Figure 6

Metformin worsens hindlimb atrophy and hyperactive behavior and induces caspase 3 activation and tau cleavage. a-c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled (a), speed (b) and number of rearings (c) are shown. Bars represent the average values ± SEM. *p < 0.05, Student t-test. n = 9 / group. d-g, k-s. P301S mice were treated with or without metformin (Met) for 4 months starting from 1 month of age. d-f. Open field test. Distance travelled (d), speed (e) and rearing (f) are shown. Bars represent the average values ± SEM. *p < 0.05, Student t-test. Panels d-f: Ctr, n = 11; Met, n = 9. g Hind limb extension test in P301S mice treated with or without metformin. Data points represent the average score ± SEM. *p < 0.05, two-way ANOVA followed by Holm-Sidak multiple comparison test. A.U., arbitrary units. Ctr, n = 12; Met, n = 13. h-j. Primary cortical neurons were treated with or without 0.5 mM metformin for 24 h. h-i. Western blot (h) and quantitative analyses (i) of cleaved caspase 3 (c-caspase 3), c-Tau, c-PARP and PSD95. Protein levels were normalized to actin. Bars represent the average values ± SEM. *p < 0.05, **p < 0.01, Student t-test. n = 3/group. j. Quantification of clusters with overlapping synapsin-I and PSD95 immunoreactivity. Bars represent the average ± SEM. **p < 0.01, Student t-test. n = 11/group. k Representative confocal maximal projection images of active c-caspase 3 immunoreactivity (green) in the prefrontal cortex of untreated and metformin-treated P301S mice. Nuclei are counterstained with Hoechst 33342. Scale bar 10 μm. l Quantitative analysis of active c-caspase 3. c-caspase 3 was analyzed by western blot and protein levels were normalized to tubulin. Bars represent the average ± SEM. n = 7 / group. *p < 0.05, Student t-test. m Western blot analysis of cleaved-tau (c-Tau) in the soluble and sarkosyl-insoluble fraction of metformin-treated and untreated P301S mouse cortex. n-o Quantitative analysis of cleaved Tau (c-Tau) in the soluble (n) and insoluble (o) cortical fraction. c-Tau was analyzed by western blot and protein levels were normalized to actin (n) or MemCode (o). Bars represent the average ± SEM. Panel n: n = 7 / group. Panel o: n = 3/group. p Western blot analysis of synapsin I, PSD95, synaptophysin, tubulin and GAPDH in the cortex of P301S mice treated with or without metformin. q-s. Quantitative analysis of synapsin I (q), PSD95 (r) and synaptophysin (s). Protein levels were analyzed by western bot and normalized to tubulin or GAPDH. Bars represent the average ± SEM. *p < 0.05, Student t-test. Panel q: n = 7 / group. Panel r: Ctr, n = 13; Met, n = 10. Panel s: Ctr, n = 13; Met, n = 11