Cytokinin Plant Hormones Have Neuroprotective Activity in In Vitro Models of Parkinson's Disease

Abstract

Cytokinins are adenine-based phytohormones that regulate key processes in plants, such as cell division and differentiation, root and shoot growth, apical dominance, branching, and seed germination. In preliminary studies, they have also shown protective activities against human neurodegenerative diseases. To extend knowledge of the protection (protective activity) they offer, we investigated activities of natural cytokinins against salsolinol (SAL)-induced toxicity (a Parkinson's disease model) and glutamate (Glu)-induced death of neuron-like dopaminergic SH-SY5Y cells. We found that kinetin-3-glucoside, cis-zeatin riboside, and N⁶-isopentenyladenosine were active in the SAL-induced PD model. In addition, trans-, cis-zeatin, and kinetin along with the iron chelator deferoxamine (DFO) and the necroptosis inhibitor necrostatin 1 (NEC-1) significantly reduced cell death rates in the Glu-induced model. Lactate dehydrogenase assays revealed that the cytokinins provided lower neuroprotective activity than DFO and NEC-1. Moreover, they reduced apoptotic caspase-3/7 activities less strongly than DFO. However, the cytokinins had very similar effects to DFO and NEC-1 on superoxide radical production. Overall, they showed protective activity in the SAL-induced model of parkinsonian neuronal cell death and Glu-induced model of oxidative damage mainly by reduction of oxidative stress.

Keywords: Parkinson’s disease; cytokinin; cytotoxicity; glutamate; neuron-like SH-SY5Y cells; neuroprotection; oxidative stress; phytohormone; salsolinol.

Figure 1

(A) Fluorescent micrographs of SH-SY5Y cells with membranes stained using a Neurite outgrowth kit (Invitrogen™): Control, undifferentiated cells (exposed to mock treatment solution: <0.1% DMSO); Cells differentiated by exposure to 10 µM all-trans retinoic acid (ATRA) for 48 h. Bars = 50 μm. (B) Proliferation rates of undifferentiated and differentiated SH-SY5Y cells: numbers of viable cells after 48 h exposure to <0.1% DMSO and 10 µM ATRA, respectively. Data were obtained from five independent experiments with triplicate cultures: asterisks show the significance of differences in numbers of viable cells (as percentages of numbers of undifferentiated cells) between the cultures: * p < 0.05.

Figure 2

(A) Neuroprotective activity of cytokinins and N-acetylcystein (NAC) in SAL-induced model of PD on neuron-like SH-SY5Y cells. The dashed line shows the NAC effect threshold at which cytokinins were selected for further testing; the dotted line then counts the number of living cells in the Calcein AM assay after treating the cells with 500 µM SAL; healthy control cells (CTR, DMSO < 0.1%). Triplicates in at least three separated days. (B) Normalized SH-SY5Y cell death after propidium iodide staining. Triplicates in at least five independent days. * P compared with vehicle with 500 µM SAL, # P compared with vehicle without 500 µM SAL.

Figure 3

(A) Microphotographs showing SAL-induced oxidative stress and oxidative stress-reducing activities of cytokinins in human differentiated neuron-like SH-SY5Y cells visualized by fluorescence microscopy following dihydroethidium (DHE) labelling. Bars = 50 μm. The images show cells treated with DMSO solution (controls), 500 µM salsolinol (SAL) alone, and combinations of 500 µM SAL and 1000 µM NAC (+NAC), 0.1 µM cZR (+cZR); 10 µM K3G (+K3G), 1µM iPR (+iPR) for 24 h before staining with DHE. (B) SAL-induced superoxide radical formation and cytokinin or N-acetylcysteine (NAC) protective activity. The graph shows the quantification of DHE stained cells using Infinite M200 Pro microplate reader (Tecan, Austria). Triplicates in at least five independent days. * P compared with vehicle with 500 µM SAL, # P compared with vehicle without 500 µM SAL.

Figure 4

Caspase-3/7 activity in the SAL-induced model of PD. Triplicates in at least four independent days. * P compared with vehicle with 500 µM SAL, # P compared with vehicle without 500 µM SAL.

Figure 5

(A) Death rate of neuron-like SH-SY5Y cells in glutamate (Glu)-induced model of cell death; (B) Glu-induced toxicity (LDH-release) of neuron-like SH-SY5Y cells. Triplicates in at least three independent days.* P compared with vehicle with 160 µM Glu, # P compared with vehicle without 160 µM Glu.

Figure 6

(A) Glu-induced oxidative stress (OS) and OS-reducing activity of indicated compounds in human neuron-like SH-SY5Y cells visualized by fluorescence microscopy following dihydroethidium (DHE) labelling. Bars = 50 μm. Images show neuron-like SH-SY5Y cells treated by DMSO (Control), 160 mM glutamate (Glu) alone, and together with: 10 µM deferoxaine (+DFO), 50 µM necrostatin-1 (+NEC-1), 0.1 µM tZ (+tZ) and 0.1µM cZ (+cZ) for 4 h, then stained by DHE. (B) Glu-induced superoxide radical formation in neuron-like SH-SY5Y cells after 4 h. The graph displays the quantification of DHE stained cells using Infinite M200 Pro microplate reader (Tecan, Austria). Triplicates in five independent days. * P compared with vehicle with 160 µM Glu, # P compared with vehicle without 160 µM Glu.

Figure 7

Caspase-3/7 activity in Glu-model of oxidative damage of neuron-like SH-SY5Y cells. Triplicates in at least four independent days. * P compared with vehicle with 160 µM Glu, # P compared with vehicle without 160 µM Glu.