Fig 2: Increased ABAT expression leads to reduced H3K4ac through HDAC3-mediated histone deacetylation(A and B) H3K4ac expression analysis in neural cells depicts significantly decreased levels in neurons (n = 3) compared with E12.5 NSC (n = 48), P2 cerebellar SC (n = 34), P2 hindbrain SC (n = 35), and astrocytes (n = 3). Scale bar, 50 µm; one-way ANOVA followed by Tukey’s multiple comparison test.(C and D) ABAThigh cells in D425 (n = 13), CHLA-01-MED (n = 17), and CHLA-01R-MED (n = 27) display significantly decreased H3K4ac expression compared with their ABATlow counterparts (n = 50 for all cell lines). Scale bar, 20 µm; t test.(E–H) ABAT OE cells in both in vitro (n = 50) and in vivo (n = 60) conditions have significantly lower H3K4ac expression compared with ABAT KD cells (n = 50 in vitro and n = 60 in vivo). Scale bar, 50 µm; t test.(I and J) LMs show significantly decreased H3K4ac (n = 160) compared with cerebellar tumors (n = 160). Scale bar, 50 µm; t test.(K) Histone deacetylase (HDAC) activity analysis reveals significantly increased HDAC activity in ABAT OE cells (n = 3).(L and M) H3K4ac deacetylase HDAC3 is significantly higher in ABAT OE cells (n = 50) compared with ABAT KD cells (n = 50). Scale bar, 50 µm; t test.n = nuclei (B, D, F, H, and J) or cells quantified (M) or replicates (K). Histogram data depict mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S5.

Fig 3: ABAT is localized in GABAergic neural cell mitochondria and is associated with a differentiated phenotype(A) ABAT topography and in situ hybridization (ISH) data from embryonic and post-natal stages reveal increased ABAT expression in the developed cerebellum. Scale bar, 250 µm.(B) Single-cell sequencing data from a developing mouse brain at different developmental stages show that ABAT expression is highest in GABA interneuron and progenitor populations.(C) ABAT expression within the GABA interneuron population is significantly associated with a higher differentiation score. p < 0.0001. Mann-Whitney U test.(D–F) Mouse neurons have the highest ABAT expression and mitochondrial localization among neural cells. Scale bar, 50 µm; one-way ANOVA followed by Tukey’s multiple comparison test, n = 6.NSC, neural stem cell; SC, stem cell. n = cells quantified. Histogram data depict mean ± SD. ****p < 0.0001. See also Figure S1.

Fig 4: MB tumors express heterogenous ABAT, which increases in nutrient-scarce microenvironments(A) Representative ABAT immunofluorescence staining reveals that 6/18 tumors exhibit mixed ABAT expression. Scale bar, 50 µm.(B and C) ABAT expression and mitochondrial localization analysis of NC (n = 6) and ABAThigh/ABATlow regions of interest (n = 36) in MB tumors show significantly higher ABAT expression and mitochondrial localization in NC and ABAThigh MB. One-way ANOVA.(D) D425, CHLA-01-MED, and CHLA-01R-MED cells exhibit heterogeneous ABAT expression. Scale bar, 50 µm.(E and F) ABAThigh cells display a significant increase in ABAT expression and mitochondrial localization. t test, n = 6.(G) Flow cytometry analysis reveals that ABAThigh cells are a rare subset of cells in the tumor bulk.(H) D425 cells in artificial CSF (aCSF) medium display increased ABAT expression. Scale bar, 50 µm.(I) aCSF cells display similar ABAT levels compared with ABAThigh cells in control medium. Two-way ANOVA, n = 6.(J) ABAT flow cytometry analysis reveals that aCSF cells have higher overall ABAT expression.(K) Increasing aCSF concentration in medium results in a stepwise increase in ABAT expression. One-way ANOVA.(L) BrdU incorporation analysis reveals that aCSF cells have a significant decrease in proliferation rate. t test, n = 3.n = cells/regions of interest (ROIs) quantified (B, C, F, and I) or replicates (L). Histogram data depict mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One- and two-way ANOVAs followed by Tukey’s multiple comparison test (B, C, I, and K). See also Figure S3.

Fig 5: Higher ABAT expression increases GABA metabolism, promotes survival in nutrient-poor conditions, and induces an OXPHOS metabolic phenotype(A) Magnetic resonance spectroscopy analysis indicates that GABA and the GABA shunt metabolites glutamate and succinate are present in the MB tumor microenvironment.(B and C) Cells given exogenous GABA show accumulation of GABA in ABATlow cells. Scale bar, 20 µm; t test, n = 6.(D) D283 and D425 ABAT OE cells show an increase in the GABA catabolism product succinate after exogenous supraphysiological GABA treatment, whereas ABAT KD cells show no significant change. t test, n = 3.(E) ABAT OE cells in aCSF show a significant increase in succinate. t test, n = 3.(F and G) Exogenous treatment of supraphysiological GABA levels shows no significant change in proliferation rate, excluding significance at 200 µM in D425 ABAT OE cells and at 2 mM in D425 ABAT OE aCSF cells. One-way ANOVA, n = 3.(H) Increasing aCSF concentration leads to increased MB sensitivity to ABAT inhibitor NEO216. n = 3.(I) Optical redox ratio analysis using a two-photon confocal microscope shows ABAT OE cells (n = 6) have higher OXPHOS utilization than ABAT KD cells (n = 7). t test.(J) D425 aCSF cells (n = 9) display a optical redox ratio similar to that of neurons (n = 9), a higher ratio compared with cerebellar (n = 7) and hindbrain (n = 10) stem cells, and a significantly higher ratio compared with D283 (n = 8) and D425 (n = 8) cells in control medium. One-way ANOVA.n = number of cells quantified (C), replicates (D–H), or images analyzed (I and J). Histogram data depict mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA followed by Tukey’s multiple comparison test (F, G, and J). See also Figure S4.