The Role of Netrin-1 in Improving Functional Recovery through Autophagy Stimulation Following Spinal Cord Injury in Rats

Abstract

Our previous findings indicated that treatment with Netrin-1 could improve functional recovery through the stimulation of autophagy, by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway in rats following spinal cord injury (SCI). However, the underlying mechanisms were not elucidated. The purpose of this study was to investigate the underlying mechanisms by which Netrin-1 promotes autophagy and improves functional recovery after SCI. Following controlled SCI in Sprague-Dawley rats, we observed that the autophagic flux in neurons was impaired, as reflected by the accumulation of light chain 3-II (LC3-II)-positive and LC3-positive autophagosomes (APs), accompanied by the accumulation of the autophagic substrate, Sequestosome 1 (SQSTM1; also known as p62). Our results showed that treatment with Netrin-1 increases the levels of the lysosomal protease cathepsin D (CTSD) and lysosomal-associated membrane protein 1 (LAMP1), through the regulation of the nuclear localization of Transcription factor EB (TFEB) via the AMPK/mTOR signaling pathway. In addition, this enhancement of lysosomal biogenesis correlated strongly with the restoration of autophagic flux, inhibition of neural apoptosis and improved functional recovery. Suppression of lysosomal biogenesis via the inhibition of the nuclear translocation of TFEB by Compound C abolished this restoration of autophagic flux and the functional recovery effects of Netrin-1 following SCI. Taken together, these results indicate that Netrin-1 enhances lysosomal biogenesis by regulating the nuclear translocation of TFEB via the AMPK/mTOR signaling pathway. Furthermore, the enhancement of lysosomal biogenesis by Netrin-1 following SCI promotes autophagic flux and improves functional recovery in rats. Thus, the regulation of lysosomal biogenesis by modulating the nuclear localization of TFEB might be a novel approach for the treatment of SCI.

Keywords: AMPK; Netrin-1; apoptosis; autophagic flux; mTOR; spinal cord injury; transcription factor EB.

Figure 1

Netrin-1-promotes transcription factor EB (TFEB) nuclear translocation via the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signal pathway after spinal cord injury (SCI). (A) Western blots of p-AMPK, p-Acetyl-CoA Carboxylase (p-ACC), p-mTOR, p-P70S6K, TFEB, H3 and β-tubulin in each group. (B,C,E) Quantification of p-AMPK, p-ACC, p-mTOR, p-P70S6K, TFEB, H3 and β-tubulin in each group (n = 5). (D) Double staining for NeuN (green)/TFEB (red) of sections from the spinal cord in each group. (F) Quantification of the nuclear-translocation neurons of TFEB in each group (n = 5); *P < 0.05 vs. SCI group, **P < 0.01 vs. SCI group, ^&P < 0.05 vs. Netrin-1 group ^&&P < 0.01 vs. Netrin-1 group.

Figure 2

Netrin-1 enhances lysosomal biogenesis after SCI. (A) Western blots of lysosomal-associated membrane protein 1 (LAMP1), ATP6V1A, CTSD (Pre-CTSD and Mature-CTSD) and β-tubulin in each group. (B,C,E) Quantification of LAMP1, ATP6V1A, CTSD (Pre-CTSD and Mature-CTSD) and β-tubulin in each group (n = 5). (D) Double staining for NeuN (green)/LAMP1 (red) of sections from the spinal cord in each group. (F) Quantification of the number of LAMP1-positive neurons in each group (n = 5); *P < 0.05 vs. SCI group, **P < 0.01 vs. SCI group, ^&P < 0.05 vs. Netrin-1 group ^&&P < 0.01 vs. Netrin-1 group.

Figure 3

Netrin-1 promotes autophagy flux after SCI. (A) Western blots of light chain 3 (LC3)-I, LC3-II, p62 and β-tubulin in each group. (B,C) Quantification of LC3-I, LC3-II, p62 and β-tubulin in each group (n = 5). (D) Double staining for NeuN (green)/LC3 (red) of sections from the spinal cord in each group (white arrows: LC3-positive neurons). (E) Quantification of the number of LC3-positive neurons in each group (n = 5). (F) Double staining for NeuN (green)/p62 (red) of sections from the spinal cord in each group. (G) Quantification of the number of p62-positive neurons in each group (n = 5). *P < 0.05 vs. SCI group, **P < 0.01 vs. SCI group; ^&&P < 0.01 vs. Netrin-1 group.

Figure 4

Netrin-1 attenuates neural apoptosis after SCI. (A) Western blots of C-caspase 3, Bax, Bcl-2 and β-tubulin in each group. (B,C) Quantification of C-caspase 3, Bax, Bcl-2 and β-tubulin in each group (n = 5). (D) Double staining for NeuN (green)/ C-caspase 3 (red) of sections from the spinal cord in each group. (E) Quantification of the number of C-caspase 3-positive neurons in each group (n = 5); **P < 0.01 vs. SCI group; ^&&P < 0.01 vs. Netrin-1 group.

Figure 5

Netrin-1 decreases the loss of neurons after SCI. (A) Nissl staining of sections from the spinal cord in each group at 28 days after SCI, Black arrows: surviving neurons. (B) Quantification of surviving neurons in each group (n = 5); **P < 0.01 vs. SCI group; ^&&P < 0.01 vs. Netrin-1 group.

Figure 6

Netrin-1 improves functional recovery after SCI. Basso, Beattie and Bresnahan (BBB) scores of rats in each group were evaluated at 0, 1, 3, 7, 14, 21 and 28 days after operation. **P < 0.01 vs. SCI group.