Article Figures & Data
Figures
- Figure 1.
Mitochondria in myelinated and demyelinated axons of DRG neurons. A, Representative live imaging of myelinated DRG cultures infected by mito-DsRed2 lentivirus. An asterisk shows the cell body of a DRG neuron. The arrows indicate predicted nodes of Ranvier. Mitochondria labeled by fluorescent red are distributed in the axon as well as the cell body. Note that surrounding Schwann cells are not labeled. B, Colabeling of mitochondria with DsRed2 (red), Na+ channel (green), and MBP (blue) by immunostaining. C, An electron micrograph of a myelinated DRG axon. Multiple elongated mitochondria were clustered at single sites (arrowhead); their spatial separation and cristae are visible at higher magnification (inset). This arrangement of mitochondria establishes that many mitochondrial stationary sites contain multiple mitochondria. D–I, Myelination and demyelination in DRG cultures. Immunostaining of MBP (green) and neurofilament (NF) (red) in control culture (D–F) and culture 2 d after lysolecithin (Lyso) treatment (G–I). The insets show magnified images of the areas marked with rectangles in F and I. Note that continuous MBP-positive myelin sheath in control (D–F) becomes discontinuous and fragmented after Lyso treatment (G–I). Scale bars: C, 500 nm; C, inset, 250 nm; others, 10 μm.
- Figure 2.
Mitochondrial behavior before and after demyelination. A, B, Single frames from the time-lapse images. A′, B′, Kymographs of the time-lapse images. C, Sizes of stationary mitochondrial site in myelinated (control; gray bars) and demyelinated (Lyso; black bars) axons. The size of stationary mitochondrial sites is significantly increased (p < 0.01) in the demyelinated axons [n = 185 mitochondria (control); n = 182 mitochondria (lysolecithin treated)].
- Figure 3.
Lysolecithin does not alter mitochondrial behavior in unmyelinated axons. A, B, Single frames from the time-lapse images of untreated (A) and lysolecithin-treated (B) unmyelinated axons. A′, B′, Representative kymographs of the time-lapse images from unmyelinated axons with (B′) or without (A′) lysolecithin treatment. C, Sizes of stationary mitochondrial sites in unmyelinated axons with (Lyso) or without (control) lysolecithin treatment. The sizes are similar in both groups. n = 180 mitochondria (control); n = 181 mitochondria (Lyso).
- Figure 4.
Mitochondrial transport velocity and stationary mitochondrial sites are similar in myelinated and remyelinated axons. A, Immunostaining for MBP in control, demyelinated [2 d after lysolecithin treatment (Lyso 2d)] and remyelinated [14 d after lysolecithin treatment (Lyso 14d)] cultures. Continuous tubular MBP-positive myelin (control) becomes fragmented in Lyso 2d, but is replaced in Lyso 14d. Scale bars, 10 μm. B, Size changes of stationary mitochondrial sites in control (gray bars), demyelinated (black bars), and remyelinated (white bars) axons. The sizes of stationary mitochondrial sites increased after demyelination (p < 0.01) but were similar in myelinated and remyelinated axons. n = 124 (control), 128 (demyelination), and 127 (remyelination) mitochondria. C, Mean velocity of motile mitochondria in control, demyelinated, and remyelinated axons. After demyelination, the mean velocity of motile mitochondria was significantly increased (*p < 0.01). The mitochondrial transport velocity was similar in myelinated and remyelinated axons. n = 158 (control), 194 (demyelination), and 183 (remyelination) mitochondria. Error bars indicate SD.
- Figure 5.
Expression of ATF3 protein and its effect on mitochondrial transport in DRG axons after demyelination. A, ATF3 (green) and neurofilament (red) distribution in myelinated DRG neurons with [Lyso(+)] or without [Lyso(−)] lysolecithin treatment. Note dense nuclear ATF3 immunostaining is observed in DRG neurons after lysolecithin treatment (arrows). B, Immunostaining for ATF3 in unmyelinated DRG cultures with [Lyso(+)] or without [Lyso(−)] lysolecithin treatment. Immunoreactivity of ATF3 is not increased after lysolecithin treatment of unmyelinated DRG cultures. C, D, Sizes of stationary mitochondrial sites in control shRNA- and ATF3 shRNA-treated axons with (D) or without (C) lysolecithin (Lyso) treatment. The sizes of stationary mitochondria are similar in control- and ATF3-shRNA treated cultures (n = 139 mitochondria for control-shRNA without lysolecithin, 147 for ATF3-shRNA without lysolecithin, 181 for control-shRNA with lysolecithin, and 180 for ATF3-shRNA with lysolecithin). E, Mean velocity of motile mitochondria in control shRNA- and ATF3 shRNA-treated axons. After demyelination, the increase in mitochondrial transport velocity was still observed under control-shRNA treatment but abolished under ATF3-shRNA treatment (n = 10 axons for control-shRNA without lysolecithin, 20 for ATF3-shRNA without lysolecithin, 11 for control-shRNA with lysolecithin, and 21 for ATF3-shRNA with lysolecithin; *p < 0.01). Error bars indicate SD. F, Immunostaining for nitrotyrosine (Nitro; green) in the demyelinated axons of DRG neurons infected with mito-DsRed (red) and treated with lysolecithin [Lyso(+)]. The neurons were also treated with control- or ATF3-shRNA. Immunoreactivity of nitrotyrosine in mitochondria was increased in ATF3 shRNA-treated DRG axons (arrowheads). Scale bars: A, B, 20 μm; F, 5 μm.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Movie 1
- supplemental material - Supplemental Movie 2
