Figure 1.
6-OHDA-induced oxidative stress causes cell death in N27 dopaminergic neuronal cell models.
N27 dopaminergic cells were treated with 6-OHDA (100 µM) for 0–8 h and assayed for cytotoxicity using Sytox green fluorescence assay. The fluorescence was measured by using a fluorescence plate reader. Non-linear regression was performed from two or more independent experiments (A), and visualized by fluorescence microscopy (B).
Figure 2.
PKD1 is activated in N27 cells during 6-OHDA-induced oxidative stress.
N27 dopaminergic neuronal cells were treated with 6-OHDA (100 µM) for 1–7 h and probed for PKD1 activation loop phosphorylation pS744/pS748 and native PKD1 expression (A). A comparative time course graph depicting the reciprocal relationship between PKD1 activation and cytotoxicity during 6-OHDA exposure (B). Cytoplasmic and nuclear extracts from control and 6-OHDA-treated N27 cells were prepared and probed for PKD1 activation loop phosphorylation pS744/pS748 and native PKD1 expression (C). Lamin B (nuclear fraction) or α-Tubulin (cytoplasmic fraction) was used as loading control.
Figure 3.
PKCδ-dependent S916-phosphorylation of PKD1 precedes PKD1 S744/S748 active loop phosphorylation in N27 dopaminergic cells.
N27 dopaminergic cells were treated with 100 µM 6-OHDA for 1–7 h and monitored for PKD1 S916 phosphorylation (A). N27 cells transiently expressing PKD1S916A mutant prevent PKD1 activation during oxidative stress, as seen by Western blotting for PKD1 S744/S748 phosphorylation (B).
Figure 4.
PKD1 activation is essential for cell survival during 6-OHDA-induced oxidative stress in N27 dopaminergic neurons.
N27 cells were co-treated with the PKD1 inhibitor kb-NB 14270 and monitored for cell death during 6-OHDA treatment for 4 h (A). N27 dopaminergic cells were transfected with 1 µM PKD1 siRNA and non-specific siRNA (B) and treated with 100 µM 6-OHDA for 9 h and monitored for cytotoxicity using Sytox green assay. Fluorescence measurements for the incorporation of Sytox green dye were performed using a fluorescence plate reader. **, p<0.01 denotes a significant difference between non-specific siRNA-6-OHDA and PKD1 siRNA-6-OHDA treated groups (C).
Figure 5.
Modulation of PKD1 offers neuroprotection in N27 dopaminergic neuronal cells.
N27 dopaminergic cells transiently transfected with 5 µg of full-length PKD1 plasmid (PKD1WT) and 5 µg of vector plasmid were treated with or without 100 µM 6-OHDA and monitored for cytotoxicity at various time points using Sytox green assay. ***, p<0.001 denotes a significant difference between treatment groups from n≥6 (A). N27 dopaminergic cells transiently transfected with 5 µg of constitutively active PKD1S744E/S748E mutant and 5 µg of vector plasmid were treated with or without 100 µM 6-OHDA and monitored for cytotoxicity at various time points using Sytox green assay. ***, p<0.001 denotes a significant difference between treatment groups from n≥6 (B).
Figure 6.
PKD1 is highly expressed in primary dopaminergic neurons and activated during 6-OHDA-induced oxidative stress.
(A) Primary dopaminergic neurons stained for tyrosine hydroxylase (TH) show co-localization of native PKD1 with TH in the cytosol. TH-Green, PKD1 native-Red, Nucleus-Blue, Merge-Yellow. Nuclei were stained with Hoechst dye. (B) Immunofluorescence analysis of primary dopaminergic neurons stained for TH shows translocation of activated PKD1 to the nucleus during 6-OHDA exposure. TH-Green, PKD1pS744/S748-Red, Nucleus-Blue, Merge-Pink. Nuclei were stained with Hoechst dye. White arrows indicate the TH+ neurons, in which activated PKD1 translocates into nucleus after 6-OHDA treatment. (C) Primary dopaminergic neurons staining for TH show the presence of PKD1pS916 in both the cytosol and nucleus during 6-OHDA exposure. TH-Green, PKD1pS916-Red, red/yellow-Merge.
Figure 7.
A proposed model for PKD1 neuroprotection in dopaminergic neurons during early stages of oxidative insult.
Oxidative insult rapidly induces PKD1 activation loop phosphorylation at pS744/pS748, which then translocates from the cytoplasm to the nucleus. The PKD1 activation and nuclear translocation lead to counteracting oxidative injury and subsequent initiation of cell survival processes.