Metformin attenuates Alzheimer's disease-like neuropathology in obese, leptin-resistant mice

Abstract

Diabetes increases the risk of Alzheimer's disease (AD). The pathological hallmarks for AD brains are extracellular amyloid plaques formed by β-amyloid peptide (Aβ) and intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. This study was designed to determine AD-like brain changes in mice modeling for type 2 diabetes. The effects of metformin on these changes also were studied. Seven-week old male db/db mice received intraperitoneal injection of 200 mg kg⁻¹ d⁻¹ metformin for 18 weeks. They were subjected to Barnes maze at an age of 21 weeks and fear conditioning at an age of 24 weeks to assess their cognitive functions. Hippocampus was harvested after these tests for biochemical evaluation. The db/db mice had more tau phosphorylated at S396 and total tau in their hippocampi than their non-diabetic control db+mice. Activated/phosphorylated c-jun N-terminal kinase (JNK), a tau kinase, was increased in the db/db mouse hippocampus. Metformin attenuated the increase of total tau, phospho-tau and activated JNK. The db/db mice had increased Aβ levels. Metformin attenuated the reduction of synaptophysin, a synaptic protein, in the db/db mouse hippocampus. Metformin did not attenuate the impairments of spatial learning and memory as well as long-term hyperglycemia in the db/db mice. Our results suggest that the db/db mice have multiple AD-like brain changes including impaired cognitive functions, increased phospho-tau and Aβ as well as decreased synaptic proteins. Activation of JNK may contribute to the increased phospho-tau in the db/db mice. Metformin attenuates AD-like biochemical changes in the brain of these mice.

Conflict of interest statement

Duality of interest: The authors declare that there is no duality of interest associated with this manuscript.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Fig. 1. Performance of mice on Barnes maze and fear conditioning tests

Seven-week old male db/db mice received or did not receive normal saline or 200 mg kg−1 d−1 metformin in saline for 18 weeks. They were then tested with Barnes maze and fear conditioning tasks. Two forms of Barnes maze tests, cued target and hidden target, were performed. The time for animals to find the target hole during the training sessions and the probe trials is shown in panels A and B. The percentage of the time spent in the zone of each hole in the total time spent in the zones of all 20 holes during the probe trial is shown in panels C, D and E. The percentage of the time with freezing behavior in the total observation time during the fear conditioning tests is shown in panel F. Results are median with 95% confidence interval (panels A and B) or means ± SD (panels C, D, E and F) (n = 8 – 11). * P < 0.05 compared with db+ mice; ^ P < 0.05 compared with the values of the same animals on the first training day. Statistical analysis was performed by Kruskal-Wallis analysis of variance on ranks for data presented in panels A and B and by one way analysis of variance for the fear conditioning data when comparisons were performed among groups. One way repeated measures analysis of variance was used to compare values of the same animals on different training days. db/db + S: db/db mice treated with saline; db/db + M: db/db mice treated with metformin; S-Probe: short-term probe trial; L-Probe: long-term probe trial.

Fig. 2. Expression of phosphorylated tau and total tau in mouse hippocampus

A representative Western blot is shown in panel A and the graphic presentation of protein abundance quantified by integrating the volume of autoradiograms from 6 – 10 mice for each experimental condition is shown in panels B and C. In panel B, the results of phosphorylated tau and total tau were normalized by the corresponding actin data and then expressed as fold change over the mean value of the db+ control mice in the same experiment. In panel C, the results of phosphorylated tau were normalized by the corresponding actin data. These results were then normalized by the total tau. The final results are expressed as fold change over the mean value of the db+ control mice in the same experiment. All results are presented as means ± SD. * P

Fig. 3. Expression of tau phosphorylated at S396 in mouse hippocampus

Hippocampus from 24-week old male db+ (panel B) and db/db (all other panels) mice was harvested for immunofluorescent staining of tau phosphorylated at S396 (red), NeuN (green), CD11b (green), GFAP (green) and MAP2 (green). Both db+ and db/db mice did not receive any injections. NeuN and MAP2 are proteins expressed in neurons. CD11b and GFAP are proteins expressed in microglia and astrocytes, respectively. An arrow in panel A indicates positive staining for tau phosphorylated at S396. The bar represents 200 μm in panels A and B and 20 μm in other panels (panels C to N).

Fig. 4. Expression of JNK in mouse hippocampus

A representative Western blot is shown in panel A and the graphic presentation of protein abundance quantified by integrating the volume of autoradiograms from 6 – 10 mice for each experimental condition is shown in panels B, C and D. In panels B and C, the results of phosphorylated JNK and total JNK were normalized by the corresponding GAPDH data and then expressed as fold change over the mean value of the db+ control mice in the same experiment. In panel D, the results of phosphorylated JNK were normalized by the corresponding GAPDH data. These results were then normalized by the total JNK. The final results are expressed as fold change over the mean value of the db+ control mice in the same experiment. All results are presented as means ± SD. * P

Fig. 5. Expression of GSK-3β, cdk5 and PP2A C subunit in mouse hippocampus

A representative Western blot is shown in panel A and the graphic presentation of protein abundance quantified by integrating the volume of autoradiograms from 6 – 10 mice for each experimental condition is shown in panels B, C, D and E. In panels B, D and E, the results of phosphorylated GSK-3β, cdk5 and PP2A C subunit were normalized by the corresponding GAPDH or actin data and then expressed as fold change over the mean value of the db+ control mice in the same experiment. In panel C, the results of phosphorylated GSK-3β were normalized by the corresponding GAPDH data. These results were then normalized by the total GSK-3β. The final results are expressed as fold change over the mean value of the control db+ mice in the same experiment. All results are presented as means ± SD. * P

Fig. 6. Expression of neuronal proteins in mouse hippocampus

A representative Western blot is shown in the top panel and the graphic presentation of protein abundance quantified by integrating the volume of autoradiograms from 6 – 8 mice for each experimental condition is shown in the bottom panel. Values in graphs are expressed as fold change over the mean value of the db+ mice in the same experiment and presented as means ± SD. * P

Fig. 7. Expression of Aβ1-42, APP, BACE1, NEP and IDE in mouse hippocampus

For panel A, results are means ± SD (n = 6 – 8). For panels B, C, D, and E, a representative Western blot is shown in the top panel and the graphic presentation of protein abundance quantified by integrating the volume of autoradiograms from 6 – 8 mice for each experimental condition is shown in the bottom panel. Values in graphs are expressed as fold change over the mean value of the db+ mice in the same experiment and presented as means ± SD. * P