Akt/mTOR Pathway Agonist SC79 Inhibits Autophagy and Apoptosis of Oligodendrocyte Precursor Cells Associated with Neonatal White Matter Dysplasia

Abstract

White matter dysplasia (WMD) in preterm infants due to intrauterine inflammation is caused by excessive apoptosis of oligodendrocyte precursor cells (OPCs). In recent years, studies have found that excessive autophagy and apoptosis are highly interconnected and important in infection and inflammatory diseases in general. Therefore, in this study, we aimed to confirm whether regulation of autophagy by using the Akt phosphorylation agonist SC79 can inhibit abnormal apoptosis of OPCs and promote myelin maturation and white matter development in neonatal rats with WMD. We investigated the effect of inflammation on oligodendrocyte development in P0 neonatal rats by intracerebellar injection of LPS, and collected brain tissue at P2 and P5. Immunohistochemical and immunofluorescence staining were used to evaluate white matter damage, while immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling analysis (TUNEL), and western blotting were used to evaluate autophagy and apoptosis. First, we observed that white matter development was arrested and white matter fiber maturation was impaired in LPS-inflicted pups compared with those in the sham-operated group. Second, treatment with SC79 reduced the levels of LC3II, caspase 3, caspase 9, and Bax/Bcl-2 and increased the levels of p62, p-Akt, and p-mTOR in the brain tissue of neonatal rats. Finally, SC79 treatment inhibited OPC apoptosis by increasing the binding of Beclin 1 to Bcl-2, which promoted OPC differentiation and maturation. However, the opposite results were observed after rapamycin administration. Taken together, our results suggest that SC79 can inhibit the abnormal apoptosis of OPCs caused by excessive autophagy through the Akt/mTOR pathway and that SC79 is a potential therapeutic agent for WMD in preterm infants.

Keywords: Apoptosis; Autophagy; Oligodendrocyte precursor cells; SC79; White matter dysplasia.

Fig. 1

White matter maturation disorders in neonatal rats after intracerebroventricular injection of LPS. a White matter of rats in the sham and LPS groups stained with H&E at P2 and P5. Scale bar = 50 μm. a, b Immunohistochemical analysis of CNPase expression at P2 and P5 and the AOD (∗∗∗∗P < 0.0001 at P2, ∗∗∗P < 0.001 at P5; n = 6). a, c Mean signal intensity of MBP (∗∗∗∗P < 0.0001 at P2, ∗∗P < 0.01 at P5; n = 6). Data are represented as mean ± SEM. Scale bar = 50 μm

Fig. 2

LPS increased OPC apoptosis and up-regulated apoptotic protein expression. a, b TUNEL staining of tissues from sham-operated and LPS-treated groups. TUNEL-positive cells are indicated in green. O4+ cells are indicated in red. (n = 6; ∗∗P < 0.01, P2). c, d Western blotting indicating relative expression of apoptosis-related proteins (Compared with the grey value of β-actin ), including Bax/Bcl-2, cleaved caspase-3, and cleaved caspase-9, in the brain tissue of rats in the sham-operated and LPS groups (n = 6; ∗P < 0.05). Data are represented as mean ± SEM. Scale bar = 50 μm

Fig. 3

Autophagy protein expression and activation in the brain tissue of neonatal rats at P2. a, b Relative expression of autophagy-related proteins (Compared with the grey value of β-actin ) in the brain tissue of neonatal rats in the sham-operated and LPS groups evaluated with western blotting. LC3-II (∗P < 0.05), LC3-II/I (∗P < 0.05), p62 (∗∗P < 0.01), p-Akt/Akt (∗P < 0.05), p-mTOR/mTOR (∗∗P < 0.01). n = 6; data are presented as mean ± SEM

Fig. 4

Autophagy activation in white matter. a–c Fluorescent staining and colocalization analysis of anti-apoptotic protein Bcl-2 (red) and autophagy initiation protein Beclin 1 (green) in the periventricular white matter of neonatal rats. d–f Confocal microscopy and colocalization analysis of Bcl-2 (red), Beclin 1 (green), and OPC marker O4 (yellow) in the periventricular white matter of neonatal rats. n = 6; data are presented as mean ± SEM. Scale bar = 50 μm

Fig. 5

Effect of SC79 treatment on OPC maturation and myelination. a, c Immunohistochemical analysis and AOD of CNPase expression in the periventricular white matter of neonatal rats at P5 (n = 6; ∗P < 0.05, ∗∗P < 0.01). b, d Immunofluorescence and mean fluorescence intensity of MBP in the periventricular white matter of neonatal rats at P5 (n = 6; ∗∗P < 0.01, ∗∗∗∗P < 0.0001). Data are presented as mean ± SEM. Scale bar = 50 μm. f Neonatal rat body weight at the postnatal time points P0, P2, and P5 (n = 8)

Fig. 6

Effect of SC79 treatment on the Akt/mTOR signaling pathway. a–g Western blotting was used to quantify Beclin 1, LC3-II, LC3-II/I, p62, p-Akt/Akt, and p-mTOR/mTOR expression in the brain tissue of neonatal rats in the Sham, LPS, LPS+SC79, and LPS+RAPA groups at P2 (Compared with the grey value of β-actin ) (∗P < 0.05; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). n = 6; data are presented as mean ± SEM

Fig. 7

Effect of SC79 treatment on OPC apoptosis. a–d Western blot analysis of apoptotic protein caspase-9, caspase-3, and Bax/Bcl-2 expression in neonatal rat brain tissue for the Sham, LPS, LPS+SC79, and LPS+RAPA groups (Compared with the grey value of β-actin ) (∗∗P < 0.01). e, f Double fluorescence staining and quantification with anti-O4 antibody (red) and TUNEL (green). Double-positive O4+ and TUNEL+ cells were considered apoptotic OPCs (∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001). n = 6; data are represented as mean ± SEM. Scale bar = 50 μm

Fig. 8

Effect of SC79 treatment on autophagy activation in white matter. a, b Immunofluorescence staining of the anti-apoptotic protein Bcl-2 (red) and autophagy initiation protein Beclin 1 (green), and their colocalization in the periventricular white matter of neonatal rat brain tissue. n = 6. Scale bar = 50 μm