Extended Data Fig. 1: The mitochondrial localization of Hexokinase 1 depends on glucose metabolism.

a, Experimental scheme detailing the sequence of plating, transfection, imaging, and alteration of extracellular glucose levels in cultured rat hippocampal neurons. b, Evaluation of HK1-shRNA knock-down efficiency in Neuro-2a cells. HK1-shRNA and shRNA resistant eGFP tagged HK1 (HK1-GFP) were expressed for 48–72 hrs, and whole cell lysate (Input) were probed with anti-HK1 and anti-Tubulin (loading control) antibodies. Asterisk indicates endogenous HK1. c, Quantification of endogenous HK1 (left) and eGFP-tagged HK1 (WT-HK1-GFP) (right) expression levels as shown in (b). All values are shown as mean ± SEM. n = 4 (two-tailed Mann-Whitney U test). d, Experimental scheme detailing the sequence of plating, transfection, and imaging conditions for the 0 mM glucose experiments in cultured rat hippocampal neurons. e, Axonal localization of HK1 in cultured rat hippocampal neurons transfected with HK1-shRNA, shRNA-resistant eGFP-tagged HK1 (pseudo-color, fire), and Mito-DsRed (gray). Representative axonal images were captured at 5 mM glucose, following a 2-hour exposure to 0 mM glucose, and at 5 mM glucose after 2 hours exposure to 0 mM glucose (1 mM lactate and pyruvate), as depicted in (d). Scale bar represents 5 µm. f, The mitochondrial (Mito) and cytoplasmic (Cyto) HK1 intensity ratios were quantified along axons. Data are presented as a violin plot with individual data points and associated p-value. n = 94–117 mitochondria, 9-10 axons from three biological replica (one-way ANOVA with post hoc Tukey’s multiple comparison test). g, Axonal localization of HK1 in cultured rat hippocampal neurons transfected with HK1-shRNA, shRNA-resistant eGFP-tagged HK1 (pseudo-color, fire), and Mito-DsRed (gray). Representative images of axons were captured after 2 hours in 2 mM glucose, and subsequently following 2 hours in 5 mM glucose after initial exposure to 2 mM glucose. The switch from 2 mM to 5 mM glucose was performed in the presence of either vehicle or OSMI-4 treatments. Scale bar represents 5 µm. h, Quantification of mitochondrial (Mito) and cytoplasmic (Cyto) HK1 intensity ratios along axons. Data are presented as a violin plot with individual data points and associated p-value. n = 65–79 mitochondria, 10, 11 axons (unpaired two-tailed t-test). i, Blood glucose measurements from the Fasted and Re-fed mice used for comparing subcellular localization of Hexokinase 1 as shown in Fig. 1. n = 3 mice for each condition, three biological replicas. All values are shown as mean ± SEM (Mann-Whitney U test). j, HK1 distribution pattern in the CA3 region of the hippocampus in Ad-lib, and after 6 hours fasting (6hrs Fasted) states. Scale bar represents 10 µm. k, Co-localization analysis to measure the percent intensity of HK1 on mitochondria for each condition. Data are presented as violin plots with individual data points and associated p-values. n = 9 hippocampal CA3 regions, 9 mice from three biological replica (unpaired two-tailed t-test).

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