ABSTRACT
Title
Effect of Nicotine on Dopamine Metabolism in PC12 TET-ON Cells Lines Treatedwith Doxycycline
Authors
Y. Spissu
Doctoral School of Biological Sciences, Neuroscience Section
Dept. of Neuroscience, Medical School - University of Sassari, Italy
Doctoral School of Biological Sciences, Neuroscience Section
Dept. of Neuroscience, Medical School - University of Sassari, Italy
Abstract
Most Parkinson’s disease (PD) cases occur sporadically and several genes associated with monogenetic forms of the disease mimicking clinical symptoms of sporadic PD have been identified. Many more amino acid residue substitutions have also been found to be associated with the disease in leucine-rich repeat kinase 2 (LRRK2) gene. LRRK2 contains 51 exons and encodes a large 2527-aa protein, which consists of several functional domains, including a Ras-like small GTPase domain and a MAP kinase-like domain. Interestingly, multiple amino acid substitutions of the same residue R1441 (R1441C, R1441G, and R1441H) in the highly conserved GTPase domain and multiple mutations (I2012T, G2019S, and I2020T) in the kinase domain have been identified [1].For the study of developmental processes and high-order brain functions,animal models of Parkinson’s disease (PD) was created using the tTA- or the rtTA-based regulatory systems [2]. Since PC12 cells are a reliable model for studying in vitro dopamine (DA) metabolism, we deemed of interest the study of DA metabolism in PC12 cells with human LRRK2, to add information in order to distinguish between the effects of transgene expression using TET systems, activated in presence of Doxycycline (DOX), and the potential effects of Nicotine (that increases intracellular free Ca2+ concentration [Ca2+]i stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH)) on their dopaminergic metabolism.
At the start of experiment, 140x103 cells/cm2 were plated and treated 24h later (time 0) with DOX (1.0 µg/ml); after 48h, through microdialysis procedure [3], was treated with Nicotine 5 mM for 1h and DA and metabolites (DOPAC, HVA and 3-MT) were assayed by HPLC-EC2.Four PC12 TET-ON cells lines were used for experiments and in 3 PC12 TET-ON cell lines, expressing human LRRK2, pathological mutation was induced as follow [1]: PC 12-ON, PC 12 ON-LRRK2 WT, PC 12 ON-LRRK2 2019 and PC 12 ON-LRRK2 1441.
PC12 TET-ON cell cultures (PC12-0N, PC12 ON-LRRK2 WT, PC12 ON-LRRK2 1441 and PC12 ON-LRRK2 2019), were incubated for 48 h with 1.0 μg/ml of DOX. When PC 12-ON where incubated in presence or in absence of DOX Nicotine-induced DA release (DA+ 3-MT) didn’t change. An increase in DA + 3-MT release was observed after Nicotine treatment. In PC 12 ON-LRRK2 WT, as well as in PC 12-ON, the Nicotine-induced DA release was lower than the control after incubation without DOX while was higher after incubation with DOX. Similar results were obtained in PC 12 ON-LRRK2 2019 line vs control. Different results were obtained in PC 12 ON-LRRK2 1441 where Nicotine-induced DA + 3-MT release was lower in the presence of DOX vs control. The sum of metabolites DOPAC and HVA decreased in all cell lines after Nicotine treatment in presence or in absence of DOX.
1) Tong Yet al. (2009) - PNAS 106(34):14622–14627.
2) Iaccarino C et al. (2007) - Hum Mol Genet. 16:1319-2619.
3) Rocchitta G. Et al. (2005) - J Pineal Res. 39(4):409-1.
At the start of experiment, 140x103 cells/cm2 were plated and treated 24h later (time 0) with DOX (1.0 µg/ml); after 48h, through microdialysis procedure [3], was treated with Nicotine 5 mM for 1h and DA and metabolites (DOPAC, HVA and 3-MT) were assayed by HPLC-EC2.Four PC12 TET-ON cells lines were used for experiments and in 3 PC12 TET-ON cell lines, expressing human LRRK2, pathological mutation was induced as follow [1]: PC 12-ON, PC 12 ON-LRRK2 WT, PC 12 ON-LRRK2 2019 and PC 12 ON-LRRK2 1441.
PC12 TET-ON cell cultures (PC12-0N, PC12 ON-LRRK2 WT, PC12 ON-LRRK2 1441 and PC12 ON-LRRK2 2019), were incubated for 48 h with 1.0 μg/ml of DOX. When PC 12-ON where incubated in presence or in absence of DOX Nicotine-induced DA release (DA+ 3-MT) didn’t change. An increase in DA + 3-MT release was observed after Nicotine treatment. In PC 12 ON-LRRK2 WT, as well as in PC 12-ON, the Nicotine-induced DA release was lower than the control after incubation without DOX while was higher after incubation with DOX. Similar results were obtained in PC 12 ON-LRRK2 2019 line vs control. Different results were obtained in PC 12 ON-LRRK2 1441 where Nicotine-induced DA + 3-MT release was lower in the presence of DOX vs control. The sum of metabolites DOPAC and HVA decreased in all cell lines after Nicotine treatment in presence or in absence of DOX.
1) Tong Yet al. (2009) - PNAS 106(34):14622–14627.
2) Iaccarino C et al. (2007) - Hum Mol Genet. 16:1319-2619.
3) Rocchitta G. Et al. (2005) - J Pineal Res. 39(4):409-1.