Fig 1.
Experimental approach to study ATP homeostasis in the optic nerve of Plpnull/y mice.
Plpnull/y mice were crossbred with ThyAT mice expressing the fluorescent ATP sensor ATeam1.03YEMK in neurons. (A) Scheme of the experimental setup allowing imaging of the ATP sensor while simultaneously stimulating and recording from optic nerves (modified from [8]). (B) Confocal image of an acutely recorded optic nerve from a ThyAT/Plpnull/y mouse showing axonal swellings (red arrowheads). Shown is the YFP channel of the ATP sensor. Scale bar: 10 μm. (C) Electron microscopic image of an axonal swelling in an optic nerve axon of a Plpnull/y mouse. Scale bar: 500 nm. (D) Compound action potential (CAP) of optic nerves of wild-type (control [Ctrl]) and Plpnull/y mice. Shown is the mean ± SEM of n = 10 and 8 optic nerves from N = 10 and 8 mice for wild type and Plpnull/y, respectively. (E) Excitability of optic nerve measured at different stimulus intensities (0–1 mA). Shown is the mean ± SEM of n = 10 and 8 optic nerves from N = 10 and 8 mice for Ctrl and Plpnull/y, respectively. ***p < 0.001, Student t test. (F) Same data as in (E), but normalized to the maximal CAP. Data underlying this figure can be found in S1 Data.
Fig 2.
Axoplasmic ATP concentration is reduced in Plpnull/y mice under basal conditions.
(A) ATP sensor ratio at baseline conditions. n = 10 and n = 8 nerves from N = 10 and 8 mice (age 10 weeks) for ThyAT/Plpwt/y (control [Ctrl]) and ThyAT/Plpnull/y (Plpnull/y), respectively. (B) Fluorescence decay of the donor fluorophore of the ATP sensor in optic nerve axons of Ctrl and Plpnull/y mice under baseline incubation conditions. A slower decay and, consequently, an increase of the fluorescence lifetime correspond to a decrease in ATP concentration. The inset shows the instrument response function (IRF). Shading represents SEM. (C) Average fluorescence decay of the same axons as in (B) but after depletion of ATP by mitochondrial blockage and glucose deprivation (MB+GD). (D) Quantification of the fluorescence lifetime (ns) of the ATP sensor in axons under baseline conditions, indicating a lower ATP concentration in Plpnull/y nerves. (E) Quantification of the fluorescence lifetime
(ns) of the ATP sensor in axons exposed to MB+GD, indicating that the longest fluorescent lifetime is obtained for each nerve under this energy-depriving condition. The average fluorescence decay of n = 6–9 nerves from N = 4–5 mice (10 weeks old) is shown in (B–E). (F) The variability of the ATP concentration within individual axons was analyzed by calculating the coefficient of variation (CV) of the fluorescence lifetime of all pixels within an axon; 88 and 79 axons from n = 9 and 8 nerves from N = 5 and 4 animals were analyzed for control and Plpnull/y nerves, respectively. (G) CV of the ATP sensor fluorescence lifetime of axons within a nerve. 8 to 10 axons each from n = 9 and 8 nerves from N = 5 and 4 animals were included in the analysis for control and Plpnull/y nerves, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; Student t test (A, D, E, G), Welch’s test (F). Data underlying this figure can be found in S1 Data.
Fig 3.
During electrical stimulation the decrease in compound action potential (CAP), but not of ATP, is larger in Plpnull/y mice.
Optic nerves from control (Ctrl) or Plpnull/y mice were challenged by electrical stimulation increasing every 30 s stepwise from 1 Hz to 100 Hz. (A) Time course of the ATP sensor signal normalized to baseline (set as 1) and MB+GD (mitochondrial blockage and glucose deprivation; set as 0). (B) Quantification of the ATP sensor signal for the last 15 s during each stimulation with the indicated frequencies. Same nerves as in (A). (C) Dynamics of CAPs normalized to baseline (set as 1) and MB+GD (set as 0). (D) Quantification of the CAPs for the last 15 s during each stimulation with the indicated frequencies. Same nerves as in (C). Shown is the mean ± SEM. Dots in (B) and (D) indicate data points from single nerves; n = 6 (ATP Ctrl), 7 (CAP Ctrl), and 5 (ATP/CAP Plpnull/y) nerves from N = 6, 7, and 5 mice, respectively. **p < 0.01; Student t test (B and D). Data underlying this figure can be found in S1 Data.
Fig 4.
ATP levels in optic nerves from Plpnull/y mice recover faster and more completely from glucose deprivation (GD).
Optic nerves from Plpwt/y (control [Ctrl]) or Plpnull/y mice were challenged by GD for 30 min (indicated by the light blue box). Afterwards, glucose (10 mM) was reperfused for an additional 45 min. (A) Time course of the ATP sensor signal in Ctrl and Plpnull/y mice. (B) Rate of ATP sensor signal decay. (C) Delay time of the onset of recovery of the ATP sensor signal after the start of reperfusion with glucose. (D) Amplitude of recovery of the ATP sensor signal after reperfusion with glucose. (E) Time course of compound action potentials (CAPs) in Ctrl and Plpnull/y mice. (F) Rate of CAP decay. (G) Delay time of the onset of recovery of CAP after the start of reperfusion with glucose. (H) Amplitude of recovery of CAP after reperfusion with glucose. n = 5 (Ctrl; B, D, F, H), 7 (Ctrl; C, G), and 5 (Plpnull/y) optic nerves from N = 5, 7, and 5 mice, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; Student t test. Data underlying this figure can be found in S1 Data.
Fig 5.
Structure of myelin in Plpnull/y optic nerves.
(A–C) Electron micrographs of high-pressure frozen optic nerves of a 10-week-old control (Ctrl) (A) and Plpnull/y (B and C) mouse. Asterisks indicate unmyelinated axons; white arrows highlight an axonal spheroid (B) and cytosolic channels (C). The scale bars correspond to 500 nm. (D) Genotype-dependent quantification of the number of myelinated axons in optic nerves dissected from Ctrl and Plpnull/y mice; 100% refers to all axons. (E) Genotype-dependent quantification of the percentage of axon–myelin units comprising axonal swellings; 100% refers to all myelinated axons.(F) Genotype-dependent quantification of the percentage of axon–myelin units with myelin containing cytosolic channels; 100% refers to all myelinated axons. In (D–F), n = 4 and 4 nerves from N = 4 and 4 mice for Ctrl and Plpnull/y, respectively. *p < 0.05, ***p < 0.001; Student t test. (G) 3D rendering of the structure of an axonal segment and the inner layers of the myelin sheaths obtained using focused ion beam scanning electron microscopy (FIB-SEM). For simplicity, the outermost layer of myelin is not visualized in the 3D reconstruction. Cytosolic channels are colored yellow, the axon is in light blue, and organelle-like structures are in purple. The scale bar corresponds to 200 nm. See S1 Movie for an animated visualization of this 3D structure. Data underlying this figure can be found in S1 Data.
Fig 6.
Abundance and localization of GLUT1 and MCT1 in myelin.
(A) Immunoblot analysis of myelin purified from the brains of wild-type (control [Ctrl]) and Plpnull/y mice probed for glucose transporter 1 (GLUT1) and monocarboxylate transporter 1 (MCT1). β-actin (ACTB) was detected as a loading control; PLP/DM20 was detected as genotype control. Blot represents N = 4 individual mice for each genotype. (B and C) Genotype-dependent quantification of the immunoblots for GLUT1 (B) and MCT1 (C). The abundance of both proteins is significantly increased in myelin purified from Plpnull/y brains (N = 4 mice for both genotypes). (D and E) Quantitative RT-PCR analysis of the abundance of Glut1 (D) and Mct1 (E) mRNAs in the corpus callosum dissected from wild-type (Ctrl) and Plpnull/y mice (N = 4 mice for both genotypes). In (B–E): **p < 0.01, ***p < 0.001; Student t test. (F–I) Cryo-immuno electron microscopy to assess the localization of GLUT1 (F and G) and MCT1 (H and I) protein in the myelin of optic nerves dissected from wild-type (F and H) and Plpnull/y (G and I) mice. White arrows point at gold particles indicating localization of the respective proteins. The scale bars in (F–I) correspond to 200 nm. White boxes are 2.6× zoomed-in magnification of the corresponding box highlighting the gold particles. Original, uncropped Western blots are available in S1 Raw Images. Data underlying this figure can be found in S1 Data.