Sub-toxic pharmacological effects of copper upon CAD monoaminergic neurons in culture

The pathogenesis of Parkinson disease has been associated with the loss of dopaminergic neurons in the pars compacta of the substantia nigra as well as the presence of ubiquinated alpha synuclein protein deposits in the cytoplasm of the neurons, resulting in reduced dopamine level. The increased concentrations of heavy metal ions such as copper (Cu) in the cerebrospinal fluid of patients with PD and the cellular toxicity in dopaminergic neurons associated with Cu, as well as the formation of reactive oxygen species (ROS) is stimulating research interest in exploring a potential link with environmental contaminants as a possible contribution to the aetiology of PD [Valko M et al., 2015]. However, most studies have focused on mechanisms underlying copper-induced neurotoxicity, but have somewhat undervalued the sub-toxic pharmacological effects of such heavy metals.

This investigation is directed towards elucidating the effect of sub-toxic pharmacological effects of Cu on the cellular functionality of neuronal cells. Catecholaminergic a-differentiated (CAD) cells were used as this cell line exhibits biochemical and morphological characteristics similar to primary dopaminergic neurons and provides a useful tool for studying the effect of Cu ions on both differentiated and undifferentiated cells. As a first step, a dose-dependent effect of Copper (sulphate) on the viability of undifferentiated cells was investigated. Following a 24 hours incubation the mean IC50for Cu ions was found out to be 460 µM (n = 12 replicate values). Using lower concentrations(10 µM and 40 µM)of Cu, over a 24 hour time-course, no significant effect was observed on mitochondria function (using the MTT assay) and cytotoxicity (LDH release assay); confirming that these are non-toxic doses on undifferentiated CAD cells. To further investigate the pharmacological effects of these non-toxic levels of copper, calcium imaging on live undifferentiated and differentiated CAD cells (6 days serum-free) were carried out using Calcium Green-1 AM and the LSM 880 with Airyscan-Zeiss confocal microscope. Interestingly, there was no apparent effect of Cu at 40 µM on calcium signals with undifferentiated cells up to 10 minutes exposure, however there was a robust delayed reduction of calcium response in differentiated neuronal cell processes, with a parallel modest effect upon cell body signal (Figure 1). This correlates with recent studies which showed copper levels are distributed diffusely in neurons, but are higher in the processes relative to other metal ions, such as Zn [Colvin R et al., 2015]

Figure 1: Effects of sub-toxic (40 µM) copper ions upon calcium signals in differentiated CAD cells

This project is funded by the Nigerian Petroleum Technology Development Fund (PTDF).

Colvin R et al. (2015) Metallomics. 7(7): 1111-23

Valko M. et al. (2015) Arch Toxicol. In press. Sep 7 published online