Figure 7. Expression of mHtt causes the... | Rockefeller University Press

Figure 7.

$Figure 7. Expression of mHtt causes the activation and nuclear enrichment of AMPK-α1 through a CaMKII-mediated pathway. (A) Total lysates of STHdhQ7 and STHdhQ109 cells were assessed by Western blot analyses. Results were normalized to those of actin. a, P < 0.05 versus untreated STHdhQ109 cells. (B) Cytosolic and nuclear fractions were harvested from STHdhQ7and STHdhQ109 cells and assessed by Western blot analyses. PARP and α-tubulin were markers for the nuclear and cytoplasmic fractions, respectively. a, P < 0.05 versus untreated STHdhQ7 cells; b, P < 0.05 versus untreated STHdhQ109 cells. (C) Total lysates of STHdhQ7 and STHdhQ109 cells were assessed by Western blot analyses. Results were normalized to those of actin. a, P < 0.05 versus untreated STHdhQ7 cells. Molecular mass is indicated in kilodaltons. (D) Total striatal lysates of 12-wk-old mice were assayed for CaMKII activity. Values are expressed as percentages of the CaMKII activity in WT mice. a, P < 0.05 between WT and R6/2 mice. (A–D) Data are presented as the mean ± SEM of three independent experiments. (E and G) 10-wk-old mice (n = ∼3–6 for each condition) were intrastriatally injected with KN62 (10 µg per mouse) or saline. 1 d after injection, brain sections were harvested to analyze the expressions of AMPK-α-p (E, green) and AMPK-α1 (G, green). Nuclei were stained with Hoechst (blue). The numbers shown in the bottom left corners of the merged images are the percentages of cells expressing nuclear AMPK-α-p (E) or AMPK-α1 (G) and were identified by nuclear AMPK-α-p (E) or AMPK-α1 (G) normalized to total AMPK-α-p or AMPK-α1–positive cells. Data are presented as the mean ± SEM. At least 200 cells from each animal were analyzed. Thin arrows mark cells in which AMPK-α-p is located mostly in the cytoplasmic region. Thick arrows mark cells with nuclear enrichment of AMPK-α-p. Bars, 20 µm. (F) The histogram shows the fluorescence intensity of AMPK-α-p (n = 6 for each condition). Data are presented as the mean ± SEM. a, P < 0.05 between WT and R6/2 mice; b, P < 0.05 versus R6/2-saline mice.$

Expression of mHtt causes the activation and nuclear enrichment of AMPK-α1 through a CaMKII-mediated pathway. (A) Total lysates of STHdh^Q7 and STHdh^Q109 cells were assessed by Western blot analyses. Results were normalized to those of actin. a, P < 0.05 versus untreated STHdh^Q109 cells. (B) Cytosolic and nuclear fractions were harvested from STHdh^Q7and STHdh^Q109 cells and assessed by Western blot analyses. PARP and α-tubulin were markers for the nuclear and cytoplasmic fractions, respectively. a, P < 0.05 versus untreated STHdh^Q7 cells; b, P < 0.05 versus untreated STHdh^Q109 cells. (C) Total lysates of STHdh^Q7 and STHdh^Q109 cells were assessed by Western blot analyses. Results were normalized to those of actin. a, P < 0.05 versus untreated STHdh^Q7 cells. Molecular mass is indicated in kilodaltons. (D) Total striatal lysates of 12-wk-old mice were assayed for CaMKII activity. Values are expressed as percentages of the CaMKII activity in WT mice. a, P < 0.05 between WT and R6/2 mice. (A–D) Data are presented as the mean ± SEM of three independent experiments. (E and G) 10-wk-old mice (n = ∼3–6 for each condition) were intrastriatally injected with KN62 (10 µg per mouse) or saline. 1 d after injection, brain sections were harvested to analyze the expressions of AMPK-α-p (E, green) and AMPK-α1 (G, green). Nuclei were stained with Hoechst (blue). The numbers shown in the bottom left corners of the merged images are the percentages of cells expressing nuclear AMPK-α-p (E) or AMPK-α1 (G) and were identified by nuclear AMPK-α-p (E) or AMPK-α1 (G) normalized to total AMPK-α-p or AMPK-α1–positive cells. Data are presented as the mean ± SEM. At least 200 cells from each animal were analyzed. Thin arrows mark cells in which AMPK-α-p is located mostly in the cytoplasmic region. Thick arrows mark cells with nuclear enrichment of AMPK-α-p. Bars, 20 µm. (F) The histogram shows the fluorescence intensity of AMPK-α-p (n = 6 for each condition). Data are presented as the mean ± SEM. a, P < 0.05 between WT and R6/2 mice; b, P < 0.05 versus R6/2-saline mice.