Rapamycin Enhances Mitophagy and Attenuates Apoptosis After Spinal Ischemia-Reperfusion Injury

Abstract

The spinal cord is extremely vulnerable to ischemia-reperfusion (I/R) injury, and the mitochondrion is the most crucial interventional target. Rapamycin can promote autophagy and exert neuroprotective effects in several diseases of the central nervous system. However, the impact of rapamycin via modulating mitophagy and apoptosis after spinal cord ischemia-reperfusion injury remains unclear. This study was undertaken to investigate the potential role of rapamycin in modulating mitophagy and mitochondria-dependent apoptosis using the spinal cord ischemia-reperfusion injury (SCIRI) mouse model. We found that rapamycin significantly (p < 0.05) enhanced mitophagy by increasing the translocation of p62 and Parkin to the damaged mitochondria in the mouse spinal cord injury model. At the same time, rapamycin significantly (p < 0.05) decreased mitochondrial apoptosis related protein (Apaf-1, Caspase-3, Caspase-9) expression by inhibiting Bax translocation to the mitochondria and the release of the cytochrome c from the mitochondria. After 24 h following SCIRI, rapamycin treatment reduced the TUNEL⁺ cells in the spinal cord ischemic tissue and improved the locomotor function in these mice. Our results therefore demonstrate that rapamycin can improve the locomotor function by promoting mitophagy and attenuating SCIRI -induced apoptosis, indicating its potential therapeutic application in a spinal cord injury.

Keywords: apoptosis; ischemia-reperfusion injury; mitophagy; rapamycin; spinal cord.

FIGURE 1

Rapamycin treatment enhanced mitophagy after spinal cord ischemia-reperfusion injury. (A). Schematic diagram of the experimental design. Experimental groups were treated with vehicle and rapamycin (intraperitoneal injection, 1mg/kg). Mice received treatmetn at reperfusion onset and were sacrificed at 24 h after SCIRI. (B) Immunohistochemical localization of VDAC1 (mitochondrial marker) and LC3B (autophagy marker) co-staining in spinal cord at 24 h after SCIRI. VDAC1 (red), LC3B (green). The inset images represent higher magnification of the boxed area in the corresponding merged images. (C). Electron micrographs demonstrating neuron mitophagy structure in rapamycin-treated SCIRI mice. Autophagic vacuoles, autolysosomes (AL), typical mitophagy structure and swollen mitochondria (black arrow)were observed in neurons. High magnifications in (b) revealed a typical autolysosomes structure. High magnifications in (c) revealed a typical mitophagy structure, that is a degradative autophagic vacuole (Avd) including a partially degraded mitochondrial. (b) is the high magnification of lower-left box in the figure C-a and (C) is the high magnification of upper-right box in the figure C-a. M-mitochondrial, N-nuclear, V-vacuole. scale bar = 0.5 μm or 2 μm. (C) Representative western blots and quantitative graphs demonstrate the expression of LC3-I /LC3-II in the whole homogenates at 24 h after SCIRI. (D) Representative western blots and quantitative graphs demonstrate the expression of LC3-I and LC3-II in the mitochondrial fractions at 24 h after SCIRI. Data were shown as mean ± SD, n = 6. ^∗p < 0.05, ^∗∗p < 0.01 vs. Sham; ^#p < 0.05, ^##p < 0.01.

FIGURE 2

Rapamycin enhanced mitophagy via promoting p62, Parkin translocation to the mitochondria. (A–C) Representative western blots and quantitative graphs demonstrate the expression of p62 in the whole homogenates, in the cytosolic fractions and in the mitochondrial fractions at 24 h after SCIRI. Data were shown as mean ± SD, n = 6. ^∗p < 0.05, ^∗∗p < 0.01 vs; Sham, ^#p < 0.05, ^##p < 0.01. (D-F) Representative western blots and quantitative graphs demonstrate the expression of Parkin in the whole homogenates, in the cytosolic fractions and in the mitochondrial fractions at 24 h after SCIRI. Cox- IV is as inner control for mitochondria protein, β-actin is as inner control for tissue homogenate protein. Data were shown as mean ± SD, n = 6. ^∗p < 0.05, ^∗∗p < 0.01 vs. Sham; ^#p < 0.05, ^##p < 0.01.

FIGURE 3

Rapamycin treatment following spinal cord ischemic injury reduced mitochondrial release of apoptosis related proteins. (A,B) Representative western blots and quantitative graphs demonstrate the expression of Bax in cytosolic and mitochondrial fractions at 24 h after SCIRI. Data are presented as the mean ± SD, n = 6. ^∗p < 0.05, ^∗∗p < 0.01 vs. Sham; ^#p < 0.05, ^##p < 0.01. (C,D) Representative western blots and quantitative graphs demonstrate the expression of Cyt-c in cytosolic and mitochondrial fractions at 24 h after SCIRI. Data are presented as the mean ± SD, n = 6. ^∗p < 0.05, ^∗∗p < 0.01 vs. Sham; ^#p < 0.05, ^##p < 0.01. (E–H) Representative western blots and quantitative graphs demonstrate the expression of mitochondrial apoptosis related proteins (Apaf-1, cleaved Caspase-3, cleaved Caspase-9) at 24 h after SCIR. Data are presented as the mean ± SD, n = 6. ^∗p < 0.05, ^∗∗p < 0.01 vs. Sham; ^#p < 0.05, ^##p < 0.01.

FIGURE 4

Rapamycin attenuated spinal cord ischemia-reperfusion induced apoptosis. (A). These cells were stained with TUNEL and DAPI. The merged picture shows colocolization of apoptotic and nuclear markers. Scare bar = 50 μm. (B).The bar graph shows rapamycin treatment rescued neurons from apoptosis at 24 h after SCIR. Data were shown as mean ± SD, n = 6. The numbers of apoptotic cells were counted in three sections of ischemic spinal cord. ^∗p < 0.05, ^∗∗p < 0.01 vs. Sham; ^#p < 0.05, ^##p < 0.01. (C) Bar graphs show the rapamycin-treated mice have significantly higher BMS scores than the vehicle-treated mice at 24 h after injury. Data were shown as mean ± SD, n = 16. ^∗p < 0.05, ^∗∗p < 0.01 vs. Sham; ^#p < 0.05, ^##p < 0.01.