Subcellular localization of a fluorescent derivative of CuII(atsm) offers insight into the neuroprotective action of CuII(atsm)

Abstract

Copper complexes of bis(thiosemicarbazone) (Cu(II)(btsc)s) have been studied as potential anti-cancer agents and hypoxia imaging agents. More recently, Cu(II)(btsc)s have been identified as possessing potent neuroprotective properties in cell and animal models of neurodegenerative disease. Despite their broad range of pharmacological activity little is known about how cells traffic Cu(II)(btsc)s and how this relates to potential anti-cancer or neuroprotective outcomes. One method of investigating sub-cellular localization of metal complexes is through confocal fluorescence imaging of the compounds in cells. Previously we harnessed the fluorescence of a pyrene group attached to diacetyl-bis(N4-methylthiosemicarbazonato)copper(ii)) (Cu(II)(atsm)), (Cu(II)L(1)). We demonstrated that Cu(II)L(1) was partially localized to lysosomes in HeLa cancer epithelial cells. Here we extend these studies to map the sub-cellular localization of Cu(II)L(1) in M17 neuroblastoma cells. Treatment of M17 or HeLa cells led to rapid association of the Cu-complex into distinct punctate structures that partially co-localized with lysosomes as assessed by co-localization with Lysotracker and acridine orange. No localization to early or late endosomes, the nucleus or mitochondria was observed. We also found evidence for a limited association of Cu(II)L(1) with autophagic structures, however, this did not account for the majority of the punctate localization of Cu(II)L(1). In addition, Cu(II)L(1) revealed partial localization with ER Tracker and was found to inhibit ER stress induced by tunicamycin. This is the first report to comprehensively characterize the sub-cellular localization of a Cu(II)(atsm) derivative in cells of a neuronal origin and the partial association with lysosome/autophagic structures and the ER may have a potential role in neuroprotection.