S1P Released by SGPL1-Deficient Astrocytes Enhances Astrocytic ATP Production via S1PR2,4, Thus Keeping Autophagy in Check: Potential Consequences for Brain Health

Abstract

Astrocytes are critical players in brain health and disease. Sphingosine-1-phosphate (S1P), a bioactive signaling lipid, is involved in several vital processes, including cellular proliferation, survival, and migration. It was shown to be crucial for brain development. Its absence is embryonically lethal, affecting, inter alia, the anterior neural tube closure. However, an excess of S1P due to mutations in S1P-lyase (SGPL1), the enzyme responsible for its constitutive removal, is also harmful. Of note, the gene SGPL1 maps to a region prone to mutations in several human cancers and also in S1P-lyase insufficiency syndrome (SPLIS) characterized by several symptoms, including peripheral and central neurological defects. Here, we investigated the impact of S1P on astrocytes in a mouse model with the neural-targeted ablation of SGPL1. We found that SGPL1 deficiency, and hence the accumulation of its substrate, S1P, causes the elevated expression of glycolytic enzymes and preferentially directs pyruvate into the tricarboxylic acid (TCA) cycle through its receptors (S1PR_2,4). In addition, the activity of TCA regulatory enzymes was increased, and consequently, so was the cellular ATP content. The high energy load activates the mammalian target of rapamycin (mTOR), thus keeping astrocytic autophagy in check. Possible consequences for the viability of neurons are discussed.

Keywords: S1P-lyase (SGPL1); SPLIS; autophagy; glucose metabolism; sphingosine 1-phosphate (S1P).

Figure 1

Neural SGPL1 ablation triggers glucose degradation in primary cultured astrocytes. (A,B) Protein quantification of phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate dehydrogenase (PDH), lactate dehydrogenase (LDH), and isocitrate dehydrogenase (IDH) in astrocytes from control (Ctrl) or SGPL1^fl/fl/Nes (KO) mice, as indicated. (C) IDH activity measurement. (D) Protein quantification of the indicated enzymes following stimulation (+) of control astrocytes with exogenous S1P (10 nM, 24 h), (−) represents without stimulation (E) Quantification of ATP in cultured astrocytes derived from control (Ctrl) and SGPL1^fl/fl/Nes (KO) mice. For all, representative immunoblots are shown with β-actin as loading control. Bars represent means ± SEM, n ≥ 3, unpaired Student’s t-test, one-way ANOVA with Bonferroni multiple comparison test; * p < 0.05, ** p < 0.001, *** p < 0.0001, ns, not significant.

Figure 2

S1P receptors 2 and 4 mediate the effect of S1P on glucose degradation and ATP generation in SGPL1-deficient astrocytes. (A,B) Transcript quantification of the five S1P receptors (S1PR) and of the S1P transporter SPNS2 in SGPL1-deficient astrocytes relative to control cells (Ctrl) by qPCR using β-actin as a reference. (C) Protein quantification of PFK, GAPDH, and PDH following stimulation (+) of control astrocytes with a combination of specific agonists of S1PR₂ (5 µM CYM5520) and S1PR₄ (5 µM CYM50308) for 24 h. (D) Protein quantification of PFK, GAPDH, and PDH in astrocytes from control (Ctrl) or SGPL1^fl/fl/Nes (KO) mice cultured for 24 h in the absence (−) or presence (+) of the S1PR₂ antagonist JTE-013 (10 µM) and the S1PR₄ antagonist CYM-55380 (10 µM) as indicated. (E) Determination of ATP levels in control (Ctrl) or SGPL1-deficient astrocytes (KO) in the absence or presence of S1PR_2,4 antagonists as indicated. For all, representative immunoblots are shown with β-actin as loading control. Bars represent means ± SEM, n ≥ 3, unpaired Student’s t-test, one-way ANOVA with Bonferroni multiple comparison test; * p < 0.05, ** p < 0.001, *** p < 0.0001, **** p < 0.00001.

Figure 3

S1P activates mTOR and inhibits autophagy via S1PR_2,4 in SGPL1-deficient astrocytes. (A) Transcript quantification of mTOR in SGPL1-deficient astrocytes (KO) relative to controls (Ctrl) by qPCR using β-actin as reference. (B) Quantification of autophagy marker proteins p62 and LC3-II:LC3-I and (C) representative images showing the fluorescence of the RFP–GFP–LC3 construct in cultured astrocytes from control (Ctrl) and SGPL1^fl/fl/Nes (KO) mice. Red puncta represent autolysosomes, whereas yellow puncta represent autophagosomal structures. DAPI staining indicates cell nuclei in blue. Scale bar: 50 µm. (D) Protein quantification of p62, LC3-I, and LC3-II following stimulation (+) of control astrocytes with a combination of specific agonists of S1PR₂ (5 µM CYM5520) and S1PR₄ (5 µM CYM50308), (−) represents without stimulation for 24 h. For all, representative immunoblots are shown with β-actin as loading control. Bars represent means ± SEM, n ≥ 3, unpaired Student’s t-test; one-way ANOVA with Bonferroni multiple comparison test, * p < 0.05, ** p < 0.001, *** p < 0.0001.

Figure 4

Rapamycin and S1PR antagonists re-establish autophagy in SGPL1-deficient astrocytes. (A) Quantification of autophagy marker proteins p62 and LC3-II:LC3-I in astrocytes from control (Ctrl) or SGPL1^fl/fl/Nes (KO) mice cultured for 6 h in the absence (−) or presence (+) of the S1PR₂ antagonist JTE-013 (10µM) and the S1PR₄ antagonist CYM-55380 (10 µM) as indicated. (B) Quantification of the autophagic marker proteins p62 and LC3-II:LC3-I in astrocytes from control (Ctrl) and SGPL1^fl/fl/Nes (KO) mice, cultured for 5 h in the absence or presence of rapamycin (RAPA, 5 µM) as indicated. (C) Representative images showing the fluorescence of the RFP–GFP–LC3 construct expressed in astrocytes in the presence of rapamycin in control (Ctrl) and KO astrocytes. For all, representative immunoblots are shown with β-actin as loading control. Bars represent means ± SEM, n ≥ 3, one-way ANOVA with Bonferroni multiple comparison test, * p < 0.05, ** p < 0.001, *** p < 0.0001.

Figure 5

Scheme summarizing the effects of SGPL1 ablation on glucose degradation and autophagy in astrocytes. In the absence of SGPL1, accumulated S1P is released by the cells via SPNS2 and promotes the increased expression and activity of proteins involved in glucose breakdown, acting in an auto- or paracrine manner through S1PR_2,4. This leads to increased levels of ATP, the activation of mTOR, and reduced autophagy.