When cytokinin, a plant hormone, meets the adenosine A2A receptor: a novel neuroprotectant and lead for treating neurodegenerative disorders?

Abstract

It is well known that cytokinins are a class of phytohormones that promote cell division in plant roots and shoots. However, their targets, biological functions, and implications in mammalian systems have rarely been examined. In this study, we show that one cytokinin, zeatin riboside, can prevent pheochromocytoma (PC12) cells from serum deprivation-induced apoptosis by acting on the adenosine A(2A) receptor (A(2A)-R), which was blocked by an A(2A)-R antagonist and a protein kinase A (PKA) inhibitor, demonstrating the functional ability of zeatin riboside by mediating through A(2A)-R signaling event. Since the A(2A)-R was implicated as a therapeutic target in treating Huntington's disease (HD), a cellular model of HD was applied by transfecting mutant huntingtin in PC12 cells. By using filter retardation assay and confocal microscopy we found that zeatin riboside reversed mutant huntingtin (Htt)-induced protein aggregations and proteasome deactivation through A(2A)-R signaling. PKA inhibitor blocked zeatin riboside-induced suppression of mutant Htt aggregations. In addition, PKA activated proteasome activity and reduced mutant Htt protein aggregations. However, a proteasome inhibitor blocked both zeatin riboside-and PKA activator-mediated suppression of mutant Htt aggregations, confirming mediation of the A(2A)-R/PKA/proteasome pathway. Taken together, zeatin riboside might have therapeutic potential as a novel neuroprotectant and a lead for treating neurodegenerative disorders.

Figure 1. Cytokinins acting on the A_2A-R prevent serum deprivation-induced PC12 cell apoptosis.

(A) Serum-contained and serum-deprived cells were treated with or without the indicated reagent(s) for 24 h. NGF were treated in 100 ng/ml. Besides, cells pretreated with zeatin riboside (100 µM) for 3 h were treated with or without H₂O₂ (25 µM) for 24 h. Cell viability was expressed as a percentage of the results from the MTT assay with respect to the mean value of the serum-contained control group. Data points represent the mean ± SEM (n = 3∼6). *p<0.05, compared to its serum-deprived group. #p<0.05, compared to the H₂O₂-treated group. (B) Serum-deprived cells were pretreated with or without the indicated reagents for 30 min. Zeatin riboside or CGS (0.1 µM) was added for another 24 h. Cell viability was expressed as a percentage of the results from the MTT and trypan blue exclusion assays with respect to the mean value of the serum-contained control group. Data points represent the mean ± SEM (n = 3∼6). *p<0.05, compared to its serum-deprived group. (C) Serum-deprived cells were pretreated with or without 1 µM ZM or 1 µM SCH for 30 min. Zeatin riboside was added for 24 h and followed by Annexin V-FITC staining. Cells were subjected to image and cytometry analysis. Bar represents 50 µm. Data points represent the mean ± SEM (n = 3∼6). *p<0.05, compared to the serum-contained control group. (D) Serum-contained or -deprived cells in the presence or absence of zeatin riboside were harvested and subjected to a Western blot analysis. The relative optical density of the bands were quantified by densitometry relative to actin and normalized to the levels in serum-contained control group or in serum-deprived for 24 h group. Data points (mean ± SEM) represent one out of three independent experiments that gave similar results. *p<0.05, compared to its serum-containing control group. #p<0.05, compared to its 24 h serum-deprived group. AU represents arbitrary unit.

Figure 2. Zeatin riboside acting on the A_2A-R attenuates mutant Htt aggregations.

(A) pHtt-25Q-mKate- or pHtt-109Q-mKate-transfected cells were treated with 1 µM CGS or zeatin riboside for 24 h. Cells were harvested and subjected to a filter retardation assay and Western blot analysis. (B) The transfected cells were pretreated with or without 1 µM ZM for 30 min and treated with zeatin riboside for another 24 h. Cells were harvested and subjected to a filter retardation assay and Western blot analysis. (C) The transfected cells were pretreated with or without 5 µM H-89 for 30 min and treated with zeatin riboside for another 24 h. Cells were harvested and subjected to a filter retardation assay and Western blot analysis. The relative optical density of the bands (A∼C) were quantified by densitometry relative to actin and normalized to the levels under the Htt-109Q-overexpressed control condition. Data points represent the mean ± SEM. *p<0.05, compared to the mutant Htt control group. #p<0.05, compared to the zeatin riboside-treated mutant Htt group. (D) After 1 µM ZM pretreatment for 30 min, pHtt-25Q-mKate- or pHtt-109Q-mKate-transfected and pZsProSensor-co-transfected cells were treated with or without zeatin riboside for another 24 h and subjected to a confocal microscopic analysis. Bar represents 5 µm. In each group, the mKate-aggregated cells in proportion to the transfected cells were counted (100∼150 cells) These data points (mean ± SEM) represent one out of three independent experiments that gave similar results. *p<0.05, compared to the mutant Htt control group.

Figure 3. Zeatin riboside attenuates mutant Htt aggregations through increasing proteasome activity.

(A) With or without 1 µM MG 132 pretreatment for 30 min and following the presence or absence of zeatin riboside for 24 h, pHtt-25Q-mKate- or pHtt-109Q-mKate-transfected and pZsProSensor-co-transfected cells were subjected to a confocal microscopic analysis. Bar represents 5 µm. 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. #p<0.05, compared to the MG 132-treated mutant Htt group. (B) With or without 1 µM MG 132 pretreatment for 30 min, pHtt-109Q-mKate-transfected cells were treated with or without zeatin riboside 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. AU represents arbitrary unit. (C) pHtt-25Q-mKate-transfected cells were treated with or without zeatin riboside or 1 µM MG 132. pHtt-109Q-mKate-transfected cells were pretreated with or without 1 µM ZM for 30 min and then supplemented with or without zeatin riboside or 1 µM MG 132 for 24 h and subjected to a proteasome activity assay. *p<0.05, compared to the Htt-25Q control group. #p<0.05, compared to the Htt-109Q control group. These data represent one out of three independent experiments that gave similar results.

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

(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 A_2A receptor (A_2A-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.