In vitro effect of Penicillamine on neuronal and gliomal cells : possible implications on Wilson Disease patients -

Abstract

Background: Copper is an essential trace element that acts as a cofactor for many cuproenzymes. Cu is also crucial for the brain development and function. However, due to its high redox potential when present in excess, copper may damage the lipids, proteins, and DNA of the cell. Disturbance in Cu homeostasis has been implicated in many neurodegenerative disorders. Wilson’s disease is a rare autosomal recessive disorder resulting from copper accumulation in liver and brain of patients. WD is caused by mutations in ATP7B encoding a copper transporter that incorporates Cu into ceruloplasmin, a member of the multi-Cu oxidase family of enzymes. Cp maintains iron homeostasis. Copper loading into apoceruloplasmin yields the active holoceruloplasmin which has a ferroxidase activity that catalyzes iron oxidation (Fe2+ into Fe3+) with subsequent transfer onto transferrin. D-Penicillamine, the recommended copper chelating drug for treating patients with WD, has been reported to worsen the symptoms of WD patients with neurologic manifestation. Hypothesis and aims: The lifelong treatment of WD patients with PA will decrease Cu loading onto apo-Cp, reduce the level of active Cp, consequently lower ferroxidase activity, iron oxidation and loading into transferrin, leading to iron deposition in the brain. This study aims to investigate the effect of treatment with D-PA alone, Cu alone, and of PA combined with copper on neuronal and glial cells viability and expression of copper and iron binding proteins. Methods: Neuronal (PC12) and glial (U251) cells were cultured under optimal conditions and treated with Cu and-or PA, following which we determined: viability using MTT assay and Trypan blue exclusion test, ROS level using NBT reduction assay, intracellular GSH level using fluorometric glutathione detection kit, membrane integrity using LDH release assay, mitochondrial depolarization using MitoPT-JC1 assay, distribution of cell cycle phases by flow cytometry. In addition, α-fodrin cleavage and expression levels of