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[Preprint]. 2023 May 19:rs.3.rs-2859584. [Version 1] doi: 10.21203/rs.3.rs-2859584/v1

Figure 6. NAD+ supplementation depends on glycolysis to rescue APP transport in NMNAT2 KO neurons.

Figure 6

(A) Representative kymographs from DIV8 ctrl or KO neurons supplemented with Veh (vehicle) or 1 mM NAD+ since DIV5. Scale bar, 20 μm. (B) Percentages of APP-EGFP vesicles moving anterogradely, retrogradely, or pausing in distal axons of DIV8 ctrl and KO neurons supplemented with Veh or NAD+. Numbers (neurons imaged) and statistics: 56 ctrl+Veh, 62 KO+Veh, 17 ctrl+NAD+,and 49 KO+NAD+, *p= 0.0232, ##p= 0.0018. Significant comparison to ctrl+Veh and KO+Veh groups was labeled with* and #, respectively. (C) Velocity of antero- and retrograde transport of APP-EGFP in distal axons of DIV8 ctrl and KO neurons supplemented with Veh or NAD+. Numbers (neurons imaged) and statistics: 56 ctrl+Veh, 51–58 KO+Veh, 17 ctrl+NAD+, and 47–48 KO+NAD+ for anterograde velocity, (anterograde) **p= 0.0041, (retrograde) **p= 0.0013, ***p= 0.0002. (E) Schematic representation of how glycolysis and mitochondrial respiration are affected by Oligo or 2DG(+Methyl-Pyr) treatment. (F) Percentages of APP-EGFP vesicles moving anterogradely, retrogradely, or pausing in distal axons of DIV8 ctrl and KO+NAD+ neurons in the presence of vehicle, Oligo, or 2DG. Numbers (neurons imaged) and statistics: 56 ctrl+Veh, 39 ctrl+oligo, and 20 ctrl+2DG, **p=0.0059; 49 KO+NAD++Veh, 22–25 KO+NAD++Oligo,and 15–18 KO+NAD++2DG, *p= 0.0291, #p= 0.0252. Significant comparisons to Veh-treated and Oligo-treated groups were labeled with * and #, respectively. (G) Velocity of antero- and retrograde transport of APP-EGFP in distal axons of DIV8 ctrl and KO+NAD+ neurons in the presence of vehicle, Oligo, or 2DG. Numbers (neurons imaged) and statistics: 56 ctrl+Veh, 38–39 ctrl+oligo, 10 ctrl+2DG, 47–48 KO+NAD+, 24 KO+NAD++Oligo, and 12–13 KO+NAD++2DG, **p=0.0011. All above data represent mean ± SEM, two-way ANOVA with Tukey’s multiple comparisons test or Kruskal-Wallis test with Dunn’s multiple comparisons test were used, see details in supplemented excel, ****p<0.0001, ####p<0.0001.