Protein kinase D1 (PKD1) phosphorylation promotes dopaminergic neuronal survival during 6-OHDA-induced oxidative stress.

Arunkumar Asaithambi,Huajun Jin,Anamitra Gosh,Anumantha G Kanthasamy,Muhammet Ay,Arthi Kanthasamy,Vellareddy Anantharam,Xiao-Jiang Li

doi:10.1371/journal.pone.0096947

Abstract

Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1WT) or constitutively active PKD1 (PKD1S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.

Highlights

Parkinson’s disease (PD) is a major neurodegenerative disorder affecting over a million Americans at an annual cost of several billion dollars
Overexpression of PKD1S916A but not PKD1WT blocked this effect, suggesting that Ser 916 phosphorylation is required for Protein Kinase D1 (PKD1) activation loop phosphorylation. These results suggest that C-terminal PKD1 Ser916 phosphorylation occurs at very early stages of oxidative stress to positively regulate PKD1 activation loop phosphorylation during 6-OHDA-induced oxidative stress in dopaminergic cells
We found a high-level expression of PKD1 in the cytosolic region of tyrosine hydroxylase-positive (TH+) primary dopaminergic neurons obtained from mouse mesencephalon

Summary

Introduction

Parkinson’s disease (PD) is a major neurodegenerative disorder affecting over a million Americans at an annual cost of several billion dollars. To study key signaling molecules involved in oxidative stress-induced neuronal apoptosis in PD, we used the dopaminergic system specific neurotoxicant 6-hydroxydopamine (2,4,5-trihydroxyphenylethylamine; 6-OHDA). Signaling through PKD1 is activated in response to multiple stimuli, and its activation has been shown to play important roles in diverse cellular functions, including proliferation, cytoskeletal reorganization, Golgi function, immune function, and cell survival [14,15,16,17,18,19,20,21]. We demonstrate that the 6-OHDA-activated PKD1 signaling pathway serves as a key compensatory protective mechanism in dopaminergic neurons during the early stages of oxidative insult and dopaminergic degeneration in cell culture models of PD

Materials and Methods

Results

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Discussion