Journal: Nature Metabolism

Article Title: DDHD2 provides a flux of saturated fatty acids for neuronal energy and function

doi: 10.1038/s42255-025-01367-x

Figure Lengend Snippet: a , Representative, simultaneously acquired, fluorescence images of green (iATPSnFR2) and red (HaloTag-Janelia Fluor 549) fluorescence of ratio-metric hSynapsin.iATPSnFR2.HaloTag-JF549 ATP sensor at rest, during high K + stimulation, and following a 15-min high K + stimulation in the presence of 2-DG in E16 cultured hippocampal C57BL/6J neurons at DIV18-19. Boxed areas (i–vi) are shown magnified on the right. b , Kinetic graph shows the average ratio (normalized average numbers indicated on top of each condition) of iATPSnFR2 green to HaloTag-Janelia Fluor 549 red fluorescence and maximum change in green:red normalized to the C57BL/6J/ATP sensor at t 1 . c , Representative, simultaneously acquired, fluorescence images of green (cpSFGFP) and red (HaloTag-Janelia Fluor 549) fluorescence of ratio-metric hSynapsin.cpSFGFP.HaloTag-JF549 non-responsive control at rest, during high K + stimulation, and following a 15-min high K + stimulation in the presence of 2-DG in E16 cultured hippocampal C57BL/6J neurons at DIV18-19. Boxed areas (i–vi) are shown magnified on the right. d , Kinetic graph shows the average ratio (normalized average numbers indicated on top of each condition) of cpSFGFP green to HaloTag-Janelia Fluor 549 red fluorescence and maximum change in green:red normalized to the C57BL/6J/ATP sensor at t 1 . e , Representative, simultaneously acquired, fluorescence images of green (iATPSnFR2) and red (HaloTag-Janelia Fluor 549) fluorescence of ratio-metric hSynapsin.iATPSnFR2.HaloTag-JF549 ATP sensor at rest, during high K + stimulation, and following a 15-min high K + stimulation in the presence of 2-DG in E16 cultured hippocampal Ddhd2 − / − neurons at DIV18-19. Boxed areas (i–vi) are shown magnified on the right. f , Kinetic graph shows the average ratio (normalized average numbers indicated on top of each condition) of iATPSnFR2 green to HaloTag-Janelia Fluor 549 red fluorescence and maximum change in green:red normalized to the C57BL/6J/ATP sensor at t 1 . g , Representative, simultaneously acquired, fluorescence images of green (cpSFGFP) and red (HaloTag-Janelia Fluor 549) fluorescence of ratio-metric hSynapsin.cpSFGFP.HaloTag-JF549 non-responsive control at rest, during high K + stimulation, and following a 15-min high K + stimulation in the presence of 2-DG in E16 cultured hippocampal Ddhd2 − / − neurons at DIV18-19. Boxed areas (i–vi) are shown magnified on the right. h , Kinetic graph shows the average ratio (normalized average numbers indicated on top of each condition) of cpSFGFP green to HaloTag-Janelia Fluor 549 red fluorescence and maximum change in green:red normalized to the C57BL/6J/ATP sensor at t 1 . i , Western blot of DDHD2 protein levels in C57BL/6J and Ddhd2 − / − ± lenti control infection, DDHD2 wt -myc, DDHD2 D660H -myc, DDHD2 S351A -myc and DDHD2 W103R -myc. j , Hippocampal C57BL/6J neurons, and lentivirally induced Ddhd2 − / − hippocampal neurons expressing DDHD2 wt -myc and DDHD2 W103R -myc, were AAV9-infected to express hSynapsin.iATPSnFR2.HaloTag-JF549. Quantification of the hSynapsin.iATPSnFR2.HaloTag-JF549 maximum change in green:red normalized to the C57BL/6J/ATP sensor at t 1 in E16 hippocampal C57BL/6J, and Ddhd2 − / − neurons transiently expressing DDHD2 wt -myc and DDHD2 W103R -myc ± 1 µM M:P:S-CoA for 48 h at DIV14-16. Data are presented as mean values ± SEM from individual synapses. The exact P values stated in the graphs were determined from biological replicates using one-way ANOVA Kruskal–Wallis multiple comparison test ( b ), one-way ANOVA Tukey’s multiple comparisons test ( f , d and h ) and two-tailed paired t -test ( j ). Sample sizes were N = 3 in a and b , and Ddhd2 − / − +DDHD2 wt -myc and Ddhd2 − / − +DDHD2 W103R -myc + M:P:S-CoA in j , and N = 4 in d , f and h , and C57BL/6J, Ddhd2 − / − +DDHD2 W103R -myc in j , biologically independent experiments.

Article Snippet: For the analysis of HSP54 mutant rescue with fatty acyl-CoA treatment, E16 Ddhd2 −/− neurons were infected with lentiviral control, Fuw-DDHD2 WT -myc, Fuw-DDHD2 D660H -myc, Fuw-DDHD2 S351A -myc or Fuw-DDHD2 W103R -myc at DIV 4, and the expression of DDHD2 in Ddhd2 −/− neurons was analysed at DIV 14 using anti-DDHD2 (Proteintech, 25203-1-AP; 1:1,000 dilution) in a standard western blot protocol, in comparison to endogenous Ddhd2 in C57BL/6J (and Ddhd2 −/− neurons).

Techniques: Fluorescence, Cell Culture, Control, Western Blot, Infection, Expressing, Comparison, Two Tailed Test