Fig. 2: Mitochondrial fatty acid import regulates neuronal energy balance without inducing ROS or mtDNA damage.
From: DDHD2 provides a flux of saturated fatty acids for neuronal energy and function
a–e, Seahorse XF OCR kinetics (a) and quantification of basal respiration (b), maximal respiration (c), non-mitochondrial oxygen consumption (d) and ATP production (e) in E16 C57BL/6J hippocampal neuronal cultures ± etomoxir at DIV 21–22. Injection of etomoxir, oligomycin, FCCP and Rot/AA is indicated. f, Seahorse XF ECAR glycolysis stress test kinetics in DIV 21–22 hippocampal neuronal cultures of E16 C57BL/6J treated with etomoxir or 1 µM M:P:S-CoA for 48 h. Injection of etomoxir, glucose, oligomycin and 2-DG is indicated. g, LFQ LC–MS/MS protein abundance analysis of gluconeogenesis (R-HSA-70263), glucose metabolism (R-HSA-70326), glucose breakdown (gluconeogenesis, R-HSA-70221) and glycolysis (R-HAS-70171) proteins at DIV 21–22 cortical neuron cultures of E16 C57BL/6J, Ddhd2−/− ± 1 µM S-CoA or 1 µM M:P:S-CoA treatment for 48 h, showing column-wise z-score-normalized protein abundances in each condition. h, LFQ LC–MS/MS analysis of oxidative stress (GO:0006979) and ROS metabolic process protein abundance at DIV 21–22 in E16 cortical neuron cultures of C57BL/6J, and Ddhd2−/− ± 1 µM S-CoA or 1 µM M:P:S-CoA for 48 h, showing column-wise z-score-normalized protein abundances in each condition. i,j, Representative fluorescence images (i) and quantification (j) of CMHrXRos fluorescence intensity in E16 hippocampal neuron cultures of C57BL/6J and Ddhd2−/− ± 1 µM or 10 µM M:P:S-CoA for 48 h, or menadione. k,l, qPCR analysis of mtDNA copy number and mtDNA lesion levels in cortical neuron cultures of C57BL/6J, and Ddhd2−/− treated with 1 µM M:P:S-CoA (k) or 10 µM M:P:S-CoA (l) for 48 h. Proteomics samples and proteins were clustered using Euclidean distance and complete linkage, and the colour gradients in heat maps reflect z-scores from low (blue) to high (red) abundance, each column representing a unique protein and each row a biological replicate. Data are presented as mean values ± s.e.m.; dots present averages of biological replicates. Sample sizes are N = 3 (a–f, k and l) and N = 5 (g, h, and C57BL/6J, Ddhd2−/− and Ddhd2−/− ± 1 µM M:P:S-CoA in j), N = 2 (Ddhd2−/− ± 1 µM M:P:S-CoA and Ddhd2−/− + menadione in j) biologically independent experiments in each condition. The exact P values stated in the graphs were determined from biological replicates using a two-tailed unpaired t-test (b, d, e and l), two-tailed Mann–Whitney test (c), and ordinary one-way ANOVA Kruskal–Wallis multiple-comparisons test (j) and ordinary one-way ANOVA Tukey’s multiple-comparisons test (k).
