Extended Data Figure 6: Metabolic inhibition in astrocyte causes neuronal cell death and prevents neurite outgrowth in vitro.

a, Astrocytic aconitase was inhibited by fluorocitrate (FC), which disrupted astrocyte metabolism that was accompanied by a senescence-associated β-galactosidase (SA-β-gal) signal. b, Intracellular ATP was decreased in these metabolically disrupted astrocytes (n = 2 biological replicates, n = 6 independent experiments). *P < 0.05, **P < 0.01 versus 0 mM fluorocitrate. c, Propidium iodide (PI) staining showed that fluorocitrate (0.5 mM) did not induce cell death in astrocytes. d, Metabolically disrupted astrocytes significantly decreased the mitochondrial membrane potential. Red: aggregated JC1; green: monomer JC1. Scale bar, 20 μm. e, Rat cortical neurons were co-cultured with JC1-labelled astrocytes. After 24 h co-culture, control astrocytes transferred mitochondria, which had a high-membrane potential (aggregated JC1), but metabolically disrupted astrocytes released and transferred dysfunctional mitochondria into neurons (n = 3 independent experiments). f, Metabolically disrupted astrocytes could not support neural viability under starvation in the co-culture (n = 4 independent experiments). g, Co-culture between astrocytes and neurons was conducted for 48 h to test neurite outgrowth. Immunocytochemistry showed that metabolically disrupted astrocytes prevented neurite outgrowth and increased neuronal cell death (n = 3 independent experiments). h, The LDH assay indicated that fluorocitrate (0.5 mM) did not affect cell viability in either rat cortical astrocytes (n = 4 independent experiments) or rat cortical neurons (n = 4 independent experiments). Data are mean ± s.e.m. P values are from a one-way ANOVA followed by Tukey’s test (b) or an un unpaired t-test (e–g).