Molecular cloning, epigenetic regulation, and functional characterization of Prkd1 gene promoter in dopaminergic cell culture models of Parkinson's disease

Abstract

We recently identified a compensatory survival role for protein kinase D1 (PKD1) in protecting dopaminergic neurons from oxidative insult. To investigate the molecular mechanism of Prkd1 gene expression, we cloned the 5’-flanking region (1620-bp) of the mouse Prkd1 gene. Deletion analyses revealed that the −250/+113 promoter region contains full promoter activity in MN9D dopaminergic neuronal cells. In silico analysis of the Prkd1 promoter uncovered binding sites for key redox transcription factors including Sp1 and NF-κB. Overexpression of Sp1, Sp3, and NF-κB-p65 proteins stimulated Prkd1 promoter activity. Binding of Sp3 and NF-κB-p65 to the Prkd1 promoter was confirmed using chromatin immunoprecipitation. Treatment with the Sp inhibitor mithramycin-A significantly attenuated Prkd1 promoter activity and PKD1 mRNA and protein expression. Further mechanistic studies revealed that inhibition of histone acetylation and DNA methylation upregulates PKD1 mRNA expression. Importantly, negative modulation of PKD1 signaling by pharmacological inhibition or shRNA knockdown increased dopaminergic neuronal sensitivity to oxidative damage in a human mesencephalic neuronal cell model. Collectively, our findings demonstrate that Sp1, Sp3 and NF-κB-p65 can transactivate the mouse Prkd1 promoter and that epigenetic mechanisms, such as DNA methylation and histone modification, are key regulatory events controlling the expression of pro-survival kinase PKD1 in dopaminergic neuronal cells.

Neuronal protection against oxidative insult by polyanhydride nanoparticle-based mitochondria-targeted antioxidant therapy

“…This cell line can be differentiated into post-mitotic neurons with a dopaminergic phenotype (47, 48). Undifferentiated LUHMES cells were propagated in Advanced DMEM/F12 supplemented with 1× N-2 supplement, 2 mM L-glutamine, and 40 ng/mL recombinant bFGF on multi-well plates pre-coated with 50 μg/mL poly-L-ornithine and 1 μg/mL fibronectin.…”

“…Dopamine uptake measurements were measured as described previously (47, 49) with modifications. Briefly, differentiated LUHMES cells grown in 6-well plates were washed twice with Krebs-Ringer buffer (16 mM NaH 2 PO 4 , 120 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl 2 , 1.2 mM MgSO 4 , 1.3 mM EDTA, and 5.6 mM glucose, pH 7.4), followed by incubation with 10 nM [ 3 H] dopamine for 30 minutes at 37°C.…”

Protein kinase Cδ upregulation in microglia drives neuroinflammatory responses and dopaminergic neurodegeneration in experimental models of Parkinson's disease

“…Samples were visualized using a Leica DMIRE2 confocal microscope or Nikon inverted fluorescence microscope (TE-2000U) and images were captured using a SPOT digital camera (Diagnostic Instruments Inc). PKCδ and p65 immunofluorescence in primary microglia was performed according to previously published protocols (Ay et al, 2015; Jin et al, 2015), with some modifications. Briefly, microglial cells were grown on poly-D-lysine-coated coverslips and treated 48 h later.…”

Molecular mechanisms underlying protective effects of quercetin against mitochondrial dysfunction and progressive dopaminergic neurodegeneration in cell culture and MitoPark transgenic mouse models of Parkinson's Disease

“…; Ay et al . ). The protective role of Akt has been documented for neurodegenerative diseases, including PD (Humbert et al .…”

“…; Ay et al . ), Akt (1 : 1000), phospho‐Akt (1 : 1000) (Gordon et al . ), TFAM (1 : 1000) (Gordon et al .…”