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. 2012 Jun 18;7(6):e38865. doi: 10.1371/journal.pone.0038865

Figure 4. PKA attenuates mutant Htt aggregations through increasing proteasome activity.

Figure 4

(A) With or without 1 µM H-89 pretreatment for 30 min, pHtt-109Q-mKate-transfected cells were treated with or without 10 µM FK or 100 µM db-cAMP for 24 h and subjected to a filter retardation assay and Western blot analysis. The relative optical density of the bands were quantified by densitometry relative to actin and normalized to the levels under the Htt-109Q-overexpressed control condition which was set as 1.0. Data points represent the mean ± SEM. *p<0.05, compared to the mutant Htt control group. #p<0.05, compared to the FK-treated mutant Htt group. AU represents arbitrary unit. (B) After 5 µM H-89 or 1 µM MG 132 pretreatment for 30 min, pHtt-25Q-mKate- or pHtt-109Q-mKate-transfected and pZsProSensor-co-transfected cells were treated with or without 10 µM FK for 24 h and subjected to a confocal microscopic analysis. In each group, the mKate-aggregated cells in proportion to the transfected cells were counted (100∼150 cells). Data points represent the mean ± SEM. *p<0.05, compared to the mutant Htt control group. (C) With or without 5 µM H-89 pretreatment for 30 min, pHtt-109Q-mKate-transfected cells were supplemented with or without zeatin riboside or 10 µM FK for 24 h and subjected to a proteasome activity assay. *p<0.05, compared to the control group. # p<0.05, compared to the FK-treated but without H-89-pretreated group. (D) With or without 10 µM H-89 pretreatment for 30 min, AKAR1-transfected cells were added with or without 10 µM FK or zeatin riboside and the FRET images (upper panel) were acquired and analyzed (middle panel). Bar represents 5 µm. In each group, the area under the curve (AUC) subtracting with the background level was calculated and plotted in arbitrary unit (AU) (lower panel). Data represents the mean ± SEM (n = 3∼6). *p<0.05, compared to the control group. (E) Zeatin riboside-mediated suppression of mutant Htt aggregations involves activation of the adenosine A2A receptor (A2A-R), PKA, and proteasome. Mutant Htt aggregations in turn inhibit proteasome activity. These data represent one out of three independent experiments that gave similar results.