Metformin-induced AMPK activation stimulates remyelination through induction of neurotrophic factors, downregulation of NogoA and recruitment of Olig2+ precursor cells in the cuprizone murine model of multiple sclerosis

Abstract

Purpose: Oligodendrocytes (OLGs) damage and myelin distraction is considered as a critical step in many neurological disorders especially multiple sclerosis (MS). Cuprizone (cup) animal model of MS targets OLGs degeneration and frequently used to the mechanistic understanding of de- and remyelination. The aim of this study was exploring the effects of metformin on the OLGs regeneration, myelin repair and profile of neurotrophic factors in the mice brain after cup-induced acute demyelination.

Methods: Mice (C57BL/6 J) were fed with chow containing 0.2% cup for 5 weeks to induce specific OLGs degeneration and acute demyelination. Next, the cup was withdrawn to allow one-week recovery (spontaneous remyelination). At the end of this period, mature OLGs markers, myelin-associated neurite outgrowth inhibitor protein A (NogoA), premature specific OLGs transcription factor (Olig2), anti-apoptosis marker (survivin), neurotrophic factors, and AMPK activation were monitored in the presence or absence of metformin (50 mg/kg body weight/day) in the corpus callosum (CC).

Results: Our finding indicated that consumption of metformin during the recovery period potentially induced an active form of AMPK (p-AMPK) and promoted repopulation of mature OLGs (MOG+ cells, MBP+ cells) in CC through up-regulation of BDNF, CNTF, and NGF as well as down-regulation of NogoA and recruitment of Olig2+ precursor cells.

Conclusions: This study for the first time reveals that metformin-induced AMPK, a master regulator of energy homeostasis, activation following toxic demyelination could potentially accelerate regeneration and supports spontaneous demyelination. These findings suggest the development of new therapeutic strategies based on AMPK activation for MS in the near future. Graphical abstract An overview of the possible molecular mechanisms of action of metformin-mediated remyelinationa.

Keywords: AMPK; Cuprizone; Metformin; Multiple sclerosis; Remyelination.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Graphical abstract

An overview of the possible molecular mechanisms of action of metformin-mediated remyelinationa

Fig. 1

Timelines of the experimental design (see study design part for details)

Fig. 2

Effects of metformin (50 mg/kg body weight/day) on mRNA expression of neurotrophic factors (BDNF/CNTF/NGF). First, the data normalized with the internal control (β-actin) and then indicated as fold change to the control group. Control (gray): normal mice on a regular diet and injected with vehicle during last week, metformin (black): normal mice on a regular diet and injected with metformin during last week, free recovery group (white): model mice with regular diet and injected with vehicle during last recovery week, metformin recovery group (dotted): model mice with regular diet and injected with metformin during last recovery week. Data represent the mean ± S.E.M analyzed by two-way ANOVA. ٭compared to control group, #compared to free recovery group. ٭, # (P < 0.05) with Bonferroni’s correction for multiple comparisons

Fig. 3

Effects of metformin (50 mg/kg body weight/day) on mRNA expression of the mature OLGs markers (MBP, MOG) and the anti-apoptosis marker (survivin). First, the data normalized with the internal control (β-actin) and then indicated as fold change to the control group. Control (gray): normal mice on a regular diet and injected with vehicle during last week, metformin (black): normal mice on a regular diet and injected with metformin during last week, free recovery group (white): model mice with regular diet and injected with vehicle during last recovery week, metformin recovery group (dotted): model mice with regular diet and injected with metformin during last recovery week. Data represent the mean ± S.E.M analyzed by two-way ANOVA. ٭compared to control group, #compared to free recovery group. ٭, # (P < 0.05) with Bonferroni’s correction for multiple comparisons

Fig. 4

Effects of metformin (50 mg/kg body weight/day) on population of the NogoA and Olig2 positive cells. Earliest, positive cells (immunofluorescence signals) from each independent group were counted and they mean are calculated. Then the data reported as a fold change to control group. Control (gray): normal mice on a regular diet and injected with vehicle during last week, metformin (black): normal mice on a regular diet and injected with metformin during last week, free recovery group (white): model mice with regular diet and injected with vehicle during last recovery week, metformin recovery group (dotted): model mice with regular diet and injected with metformin during last recovery week. Double staining for Olig2 and NogoA were performed and arrows indicated some positive signals in CC area. Data represent the mean ± S.E.M analyzed by two-way ANOVA. ٭compared to control group, #compared to free recovery group. ٭, # (P < 0.05) with Bonferroni’s correction for multiple comparisons

Fig. 5

Effects of metformin (50 mg/kg body weight/day) on mRNA expression of the premature OLGs marker (Olig2) and the growth inhibitory marker (NogoA). First, the data normalized with the internal control (β-actin) and then indicated as fold change to the control group. Control (gray): normal mice on a regular diet and injected with vehicle during last week, metformin (black): normal mice on a regular diet and injected with metformin during last week, free recovery group (white): model mice with regular diet and injected with vehicle during last recovery week, metformin recovery group (dotted): model mice with regular diet and injected with metformin during last recovery week. Data represent the mean ± S.E.M analyzed by two-way ANOVA. ٭compared to control group, #compared to free recovery group. ٭, # (P < 0.05) with Bonferroni’s correction for multiple comparisons

Fig. 6

Effects of metformin (50 mg/kg body weight/day) on expression of phosphor AMPK (p-AMPK) an active form of AMPK. First, the data normalized with the internal control (β-actin) and then indicated as fold change to the control group. Control (gray): normal mice on a regular diet and injected with vehicle during last week, metformin (black): normal mice on a regular diet and injected with metformin during last week, free recovery group (white): model mice with regular diet and injected with vehicle during last recovery week, metformin recovery group (dotted): model mice with regular diet and injected with metformin during last recovery week. Data represent the mean ± S.E.M analyzed by two-way ANOVA. ٭compared to control group, #compared to free recovery group. ٭, # (P < 0.05) with Bonferroni’s correction for multiple comparisons