Protein kinase D1 (PKD1) activation mediates a compensatory protective response during early stages of oxidative stress-induced neuronal degeneration

Abstract

Background: Oxidative stress is a key pathophysiological mechanism contributing to degenerative processes in many neurodegenerative diseases and therefore, unraveling molecular mechanisms underlying various stages of oxidative neuronal damage is critical to better understanding the diseases and developing new treatment modalities. We previously showed that protein kinase C delta (PKCδ) proteolytic activation during the late stages of oxidative stress is a key proapoptotic signaling mechanism that contributes to oxidative damage in Parkinson's disease (PD) models. The time course studies revealed that PKCδ activation precedes apoptotic cell death and that cells resisted early insults of oxidative damage, suggesting that some intrinsic compensatory response protects neurons from early oxidative insult. Therefore, the purpose of the present study was to characterize protective signaling pathways in dopaminergic neurons during early stages of oxidative stress.

Results: Herein, we identify that protein kinase D1 (PKD1) functions as a key anti-apoptotic kinase to protect neuronal cells against early stages of oxidative stress. Exposure of dopaminergic neuronal cells to H2O2 or 6-OHDA induced PKD1 activation loop (PKD1S744/748) phosphorylation long before induction of neuronal cell death. Blockade of PKCδ cleavage, PKCδ knockdown or overexpression of a cleavage-resistant PKCδ mutant effectively attenuated PKD1 activation, indicating that PKCδ proteolytic activation regulates PKD1 phosphorylation. Furthermore, the PKCδ catalytic fragment, but not the regulatory fragment, increased PKD1 activation, confirming PKCδ activity modulates PKD1 activation. We also identified that phosphorylation of S916 at the C-terminal is a preceding event required for PKD1 activation loop phosphorylation. Importantly, negative modulation of PKD1 by the RNAi knockdown or overexpression of PKD1S916A phospho-defective mutants augmented oxidative stress-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 or constitutively active PKD1 plasmids attenuated oxidative stress-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury.

Conclusion: Collectively, our results demonstrate that PKCδ-dependent activation of PKD1 represents a novel intrinsic protective response in counteracting early stage oxidative damage in neuronal cells. Our results suggest that positive modulation of the PKD1-mediated compensatory protective mechanism against oxidative damage in dopaminergic neurons may provide novel neuroprotective strategies for treatment of PD.

Figure 1

Relationship between PKCδ proteolytic activation and cell death during initial stages of H₂O₂ -induced oxidative stress in N27 dopaminergic neuronal cell model. N27 dopaminergic cells were treated with H₂O₂ (100 μM) for 0-180 min and assayed for ROS generation using DCFDA dye (A) and for cytotoxicity using Sytox green dye (B). Non-linear regression graph from two or more independent experiments (n = 6-8). PKCδ cleavage was monitored by Western blot in a time dependent manner for 0 - 90 min in N27 dopaminergic cells treated with H₂O₂ (100 μM) (C). N27 dopaminergic cells were treated with H₂O₂ (100 μM) for 0, 30 or 60 minutes and PKCδ kinase activity was measured using [³²P] kinase assay; the bands were quantified for the graph (D). Data quantified from B and C were used to generate a bar graph and were compared with cytotoxicity and PKCδ cleavage following H₂O₂ exposure for 60 min. **, p < 0.01 as indicated by two-way ANOVA analysis using Bonferroni post test (E). N27 cells were transfected with 1 μM PKCδ siRNA and non-specific siRNA for 24 h and treated with 100 μM H₂O₂ and monitored for cytotoxicity using Sytox green dye, which showed significant protection from oxidative stress. ***, p < 0.001 denotes significant difference between non-specific siRNA- H₂O₂ and PKCδ siRNA H₂O₂-treated groups from two or more independent experiments (n = 6-8). Statistics were performed by one-way ANOVA analysis using Bonferroni post test (F).

Figure 2

PKD1 is highly expressed in dopaminergic neurons and activated during initial stages of H₂O₂ -induced oxidative stress. Immunofluorescence analysis of N27 dopaminergic cells stained for native PKD1 using fluorescence and confocal microscopy. Nuclei were stained with Hoechst dye (A). Primary dopaminergic neurons staining for tyrosine hydroxylase (TH) obtained from the mouse substantia nigral region show co-localization of native PKD1 with TH. TH - Green, PKD1 native - Red, Nucleus - Blue, Yellow -Merge. Nuclei were stained with Hoechst dye (B). N27 dopaminergic neuronal cells were treated with or without H₂O₂ (100 μM) for 30, 60 or 90 min and probed for PKD1 activation loop phosphorylation pS744/pS748 and native PKD1 expression (C). N27 cells were treated with or without H₂O₂ (100 μM) and PKD1 kinase activity was measured by [³²P] kinase assay using syntide 2 substrate at 60 min (D). *, p < 0.05 denotes significant difference between untreated and H₂O₂ -treated groups.

Figure 3

PKCδ dependent phosphorylation of PKD1 activation loop. N27 dopaminergic cells were treated with H₂O₂ (100 μM) with or without 1 μM rottlerin, and the lysates were probed for PKD1 activation phosphorylation (A). N27 dopaminergic cells were transfected with 1 μM PKCδ siRNA and non-specific siRNA and monitored for PKCδ protein expression and PKD1 activation loop phosphorylation after H₂O₂ treatment (B) and PKD1 kinase activity assay was performed. **, p < 0.01 denotes significant difference between NS-siRNA-H₂O₂ and PKCδ-siRNA-H₂O₂ groups (C).

Figure 4

Proteolytically activated PKCδ-CF contributes to PKD1 phosphorylation. N27 dopaminergic cells treated with H₂O₂ (100 μM) for 30, 60 or 90 minutes were monitored for PKCδ-CF (A). N27 dopaminergic cells were treated with H₂O₂ ± DEVD-fmk (50 μM) and ± ZVAD-fmk (50 μM and 100 μM) for 60 min and monitored for PKD1 activation and PKCδ cleavage (B). N27 dopaminergic cells stably expressing the cleavage-resistant mutant of PKCδ (PKCδ^D327A) were treated with H₂O₂ and monitored for PKD1 activation (C). PKD1 activation was monitored in N27 dopaminergic cells transfected with the catalytic fragment of PKCδ (PKCδ-CF) and the regulatory fragment of PKCδ (PKCδ-RF). Additionally, the mock transfection group treated with or without H₂O₂ was also monitored for PKD1 activation (D).

Figure 5

PKD1 has a cell survival function during oxidative stress in dopaminergic neuronal cells. N27 dopaminergic cells were transfected with 1 μM PKD1 siRNA and non-specific siRNA (A) and treated with 100 μM H₂O₂ for 120 minutes and monitored for cytotoxicity using Sytox green dye. Fluorescence measurements for the incorporation of Sytox green read using a flourescence plate reader (B) and visualised by phase contrast and fluorescence microscopy (C&D). DNA fragmentation assay (E) showed increased cytotoxicity and apoptosis in PKD1 knocked down samples exposed to H₂O₂. *, p < 0.05 and ***, p < 0.001 denote significant difference between Non-Specific siRNA- H₂O₂ and PKD1 siRNA-H₂O₂ treated groups.

Figure 6

PKCδ-dependent phosphorylation of PKD1 at S916 site precedes PKD1 S744/S748 active loop phosphorylation. N27 dopaminergic cells were treated with 100 μM H₂O₂ for 10-90 min and monitored for PKD1Y469, PKD1 S916 and PKD1 S744/S748 phosphorylation (A). N27 dopaminergic cells were treated with or without 50 nM desmopressin for 1 h and monitored for PKD1 Y469 phosphorylation. The cells show PKD1 Y469 phosphorylation when exposed to desmopressin, while carbachol does not cause PKD1 Y469 phosphorylation (B). N27 cells expressing PKD1^S916A mutant blocked PKD1 activation during oxidative stress, as seen by Western blotting for PKD1 S744/S748 phosphorylation and HA expression (C). N27 dopaminergic cells were transfected with 1 μM PKCδ siRNA and non-specific siRNA and monitored for PKCδ protein expression and PKD1 S916 phosphorylation after treatment with or without H₂O₂ (D).

Figure 7

PKD1 activation acts as an early protective compensatory mechanism. A comparative time course graph based on quantifying PKD1 activation profile, PKCδ cleavage profile and cytotoxicity during H₂O₂ exposure (A). N27 dopaminergic cells transiently transfected with 5 μM full length PKD1 plasmid (PKD1^WT) and 5 μM vector plasmid were treated with or without 100 μM H₂O₂ for 150 minutes and monitored for cytotoxicity using sytox green; PKD1 protected against cytotoxicity (B). N27 dopaminergic cells transiently transfected with 5 μM PKD1^S916A plasmid and 5 μM vector plasmid were treated with or without 100 μM H₂O₂ for 150 minutes and monitored for cytotoxicity using sytox green; increased cytotoxicity was observed in the cells (C). N27 dopaminergic cells transiently transfected with 5 μM PKD1^S744E/S748E and 5 μM vector plasmid (D) were treated with or without 100 μM H₂O₂ and monitored for cytotoxicity at various time points using sytox green. ***, p < 0.001 denotes significant difference between treatment groups from n≥6.

Figure 8

Activated PKD1 translocates to nucleus during H₂O₂ -induced oxidative stress. Immunofluorescence analysis of N27 dopaminergic cells stained for activated PKD1 using fluorescence microscopy during H₂O₂ exposure show translocation to nucleus. PKD1pS744/S748 - Green, Nucleus - Blue. Nuclei were stained with Hoechst dye (A). Immunofluorescence analysis of primary dopaminergic neurons staining for TH obtained from the mouse substantia nigral region shows translocation of activated PKD1 to the nucleus during H₂O₂ exposure.TH-Green, PKD1pS744/S748 - Red, Nucleus - Blue, Merge - Pink. Nuclei were stained with Hoechst dye (B). Primary dopaminergic neurons staining for TH show presence of PKD1pS916 in both cytosol and nucleus during H₂O₂ exposure. TH - Green, PKD1pS916- Red, Nucleus - Blue, Pink - Merge in nucleus, yellow -Merge in cytosol. Nuclei were stained with Hoechst dye (C).

Figure 9

PKD1 is activated by 6-OHDA induced oxidative stress. N27 dopaminergic neuronal cells were treated with or without 6-OHDA (100 μM) for 1, 3, and 6 h and probed for PKD1 activation loop phosphorylation pS744/pS748 and PKCδ cleavage.

Figure 10

Schematic of PKCδ-PKD1 signal transduction mechanism during oxidative stress in dopaminergic neuronal cells. 1) Oxidative stress causes mitochondrial impairment; 2) activation of caspase cascade; 3) caspase-3 mediates proteolytic cleavage of PKCδ; 4) proteolytically cleaved PKCδ-catalytic fragment (CF) is active; 5) PKCδ-CF activates PKD1 by activation loop phosphorylation during the early stage of oxidative stress; 6) Fully active PKD1 regulates cell survival function in N27 dopaminergic cells.