Extended Data Fig. 2: Early axonal ATP deficits measured using ATeam in EAE.
(a) Experimental design for axonal ATP level measurement in experimental autoimmune encephalomyelitis (EAE). (b) Maximum intensity projections of in vivo multi-photon image stacks of spinal cord axons of control (left) and acute EAE (right) in AAV.PHPeB.hSyn:ATeam injected C57BL/6 mice. Top: Grayscale look-up table (LUT; λex 840 nm). Bottom: Ratiometric [ATP]axon LUT (YFP/CFP emission ratio). (c) Details from b. Top to bottom: [ATP]axon images of control axon in healthy spinal cord and normal-appearing, swollen, and fragmented axons in acute EAE. (d) [ATP]axon of single axons in healthy and EAE mice normalized to mean of controls. Mean ± s.e.m.; n = 157 axons from three control and 195 axons from four EAE mice compared by Kruskal-Wallis and Dunn’s multiple comparison test; values above 1.5 lined up on the “≥1.5” dashed line, p < 0.001, control versus control + CCCP; p = 0.001, 5.4 × 10-7, < 0.001, control versus stage 0, 1 and 2, respectively; p = 6.1 × 10-6, 0.0057, > 0.9999, EAE + CCCP versus stage 0, 1 and 2, respectively. (e) [pH]axon of single axons measured by using SypHer3s sensor in control and acute EAE mice normalized to mean of controls. Mean ± s.e.m.; n = 34 axons from two control and 34 axons from two EAE mice, compared Kruskal-Wallis and Dunn’s multiple comparison test, p > 0.9999, control versus stage 0, 1 and 2, respectively. Scale bars: 25 μm in b; 10 μm in c. **, p < 0.01; ****, p < 0.001. See source data for individual data points and further statistical parameters. Illustration created with BioRender.
