Metformin Prevents Nigrostriatal Dopamine Degeneration Independent of AMPK Activation in Dopamine Neurons

Abstract

Metformin is a widely prescribed drug used to treat type-2 diabetes, although recent studies show it has wide ranging effects to treat other diseases. Animal and retrospective human studies indicate that Metformin treatment is neuroprotective in Parkinson's Disease (PD), although the neuroprotective mechanism is unknown, numerous studies suggest the beneficial effects on glucose homeostasis may be through AMPK activation. In this study we tested whether or not AMPK activation in dopamine neurons was required for the neuroprotective effects of Metformin in PD. We generated transgenic mice in which AMPK activity in dopamine neurons was ablated by removing AMPK beta 1 and beta 2 subunits from dopamine transporter expressing neurons. These AMPK WT and KO mice were then chronically exposed to Metformin in the drinking water then exposed to MPTP, the mouse model of PD. Chronic Metformin treatment significantly attenuated the MPTP-induced loss of Tyrosine Hydroxylase (TH) neuronal number and volume and TH protein concentration in the nigrostriatal pathway. Additionally, Metformin treatment prevented the MPTP-induced elevation of the DOPAC:DA ratio regardless of genotype. Metformin also prevented MPTP induced gliosis in the Substantia Nigra. These neuroprotective actions were independent of genotype and occurred in both AMPK WT and AMPK KO mice. Overall, our studies suggest that Metformin's neuroprotective effects are not due to AMPK activation in dopaminergic neurons and that more research is required to determine how metformin acts to restrict the development of PD.

Fig 1. Body Weight and blood glucose measurements in AMPK WT and KO mice.

A–D, throughout the experiment there was no difference in body weight or volume of water consumed comparing genotype or treatment. Arrows indicate MPTP injections. E–H, Plasma analysis of insulin and glucose levels in trunk blood show no differences between genotype and treatment. During an oGTT Metformin treatment does not alter glucose clearance in AMPK WT (I & J) or AMPK KO mice (K & L). Insulin sensitivity is not altered during an ITT in AMPK WT (M) or KO (N) mice treated with Metformin. * = p<0.05. Data are represented as mean ± SEM (n = 8–10, two-way ANOVA, p<0.05).

Fig 2. Metformin is neuroprotective in AMPK WT and KO mice.

Plasma Triglycerides (A & B), NEFA (C & D) and corticosterone (E & F) are elevated in response to MPTP. Representative Western Blot images of pAMPK/AMPK in the SN (G) and Striatum (H). Protein analysis of the pAMPK/AMPK ratio showing no elevation in response to MPTP in the SN (J) and Striatum (L) in AMPK KO but an elevation in both the SN and Striatum of AMPK WT mice (I & K). Representative images of TH levels in the SN (M) and Striatum (N). In the SN there is a significant reduction in TH levels in both AMPK WT (O) and KO (P) in response to MPTP. In the Striatum Metformin elicits a neuroprotective effect in MPTP treated AMPK WT (Q) and KO (R) mice. MPTP reduced dopamine and DOPAC in both AMPK WT (S & T) and AMPK KO (V & W) mice. Metformin reduced the elevation of the DOPAC:DA ratio in MPTP treated mice compared to water alone, in AMPK WT and KO mice (U & X) a, significant compared to water/saline treated mice and b, significant compared to water/MPTP treated mice. Data are represented as mean ± SEM (n = 6–9, two-way ANOVA, p<0.05).

Fig 3. Metformin preserves cell number and volume after MPTP exposure, independent of genotype.

Stereological quantification of TH levels in the SN shows a protective effect of Metformin after MPTP exposure in both AMPK WT (A) and KO (B) mice. Overall cell volume shows a significant reduction after MPTP exposure in water treatment but not with Metformin treatment in AMPK WT (C) and KO (D). When TH cells were separated and plotted based on volume distribution, mice treated with MPTP and Metformin had a significant effect on smaller volume (1000–2000μm³) cells compared to those not treated with Metformin in both AMPK WT (E) and KO (F) mice. G, Representative image showing MPTP induced microglial activation in the SN (green = TH, red = IBA1). Stereological quantification of IBA1 (H & I) and GFAP (J & K) showing increased numbers after MPTP with a significant protective effect of Metformin in both AMPK WT and KO. L, Representative images showing MPTP induced astrocytic activation in the SN (green = TH and red = GFAP). a, significant compared to Water/saline treated mice and b, significant compared to Water/MPTP treated mice. * = p<0.05. Data are represented as mean ± SEM (n = 7–10, two-way ANOVA, p<0.05).