D-serine inhibits one-carbon metabolism

A. A heatmap shows top 25 metabolites in PCNs altered by treatment with D- and/or L-serine in the metabolomic analysis (n = 3). B. Major metabolites in the one-carbon metabolism. C. KEGG metabolic pathways enriched in D-serine treated cells compared to vehicle treated cells. D. Pearson’s correlation coefficients between glycine and other metabolites. Top 25 metabolites positively (red) and negatively (blue) correlated with glycine are shown. E, F. Concentrations of glycine (E) and L-serine (F) in PCNs were quantified using HPLC (n = 4). G. Concentrations of formate in the culture media of PCNs treated with 2 mM D- or L-serine (n = 4). Data are presented as the mean ± s.e.m. (E, F, and G). Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s post hoc-test (E, F, and G). *p < 0.05 (A). Data are the representative of two independent experiments.

D-serine inhibits cytoplasmic L-serine transport to mitochondria

A. L-serine/glycine transport and conversion across cytoplasm and mitochondria in one-carbon metabolism. B. Binding sites of D- (right) or L-serine (left), THF, and pyridoxal phosphate (PLP) in the ligand-bound SHMT2 complex systems. Arrows indicate carbon atoms on serine enantiomers that transfer to THF. C. Time series variation of the number of contact atoms within the distance cutoff of 7.0 Å from D- or L-serine observed at the initial structure. D. SHMT2 enzymatic activity determined by glycine production is shown as percent of control (with no D-serine) (n = 3). E. Relative concentrations of glycine in NPCs treated with or without 2mM D-serine and/or 50 μM THF. F. Inhibition of mitochondrial L-serine transport by D-serine was examined in semi-permeabilized cells (n = 4). G. A PCA plot shows RNA-seq results from cells treated with D-serine (pink) or control (gray). H. A volcano plot indicates differentially expressed genes in cells treated with D-serine vs controls. Gene expressions with adjusted p-values < 0.05 are highlighted in yellow (up) or blue (down). I. Bar plots show Normalized Enrichment Score for the top 10 up or down-regulated GSEA-enriched categories. Color, q-value. J. GSEA plots for a core subset of gene ontologies are displayed. Data are plotted as the mean ± s.e.m. (D, E, and F). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post hoc-test (D) or Dunnett’s post hoc-test (E).

D-serine attenuates proliferation of neural tumor cells

A, C, H. Relative proliferation of neuroblastoma cell lines (A), glioblastoma lines (C), and glioma stem cells (H) treated with D-serine at indicated doses were analyzed 48 h after treatment (n = 4, each). Cells with the vehicle treatment were used as 100% proliferation controls. B. Glycine concentration in Neuro2a cells was quantified 48 h after treatment with D- and/or L-serine (n = 3). D, E. Cell proliferation was assessed 48 h after treatment with 5 mM D-serine and formate/folates at indicated doses (n = 4). Cell proliferation was compared to that of cells treated with D-serine alone. F, G, J, K. Ex vivo slice cultures of mouse brain transplanted with U87 (F, G) or hG008 cells (J, K) expressing Venus fluorescence were treated with D-serine. Representative images of the xenograft at 0 and 7 days after treatment are shown (F, J). Increased area of fluorescence after 7 days of culture was measured (G, K). I. Sphere diameters of glioma stem cells (hG008) were measured at 7 days after treatment with D-serine at indicated doses. Data are presented as the mean ± s.e.m. (A-E, G-I, and K). Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s post-hoc test (B, D, and E) or by Student’s t-test (G and K).

Developmental transition of serine enantiomer synthesis and enantio-selective effects on immature and mature neurons

A. Light microscopic view showing primary cultured NPCs treated with 1mM D- or L-serine for 7 days in serine-free media. B. Immunofluorescent images are NPCs treated with 1 mM D-serine for 48 hours. Cleaved caspase-3 (c-Casp-3), green (left) or red (right); βIII Tubulin, red (left); GAD67, green (right); and DAPI (blue). C. Numbers of NPCs per area at DIV7 in (A) were counted (n = 4). D. Western blots indicate cleavage of caspase-3 in cultured neurons at 48 h after treatment of D-serine with indicated doses. E. Western blots indicate cleavage of caspase-3 in cultured neurons at 48 h after treatment of serine enantiomers with indicated doses starting at DIV1 (immature) or DIV7 (mature neurons or astrocytes). F. Relative cell proliferation of NPCs after 48 h treatment of 1mM serine enantiomers was analyzed (n = 4). G. H. Cleavage of caspase-3 in NPCs induced by 1mM D-serine was observed in the presence of various L-amino acids (1 mM each) (G) or L-serine at indicated doses (H). I. Cleavage of caspase-3 in NPCs triggered by 2 mM D-serine was observed in the presence or absence of 40 μM 5,10-meTHF. J. Concentrations of serine enantiomers and glycine and the ratio of serine enantiomer concentrations in the cerebrum during development (n= 3-4). K. Single-cell transcriptome profiles of enzymes involved in serine metabolic pathways during mouse brain development. Original data were from Bella et al(Bella et al., 2021). Data are plotted as the mean ± s.e.m. Statistical significance was evaluated by One-way ANOVA followed by Dunnett’s post hoc test (C). All in vitro data are the representative of at least two independent experiments.