Neuroprotective Copper Bis(thiosemicarbazonato) Complexes Promote Neurite Elongation

Laura Bica,Peter J Crouch,Anthony R White,Aphrodite Caragounis,Roberto Cappai,Brett M Paterson,Clare Duncan,Irene Volitakis,James Camakaris,Jeffrey R Liddell,Alexandra Grubman,Paul S Donnelly,Stephen D Ginsberg

doi:10.1371/journal.pone.0090070

Abstract

Abnormal biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and motor neuron disease. Recent studies have shown that metal complexing compounds behaving as ionophores such as clioquinol and PBT2 have robust therapeutic activity in animal models of neurodegenerative disease; however, the mechanism of neuroprotective action remains unclear. These neuroprotective or neurogenerative processes may be related to the delivery or redistribution of biometals, such as copper and zinc, by metal ionophores. To investigate this further, we examined the effect of the bis(thiosemicarbazonato)-copper complex, CuII(gtsm) on neuritogenesis and neurite elongation (neurogenerative outcomes) in PC12 neuronal-related cultures. We found that CuII(gtsm) induced robust neurite elongation in PC12 cells when delivered at concentrations of 25 or 50 nM overnight. Analogous effects were observed with an alternative copper bis(thiosemicarbazonato) complex, CuII(atsm), but at a higher concentration. Induction of neurite elongation by CuII(gtsm) was restricted to neurites within the length range of 75–99 µm with a 2.3-fold increase in numbers of neurites in this length range with 50 nM CuII(gtsm) treatment. The mechanism of neurogenerative action was investigated and revealed that CuII(gtsm) inhibited cellular phosphatase activity. Treatment of cultures with 5 nM FK506 (calcineurin phosphatase inhibitor) resulted in analogous elongation of neurites compared to 50 nM CuII(gtsm), suggesting a potential link between CuII(gtsm)-mediated phosphatase inhibition and neurogenerative outcomes.

Highlights

Altered biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD) and motor neuron diseases
CuII(gtsm) increased cellular copper levels resulting in enhanced neurite elongation in vitro nerve growth factor (NGF)-treated PC12 cells were used to examine the effects of
PC12 cells were treated with NGF for 48 hr and exposed to 50 nM CuII(gtsm), CuII(atsm),H2 (Cu-free ligand) or CuCl2, for 18 hr

Summary

Introduction

Altered biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD) and motor neuron diseases. Malm et al demonstrated that the metal ionophore pyrrolidine dithiocarbamate (PDTC) elevated brain Cu levels and improved cognition in APP/PS1 mice without altered Ab levels [13] We found that another bis(thiosemicarbazone) diacetyl-bis(N4-methylthiosemicarbazonato)-copper(II) (CuII(atsm)), which is closely related to CuII(gtsm), had a potent neuroprotective action in multiple models of Parkinson’s disease and motor neuron disease, two diseases not associated with the Ab peptide [14,15]. This could be enhanced by the addition of Cu or Zn and prevented by co-incubation with the cellimpermeable Cu/Zn chelator, diAmsar [17] These studies suggested that the neuroprotective action of PBT2 involved the redistribution of biometals such as Cu and/or Zn. As CuII(gtsm) and CuII(atsm) have demonstrated neuroprotective activity in vivo in murine models of neurodegeneration, we have examined whether these metal-complexes induce similar changes in cultured cells. Promotion of neurogenerative outcomes could potentially be a valuable in vitro biomarker for the neuroprotective action of these metal-complexes

Materials and Methods

Results

Discussion