Metformin Protects From Rotenone-Induced Nigrostriatal Neuronal Death in Adult Mice by Activating AMPK-FOXO3 Signaling and Mitigation of Angiogenesis

Abstract

Parkinson's disease (PD) is a neurodegenerative disease that affects substantia nigra dopamine neurons. Many studies have documented the role of oxidative stress and angiogenesis in the pathogenesis of PD. Metformin (MTF) is an antidiabetic medication and AMP-activated protein kinase (AMPK) regulator that has shown antioxidant and antiangiogenic properties in many disorders. The aim of this study is to investigate the neuroprotective effect of MTF in a mouse model of rotenone-prompted PD with a highlight on its influence on the AMPK/forkhead box transcription factor O3 (FOXO3) pathway and striatal angiogenesis. In the running study, PD was induced in mice using repeated doses of rotenone and concomitantly treated with MTF 100 or 200 mg/kg/day for 18 days. Rotarod and pole tests were used to examine the animals' motor functionality. After that, animals were sacrificed, and brains were isolated and processed for immunohistochemical investigations or biochemical analyses. Oxidant stress and angiogenic markers were measured, including reduced glutathione, malondialdehyde, the nuclear factor erythroid 2-related factor 2 (Nrf2), hemoxygenase-1, thioredoxin, AMPK, FOXO3, and vascular endothelial growth factor (VEGF). Results indicated that MTF improved animals' motor function, improved striatal glutathione, Nrf2, hemoxygenase-1, and thioredoxin. Furthermore, MTF upregulated AMPK-FOXO3 proteins and reduced VEGF and cleaved caspase 3. MTF also increased the number of tyrosine hydroxylase (TH)-stained neurons in the substantia nigra neurons and in striatal neuronal terminals. This study is the first to highlight that the neuroprotective role of MTF is mediated through activation of AMPK-FOXO3 signaling and inhibition of the proangiogenic factor, VEGF. Further studies are warranted to confirm this mechanism in other models of PD and neurodegenerative diseases.

Keywords: AMPK-FOXO3; cleaved caspase 3; metformin; oxidative stress; rotenone-induced parkinsonism; vascular endothelial growth factor.

FIGURE 1

A scheme describing the study design.

FIGURE 2

Effect of MTF on the locomotor activity of mice using (A,B) the pole test: presented as medians in boxplots, and analysis was done using the Kruskal–Wallis ANOVA and Dunn’s post hoc test with P < 0.05 as the accepted level of significance. (C) Rotarod test: results are expressed as mean ± SD and analyzed by applying one-way ANOVA followed by Bonferroni’s test with p < 0.05. #: Different from vehicle group, $: Different from rotenone group. *: Different from rotenone + MTF 100 mg/kg group at p < 0.05 (n = 6).

FIGURE 3

Effect of metformin on the striatal level of dopamine and oxidative stress markers in rotenone-parkinsonian mice. (A) MDA, (B) GSH, (C) HO-1, and (D) Dopamine. Results are expressed as mean ± SD and analyzed by applying one-way ANOVA followed by Bonferroni’s test. #: Different from vehicle group, $: Different from rotenone group. *: Different from rotenone + MTF 100 mg/kg group at p < 0.05 (n = 6).

FIGURE 4

Effect of metformin on mRNA expression of AMPK and antioxidant markers in striata of rotenone-parkinsonian mice. Column charts for (A) Nrf2, (B) thioredoxin, (C) AMPK, and (D) FOXO3. mRNA expression in each group was calculated relative to the control value. Data are expressed as mean ± SD and analyzed using one-way ANOVA followed by Bonferroni’s test. #: Different from vehicle group, $: Different from rotenone group. *: Different from rotenone + MTF 100 mg/kg group at p < 0.05 (n = 6).

FIGURE 5

Effect of metformin on striatal levels of proteins. (A) Western blotting of Nrf2, HO-1, AMPK, FOXO3, and thioredoxin versus β-actin was done using samples from brain striata. (B–F) Graphs present the densitometric analysis of western blotting of Nrf2, HO-1, AMPK, FOXO3, and thioredoxin, respectively. Data are mean ± SD for densitometric analysis of each gene relative to β-actin. Analysis was performed by applying one-way ANOVA followed by Bonferroni’s test #: Different from vehicle group, $: Different from rotenone group. *: Different from rotenone + MTF 100 mg/kg group at p < 0.05 (n = 3).

FIGURE 6

Effect of metformin on striatal levels of c-caspase 3 and VEGF proteins. (A) Western blotting of c-caspase 3 and VEGF versus β-actin done using samples from brain striata. (B,C) Graphs present the densitometric analysis of western blotting of c-caspase 3 and VEGF. Data are mean ± SD for densitometric analysis of each gene relative to β-actin. Analysis was performed by applying one-way ANOVA followed by Bonferroni’s test. #: Different from vehicle group, $: Different from rotenone group. *: Different from rotenone + MTF 100 mg/kg group at p < 0.05 (n = 3).

FIGURE 7

Immunohistochemistry staining for tyrosine hydroxylase in rotenone-parkinsonian mice. (A) The above panel shows images from the substantia nigra of the vehicle control group with multiple healthy TH-positive somas with regular shape and rounded large nuclei, and the striatum shows widespread positive regular staining. The image from the rotenone group shows a low number of healthy TH-positive cell somas and low scattered TH staining in the striatal nerve terminals. The rotenone + MTF 100 mg/kg group shows a higher number of TH-positive somas in the substantia nigra and higher striatal TH staining. The rotenone + MTF 200 mg/kg group shows improved TH staining in the cell somas and the striatal nerve terminals. (B) A column chart illustrating the number of TH-positive neurons per section in the substantia nigra pars compacta (left panel) and the striatal percent of TH-positive neurons (right panel). Results are expressed as mean ± SD and analyzed by applying one-way ANOVA followed by Bonferroni’s test. #: Different from vehicle group. $: Different from rotenone group. *: Different from rotenone + MTF 100 mg/kg group at p < 0.05 (n = 6).