6-Hydroxydopamine disrupts cellular copper homeostasis in human neuroblastoma SH-SY5Y cells.

Copper (Cu) is an essential trace element that plays an important role in maintaining neuronal functions such as the biosynthesis of neurotransmitters. In contrast, exposure to excess Cu results in cell injury. Therefore, intracellular Cu levels are strictly regulated by proteins related to Cu-trafficking, including ATP7A. Parkinson's disease (PD) is a neurodegenerative disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra. Recently, the abnormality of Cu homeostasis was demonstrated to be related to the pathogenesis of PD. However, the association between Cu dyshomeostasis and PD remains unclear. In this study, we examined the effects of 6-hydroxydopamine (6-OHDA), a neurotoxin used for the production of PD model animals, on cellular Cu trafficking in human neuroblastoma SH-SY5Y cells. 6-OHDA reduced the protein levels of the Cu exporter ATP7A and the Cu chaperone Atox1, but not CTR1, a Cu importer; however, it did not affect the expression of ATP7A and Atox1 mRNAs. The decreased levels of ATP7A and Atox1 proteins were restored by the antioxidant N-acetylcysteine and the lysosomal inhibitor bafilomycin A1. This suggests that 6-OHDA-induced oxidative stress facilitates the degradation of these proteins. In addition, the amount of intracellular Cu after exposure to CuCl2 was significantly higher in cells pretreated with 6-OHDA than in untreated cells. Moreover, 6-OHDA reduced the protein levels of the cuproenzyme dopamine ß-hydroxylase that converts dopamine to noradrenaline. Thus, this study suggests that 6-OHDA disrupts Cu homeostasis through the dysregulation of cellular Cu trafficking, resulting in the dysfunction of neuronal cells.

Dual Role for Astroglial Copper-Assisted Polyamine Metabolism during Intense Network Activity.

Astrocytes serve essential roles in human brain function and diseases. Growing evidence indicates that astrocytes are central players of the feedback modulation of excitatory Glu signalling during epileptiform activity via Glu-GABA exchange. The underlying mechanism results in the increase of tonic inhibition by reverse operation of the astroglial GABA transporter, induced by Glu-Na+ symport. GABA, released from astrocytes, is synthesized from the polyamine (PA) putrescine and this process involves copper amino oxidase. Through this pathway, putrescine can be considered as an important source of inhibitory signaling that counterbalances epileptic discharges. Putrescine, however, is also a precursor for spermine that is known to enhance gap junction channel communication and, consequently, supports long-range Ca2+ signaling and contributes to spreading of excitatory activity through the astrocytic syncytium. Recently, we presented the possibility of neuron-glia redox coupling through copper (Cu+/Cu2+) signaling and oxidative putrescine catabolism. In the current work, we explore whether the Cu+/Cu2+ homeostasis is involved in astrocytic control on neuronal excitability by regulating PA catabolism. We provide supporting experimental data underlying this hypothesis. We show that the blockade of copper transporter (CTR1) by AgNO3 (3.6 µM) prevents GABA transporter-mediated tonic inhibitory currents, indicating causal relationship between copper (Cu+/Cu2+) uptake and the catabolism of putrescine to GABA in astrocytes. In addition, we show that MnCl2 (20 µM), an inhibitor of the divalent metal transporter DMT1, also prevents the astrocytic Glu-GABA exchange. Furthermore, we observed that facilitation of copper uptake by added CuCl2 (2 µM) boosts tonic inhibitory currents. These findings corroborate the hypothesis that modulation of neuron-glia coupling by copper uptake drives putrescine → GABA transformation, which leads to subsequent Glu-GABA exchange and tonic inhibition. Findings may in turn highlight the potential role of copper signaling in fine-tuning the activity of the tripartite synapse.

Inhibiting the copper efflux system in microbes as a novel approach for developing antibiotics.

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living cells, but it can be toxic in excess. Both eukaryotic and prokaryotic cells have developed distinct copper regulation systems to prevent its toxicity. Therefore, selectively targeting the prokaryotic copper regulation system might be an initial step in developing next-generation antibiotics. One such system is the Gram-negative bacterial CusCFBA efflux system. CusB is a key protein in this system and was previously reported to play an important role in opening the channel for efflux via significant structural changes upon copper binding while also controlling the assembly and disassembly process of the entire channel. In this study, we aimed to develop novel peptide copper channel blockers, designed by in silico calculations based on the structure of CusB. Using a combination of magnetic resonance spectroscopy and various biochemical methods, we found a lead peptide that promotes copper-induced cell toxicity. Targeting copper transport in bacteria has not yet been pursued as an antibiotic mechanism of action. Thus, our study lays the foundation for discovering novel antibiotics.

Mechanistic and Structural Basis for Inhibition of Copper Trafficking by Platinum Anticancer Drugs.

Copper (Cu) is required for maturation of cuproenzymes, cell proliferation, and angiogenesis, and its transport entails highly specific protein-protein interactions. In humans, the Cu chaperone Atox1 mediates Cu(I) delivery to P-type ATPases Atp7a and Atp7b (the Menkes and Wilson disease proteins, respectively), which are responsible for Cu release to the secretory pathway and excess Cu efflux. Cu(I) handover is believed to occur through the formation of three-coordinate intermediates where the metal ion is simultaneously linked to Atox1 and to a soluble domain of Cu-ATPases, both sharing a CxxC dithiol motif. The ultrahigh thermodynamic stability of chelating S-donor ligands secures the redox-active and potentially toxic Cu(I) ion, while their kinetic lability allows facile metal transfer. The same CxxC motifs can interact with and mediate the biological response to antitumor platinum drugs, which are among the most used chemotherapeutics. We show that cisplatin and an oxaliplatin analogue can specifically bind to the heterodimeric complex Atox1-Cu(I)-Mnk1 (Mnk1 is the first soluble domain of Atp7a), thus leading to a kinetically stable adduct that has been structurally characterized by solution NMR and X-ray crystallography. Of the two possible binding configurations of the Cu(I) ion in the cage made by the CxxC motifs of the two proteins, one (bidentate Atox1 and monodentate Mnk1) is less stable and more reactive toward cis-Pt(II) compounds, as shown by using mutated proteins. A Cu(I) ion can be retained at the Pt(II) coordination site but can be released to glutathione (a physiological thiol) or to other complexing agents. The Pt(II)-supported heterodimeric complex does not form if Zn(II) is used in place of Cu(I) and transplatin instead of cisplatin. The results indicate that Pt(II) drugs can specifically affect Cu(I) homeostasis by interfering with the rapid exchange of Cu(I) between Atox1 and Cu-ATPases with consequences on cancer cell viability and migration.