) and EAP (n ≥ 3; unpaired Student t test, P_h12m = 0.0277, P_c12m = 0.0828) were determined by LC/MS/MS in the hippocampus and cerebellum of control and SGPL1^fl/fl/Nes mice at the indicated ages and calculated per mg of tissue.

Figure 2.

Autophagy is altered in SGPL1-deficient brains. (A to C) Western blots and graphs showing mean ± SEM in brain extracts from control and SGPL1^fl/fl/Nes mice for: (A) BECN1 at the indicated ages (n ≥ 3; 2-way ANOVA, P_genotype = 0.0004), (B) LC3-I and LC3-II at 12 mo of age (n ≥ 3; unpaired Student t test, P_LC3 = 0.0025) and (C) SQSTM1 at 12 mo of age (n ≥ 3; unpaired Student t test, P_SQSTM1 = 0.0412). (D) Immunoblots of SQSTM1 and LC3 from hippocampi of control and SGPL1^fl/fl/Nes mice that were fed or starved (Stv) for 24 h (n ≥ 3; 2-way ANOVA, P_{SQSTM1, genotype} < 0.0001, P_{SQSTM1, stv 24 h} = 0.0011, P_{LC3, genotype} = 0.0068). Western blots of ACTB are shown as loading control. a.u., arbitrary units.

Figure 3.

Autophagosome formation is compromised in SGPL1-deficient brains. (A) Electron micrographs from CA1 hippocampal neurons of control and SGPL1^fl/fl/Nes mice showing autolysosome-like structures (AL), lysosomes (L), and phagophore-like structures (P) (unpaired Student t test, P_{AL, 3 m} = 0.0177 P_{P, 3 m} = 0.0031, P_{AL, 12 m} = 0.0021, P_{L, 12 m} < 0.0001, P_{P, 12 m} = 0.0115). (B) Representative images of immunofluorescence analysis of the CA1 hippocampal brain region in control and SGPL1^fl/fl/Nes mice of 3 or 12 mo of age using the anti-LC3 antibody.

Figure 4.

Upregulation of lysosomal markers in SGPL1-deficient brains. Representative western blot images and graphs showing mean ± SEM in brain extracts from control and SGPL1^fl/fl/Nes mice for: (A) LAMP2 (n ≥ 3; 2-way ANOVA, P_3 m = 0.0197, P_9 m = 0.013, P_12 m = 0.0481) and (B) CTSD (with indication of intermediate and active variants) (n ≥ 3; 2-way ANOVA, total CTSD, P_time = 0.0497, P_genotype < 0.0001, and CTSD active:intermediate, P_12 m = 0.0455). Western blots of ACTB are shown in all panels as loading control. a.u., arbitrary units.

Figure 5.

Accumulation of aggregate-prone proteins and deficits in spatial learning and memory in SGPL1^fl/fl/Nes mice. Representative western blot images and graphs showing mean ± SEM in brain extracts of control and SGPL1^fl/fl/Nes mice of the indicated ages for: (A) APP-FL (full length) and APP-CTFs (C-terminal fragments) (n ≥ 3; 2-way ANOVA, P_{genotype, APP-FL} = 0.0034, P_{time, APP-CTFs} = 0.0453, P_{genotype, APP-CTFs} = 0.0359). (B) SNCA (n ≥ 3; 2-way ANOVA, P_genotype = 0.0050). Western blots of ACTB are shown in all panels as loading control. (C to E) Hidden version of the Morris water maze; TQ, target quadrant with hidden platform; OQ, other quadrants. (C) Time of quadrant occupancy (2-way ANOVA, P = 0.001); (D) number of target crossings after completion of training (2-way ANOVA, P = 0.001); (E) time spent in the target area expressed as distance from the target (2-way ANOVA, P = 0.043). (F) Fear conditioning test. Shown is the relative time of activity expressed as the activity suppression ratio. Baseline activity was determined 2 min before aversive stimulus whereas time of activity was determined 1 d after associative training in a context fear-conditioning paradigm (unpaired t test, P = 0.0053). a.u., arbitrary units.

Figure 6.

Autophagic flux is impaired in SGPL1-deficient neurons. (A and C) Images showing the fluorescence of the EGFP-mRFP-LC3 construct expressed in cultured neurons from SGPL1^fl/fl/Nes and control mice (A) (unpaired Student t test, P < 0.0001) and in cultured WT hippocampal neurons treated with vehicle (control) or THI (C) (unpaired Student t test, P < 0.0001). DAPI staining indicates cell nuclei in blue. Graph shows mean ± SEM of the percentage of red structures corresponding to autolysosomes with respect to the total number of structures (red and yellow) per cell (n = 20 cells in each of 2 different cultures) (B) Representative western blot images and graphs showing mean ± SEM in extracts from cultured hippocampal neurons from WT rats treated or not with THI for the ATG12–ATG5 complex (unpaired Student t test, P = 0.0067) and for LC3 (unpaired Student t test, P = 0.0063). a.u., arbitrary units.

Figure 7.

PE restores autophagic flux in SGPL1-deficient neurons. (A) Representative western blot images for SQSTM1 and LC3 and graphs showing mean ± SEM in extracts from cultured neurons generated from control and SGPL1^fl/fl/Nes mice and treated or not with PE as indicated (n ≥ 3; 2-way ANOVA, P_{SQSTM1, genotype} = 0.0001, P_{SQSTM1, treatment} = 0.0158, P_{LC3, genotype} = 0.0072, P_{LC3, treatment} = 0.0293). (B) Images showing the fluorescence of the EGFP-mRFP-LC3 construct expressed in cultured neurons from control and SGPL1^fl/fl/Nes mice (2-way ANOVA, P < 0.0001). DAPI staining indicates cell nuclei in blue. Graph shows mean ± SEM of the percentage of red structures corresponding to autolysosomes with respect to the total number of structures (red and yellow) per cell (n = 20 cells in each of 2 different cultures). a.u., arbitrary units.

Figure 8.

PE restores autophagic flux in neurons with pharmacologically inhibited SGPL1. Neuronal cultures derived from hippocampi of WT rats were treated with vehicle (control) or THI in the absence or presence of PE as indicated. (A) Representative western blot images for SQSTM1 and LC3 and graphs showing mean ± SEM (n ≥ 3; one-way ANOVA, P_{SQSTM1, THI} = 0.0112, P_{SQSTM1, THI+PE} = 0.0113, P_{LC3, THI} = 0.0005, P_{LC3, THI+PE} = 0.0056). (B) Images showing the fluorescence of the EGFP-mRFP-LC3 construct expressed in cultured WT neurons treated with vehicle (control) or THI in the absence or presence of PE as indicated (one-way ANOVA, P_THI < 0.0001, P_THI+PE < 0.0001). DAPI staining indicates cell nuclei in blue. Graph shows mean ± SEM of the percentage of red structures corresponding to autolysosomes with respect to the total number of structures (red and yellow) per cell (n = 20 cells in each of 2 different cultures). a.u., arbitrary units.

Figure 9.

PE restores autophagy markers in SGPL1-deficient organotypic hippocampal slices and prevents accumulation of APP and SNCA in SGPL1-deficient neurons. (A) Representative western blot images for SQSTM1 and LC3 in hippocampal slices of 12-mo-old control and SGPL1^fl/fl/Nes mice incubated with or without PE for 24 h (n ≥ 3; 2-way ANOVA, P_{SQSTM1, genotype} = 0.0041, P_{SQSTM1, treatment} = 0.0227, P_{LC3, genotype} = 0.0031, P_{LC3, treatment} = 0.0446). (B) Representative western blot images for APP and SNCA and graphs showing means ± SEM values in extracts from cultured neurons from control and SGPL1^fl/fl/Nes mice treated or not with PE (n ≥ 3; one-way ANOVA, P_APP = 0.0304, P_SNCA = 0.0204). a.u., arbitrary units.