The role of ethanolamine phosphate phospholyase in regulation of astrocyte lipid homeostasis

Abstract

Dietary lipid composition has been shown to impact brain morphology, brain development, and neurologic function. However, how diet uniquely regulates brain lipid homeostasis compared with lipid homeostasis in peripheral tissues remains largely uncharacterized. To evaluate the lipid response to dietary changes in the brain, we assessed actively translating mRNAs in astrocytes and neurons across multiple diets. From this data, ethanolamine phosphate phospholyase (Etnppl) was identified as an astrocyte-specific fasting-induced gene. Etnppl catabolizes phosphoethanolamine (PEtN), a prominent headgroup precursor in phosphatidylethanolamine (PE) also found in other classes of neurologically relevant lipid species. Altered Etnppl expression has also previously been associated with humans with mood disorders. We evaluated the relevance of Etnppl in maintaining brain lipid homeostasis by characterizing Etnppl across development and in coregulation with PEtN-relevant genes, as well as determining the impact to the brain lipidome after Etnppl loss. We found that Etnppl expression dramatically increased during a critical window of early brain development in mice and was also induced by glucocorticoids. Using a constitutive knockout of Etnppl (Etnppl^KO), we did not observe robust changes in expression of PEtN-related genes. However, loss of Etnppl altered the phospholipid profile in the brain, resulting in increased total abundance of PE and in polyunsaturated fatty acids within PE and phosphatidylcholine species in the brain. Together, these data suggest that brain phospholipids are regulated by the phospholyase action of the enzyme Etnppl, which is induced by dietary fasting in astrocytes.

Keywords: astrocyte; brain; glucocorticoid; lipid; liver; phosphatidylethanolamine.

Figure 1

Identifying dietary regulation of genes in neurons and astrocytes using translating ribosomal affinity purification (TRAP).A, graphic showing possible metabolic outcomes of phosphoethanolamine (PEtN). B, Etnppl mRNA expression using qRT-PCR of input and pulled-down (IP) neuron (Syn-Cre) and astrocyte (Aldh1l1-Cre) fractions tissue harvested from ribo-tag mice exposed to fed, fasted diet, or ketogenic diet of TRAP screen. Samples sizes [Syn-Cre: fed (n = 5), fasted (n = 7), ketogenic diet (n = 6)]. Aldh1l1-Cre: fed (n = 5), fasted (n = 4), ketogenic diet (n = 8). C, protein expression of Etnppl from fed and overnight fasted adult mice n = 3. D, Etnppl mRNA expression using qRT-PCR across ages (prenatal day 18 (E18) and postnatal day 7 and 35 (P7 and P35) and CNS regions (cortex, spine, and hippocampus)). (n = 3) Data are expressed as mean ± S.D. Represented data analyzed using multiple Student’s two-tailed t-tests. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Figure 2

Expression of Etnppl and PEtN-related genes across early development.A, Etnppl protein expression in the whole brain across ages. WT and Etnppl^KO fasted cerebellum (CB) tissue used as positive and negative controls for Etnppl protein expression repectively. B, Etnppl protein expression in the liver across ages. WT and Etnppl^KO fasted CB and liver tissue used as positive and negative controls for Etnppl protein expression repectively. C, Etnppl and PEtN-related gene mRNA expression in the whole brain using qRT-PCR across ages P3 (n = 3), P4 (n = 4), P14 (n = 3), P21 (n = 4), P28 (n = 4). D, Etnppl and PEtN-related gene mRNA expression in the liver using qRT-PCR across ages P3 (n = 3), P4 (n = 4), P14 (n = 3), P21 (n = 4), P28 (n = 4). Data (as mentioned in text) are expressed as mean ± S.D. Represented data analyzed using ordinary measures two-way analysis of variance with Sidak’s tests for multiple comparisons. Outliers were removed after using Grubb’s outlier test. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Figure 3

Examining regulation of PEtN-related genes by diet and glucocorticoids.A, mRNA expression of Etnppl and other PEtN-related genes in the whole brain from adult chow-diet-fed, fasted, and refed after fasted mice using qRT-PCR. (n = 8). B, mRNA expression of Etnppl and other PEtN-related genes in the liver from adult chow-diet-fed, fasted, and refed after fasted mice using qRT-PCR. (n = 8). C, mRNA expression of PEtN-related genes in wild-type P2 1° astrocytes after a 24- or 72-h exposure to the glucocorticoid agonist dexamethasone. [dexamethasone] = 100 nM. (n = 3). Data in A and B are expressed as mean ± S.E.M. Represented data analyzed using ordinary measures two-way analysis of variance with Sidak’s tests for multiple comparisons. Data in C is expressed as mean ± S.D. Represented data analyzed using Student’s two-tailed t-tests. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Figure 4

Impact of Etnppl loss on gene expression and metabolic substrate use in the brain and liver.A, Etnppl protein expression in cerebellum from 9-week-old, 18-h fasted Etnppl ^KO and WT mice. B, Etnppl protein expression in liver from 9-week-old, 18-h fasted Etnppl ^KO and WT mice. C, cortex mRNA expression of Etnppl, genes that are indicators of CNS health, PEtN-related genes, and other metabolically relevant genes from 9-week-old, 18-h fasted Etnppl^KO and WT mice. n = 4. D, liver mRNA expression of Etnppl, genes that are indicators of CNS health, PEtN-related genes, and other metabolically relevant genes from 9-week-old, 18-h fasted Etnppl^KO and WT mice. n = 4. E, oxidation of 1-¹⁴C oleic acid to ¹⁴CO₂ in P2 1° cortical astrocytes derived from Etnppl^KO and WT mice (n = 6). [etomoxir] = 100 μM. F, incorporation of 1-¹⁴C ethanolamine into membranes in cultured P2 1° cortical astrocytes derived from Etnppl^KO and WT mice (n = 6). [dexamethasone] = 100 nM. Data are expressed as mean ± S.D. Represented data analyzed using Student’s two-tailed t-tests. Outliers were removed after using Grubb’s outlier test. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Figure 5

Loss of Etnppl does not result in major changes to oxygen consumption or abundance of many hippocampal PEtN-related metabolites.A, seahorse assay mitochondrial stress test measuring oxygen consumption using cultured P2 1° cortical astrocytes derived from Etnppl^KO and WT mice after overnight incubation with dexamethasone and ethanolamine (EtN) (n = 6). [dexamethasone] = 100 nM, [EtN] = 5 mM. B, relative abundances of PEtN-associated metabolites in whole hippocampus from 18-h fasted 9-week-old Etnppl^KO and WT (n = 6). Data in A are expressed as mean ± S.E.M. Represented data analyzed using multiple Student’s two-tailed t-tests. Statistical significance of represented metabolites in B determined using two-stage false discovery rate (FDR) method of Benjamini, Krieger, and Yekutieli with an FDR (Q) of 10%. Fold changes in green boxes are significantly increased, fold changes in red boxes are significantly decreased, and fold changes in yellow boxes are not significantly affected by genotype. The same data are represented as mean of relative species abundance ± S.D. in adjacent graphs in panels C–E. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Figure 6

Phospholipid abundance and composition are altered in cortex after loss of Etnppl.A, volcano plot representing PC and PE species fold changes comparing Etnppl^KO with WT using 18-h fasted cortex from 9-week-old mice. n = 5. B, volcano plot representing phospholipid species fold changes comparing Etnppl ^KO with WT using 18-h fasted liver from 9-week-old mice. C, total relative phospholipid abundance in the cortex from 18-h fasted, 9-week-old Etnppl^KO and WT mice. n = 5. D, total relative phospholipid abundance in the cortex from 18-h fasted liver from 9-week-old Etnppl ^KO and WT mice. n = 5. E, relative total AA abundance in PC species in the cortex from 18-h fasted, 9-week-old Etnppl ^KO and WT mice. n = 5. F, relative total AA abundance in PE species in the cortex from 18-h fasted, 9-week-old Etnppl^KO and WT mice. n = 5. G, relative total DHA abundance in PC species in the cortex from 18-h fasted, 9-week-old Etnppl^KO and WT mice. n = 5. H, relative total DHA abundance in PE species in the cortex from 18-h fasted, 9-week-old Etnppl ^KO and WT mice. n = 5. Data are expressed as mean ± S.D. Represented data analyzed using Student’s two-tailed t-tests. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.