The Anti-Diabetic Drug Metformin Rescues Aberrant Mitochondrial Activity and Restrains Oxidative Stress in a Female Mouse Model of Rett Syndrome

Abstract

Metformin is the first-line therapy for diabetes, even in children, and a promising attractive candidate for drug repurposing. Mitochondria are emerging as crucial targets of metformin action both in the periphery and in the brain. The present study evaluated whether treatment with metformin may rescue brain mitochondrial alterations and contrast the increased oxidative stress in a validated mouse model of Rett syndrome (RTT), a rare neurologic disorder of monogenic origin characterized by severe behavioral and physiological symptoms. No cure for RTT is available. In fully symptomatic RTT mice (12 months old MeCP2-308 heterozygous female mice), systemic treatment with metformin (100 mg/kg ip for 10 days) normalized the reduced mitochondrial ATP production and ATP levels in the whole-brain, reduced brain oxidative damage, and rescued the increased production of reactive oxidizing species in blood. A 10-day long treatment with metformin also boosted pathways related to mitochondrial biogenesis and antioxidant defense in the brain of metformin-treated RTT mice. This treatment regimen did not improve general health status and motor dysfunction in RTT mice at an advanced stage of the disease. Present results provide evidence that systemic treatment with metformin may represent a novel, repurposable therapeutic strategy for RTT.

Keywords: Nrf2; PGC-1α; Rett syndrome; metformin; repurposing.

Figure 1

Metformin systemic treatment rescues defective energy status in RTT mouse brain. A 10-day long treatment with metformin (100 mg/kg) completely normalizes the reduced mitochondrial ATP production via oxidative phosphorylation (a) and ATP levels (b) in RTT mouse brain. N = 3. Data are mean ± SEM. Statistical significance was calculated by two-way ANOVA, with Tukey’s post hoc test. ** p < 0.01. WT: wild-type mice; RTT: MeCP2-308 heterozygous female mice; sal: saline; met: metformin.

Figure 2

Metformin treatment restores mitochondrial complex II and V activity in the brain of RTT mice. The activity of complex I (a) did not differ between the two genotypes and was not affected by the metformin treatment. The activity of mitochondrial respiratory chain complex II (b) and complex V (c) was reduced in RTT mouse brain compared to WT controls and metformin treatment restored WT-like levels. N = 3. Data are mean ± SEM. Statistical significance was calculated by two-way ANOVA, with Tukey’s post hoc test. * p < 0.05, ** p < 0.01. WT: wild-type mice; RTT: MeCP2-308 heterozygous female mice; sal: saline; met: metformin.

Figure 3

Metformin treatment normalizes decreased oxidative phosphorylation protein content in RTT mouse hippocampus. Protein expression levels of SDHB (subunit of Complex II) (c) and UQCRC2 (subunit of Complex III) (d) is decreased in the hippocampus of RTT mice. A trend towards a decrease was also observed for ATP5A (subunit of Complex V) (f) in RTT compared to WT controls. No genotype differences were found for NDUFB8 (subunit of Complex I) (b) and MTCO1 (subunit of Complex IV) (e). Metformin treatment normalized protein expression levels of SDHB (subunit of Complex II) (c) and ATP5A (subunit of Complex V) (f) in RTT mice. Representative blot is shown in (a). N = 9–11. Data are mean ± SEM normalized for WT, sal. Statistical significance was calculated by two-way ANOVA, with Tukey’s post hoc test. * p < 0.05, ** p < 0.01. WT: wild-type mice; RTT: MeCP2-308 heterozygous female mice; sal: saline; met: metformin; NDUFB8: NADH:Ubiquinone Oxidoreductase Subunit B8; SDHB: Succinate Dehydrogenase Complex Iron Sulfur Subunit B; UQCRC2: Ubiquinol-Cytochrome C Reductase Core Protein II; MTCO1: mitochondrially encoded cytochrome c oxidase I; ATP5A: ATP Synthase, H⁺ Transporting, Mitochondrial F1 Complex.

Figure 4

Metformin treatment rescues increased oxidative stress status in the brain and in the blood of RTT mice. (a) Blood ROS levels, measured as the intensity of formation of CP^• by EPR, were significantly higher in RTT, sal mice compared to WT controls, confirming the occurrence of a pro-oxidant status in RTT mice. Metformin normalized this value in whole blood of RTT mice to the same level of WT controls. N = 3–4. One subject of the experimental group RTT, sal was identified as outlier and thus, excluded from the analysis. (b–c) Metformin treatment reduces the abnormal accumulation of protein 4-hydroxynonenal (HNE) adducts in RTT mouse hippocampus (c). Representative blot is shown in panel (b). N = 9–11. Data are mean ± SEM normalized for WT, sal. Statistical significance was calculated by two-way ANOVA, with Tukey’s post hoc test. * p < 0.05, ** p < 0.01. WT: wild-type mice; RTT: MeCP2-308 heterozygous female mice; sal: saline; met: metformin; HNE-adducts: 4-hydroxy-2-trans-nonenal protein bound.

Figure 5

Metformin systemic treatment boosts pathways related to mitochondrial biogenesis and remodeling in RTT mouse brain. PGC-1α protein expression and the levels of its downstream target mtTFA are increased in RTT mouse hippocampus compared to WT controls (b). Metformin treatment exacerbates this genotype difference. Following metformin treatment, RTT mouse hippocampus shows an increase in mitochondrial biogenesis related protein OPA1 (c) and MFN2 (d). Representative blots are reported in (a). N = 9–11. Data are mean ± SEM normalized for WT, sal. Statistical significance was calculated by two-way ANOVA, with Tukey’s post hoc test. * p < 0.05; *** p < 0.001. WT: wild-type mice; RTT: MeCP2-308 heterozygous female mice; sal: saline; met: metformin; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; mtTFA: Mitochondrial transcription factor A; OPA1: GTPase Optic Atrophy 1; MFN2: GTPases Mitofusin 2.

Figure 6

Metformin systemic treatment boosts pathways related to antioxidant response in RTT mouse brain. Metformin treatment induces an antioxidant response selectively in the hippocampus of RTT mice through the increase of Nrf2 protein expression and activation (b), and the corresponding increase in Nrf2 downstream target protein HO-1 (c). N = 9–11. Representative blot is included in (a). Data are mean ± SEM normalized for WT, sal. Statistical significance was calculated by two-way ANOVA, with Tukey’s post hoc test. * p < 0.05; ** p < 0.01. WT: wild-type mice; RTT: MeCP2-308 heterozygous female mice; sal: saline; met: metformin; Nrf2: nuclear respiratory factor 2; HO-1: heme oxygenase-1.

Figure 7

A 10-day long treatment with metformin does not affect the compromised general health (GH) status and motor dysfunction of RTT mice. RTT female mice received higher scores compared to WT mice in the GH evaluation (a), confirming the presence of phenotypic alterations. No treatment effects were found on this parameter. Total distance moved in the open field test (b) does not significantly differ among groups. RTT mice displayed shorter latencies to fall compared to WT controls in the dowel test (c). The metformin treatment did not affect the motor performance of the experimental subjects. N = 7–8. Data are mean ± SEM. Statistical significance was calculated by two-way ANOVA. WT: wild-type mice; RTT: MeCP2-308 heterozygous female mice; sal: saline; met: metformin; GH: general health.

Figure 8

Summary Diagram. A 10-day long treatment with metformin normalizes the reduced mitochondrial ATP production and ATP levels in whole brain of a validated RTT mouse model at an advanced stage of the disease. This is achieved through the restoration of the defective activity of mitochondrial respiratory chain complex II and V and of their reduced protein content. These beneficial effects are accompanied by a strong activation of signaling pathways related to both mitochondrial biogenesis and remodeling (PGC-1α/mtTFA, OPA1 and MFN2) selectively in RTT mouse brain. Metformin also ameliorates prooxidant status and brain oxidative damage in RTT brain, through the stimulation of Nrf2 signaling and the consequent transcription of antioxidant genes such as HO-1. WT: wild-type mice; RTT: MeCP2-308 heterozygous female mice; HNE-adducts: 4-hydroxy-2-trans-nonenal protein bound; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; mtTFA: Mitochondrial transcription factor A; OPA1: GTPase Optic Atrophy 1; MFN2: GTPases Mitofusin 2; ROS: reactive oxidizing species.