Mechanisms controlling the cellular accumulation of copper bis(thiosemicarbazonato) complexes.

Copper (Cu) bis(thiosemicarbazonato) metal complexes [Cu(II)(btsc)s] have unique tumor-imaging and treatment properties and more recently have revealed potent neuroprotective actions in animal and cell models of neurodegeneration. However, despite the continued development of Cu(II)(btsc)s as potential therapeutics or diagnostic agents, little is known of the mechanisms involved in cell uptake, subcellular trafficking, and efflux of this family of compounds. Because of their high lipophilicity, it has been assumed that cellular accumulation is through passive diffusion, although this has not been analyzed in detail. The role of efflux pathways in cell homeostasis of the complexes is also largely unknown. In the present study, we investigated the cellular accumulation of the Cu(II)(btsc) complexes Cu(II)(gtsm) and Cu(II)(atsm) in human neuronal (M17) and glial (U87MG) cell lines under a range of conditions. Collectively, the data strongly suggested that Cu(II)(gtsm) and Cu(II)(atsm) may be taken into these cells by combined passive and facilitated (protein-carrier-mediated) mechanisms. This was supported by strong temperature-dependent changes to the uptake of the complexes and the influence of the cell surface protein on Cu accumulation. We found no evidence to support a role for copper-transporter 1 in accumulation of the compounds. Importantly, our findings also demonstrated that Cu from both Cu(II)(gtsm) and Cu(II)(atsm) was rapidly effluxed from the cells through active mechanisms. Whether this was in the form of released ionic Cu or as an intact metal complex is not known. However, this finding highlighted the difficulty of trying to determine the uptake mechanism of metal complexes when efflux is occurring concomitantly. These findings are the first detailed exploration of the cellular accumulation mechanisms of Cu(II)(btsc)s. The study delineates strategies to investigate the uptake and efflux mechanisms of metal complexes in cells, while highlighting specific difficulties and challenges that need to be considered before drawing definitive conclusions.

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

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.

Zinc induces depletion and aggregation of endogenous TDP-43.

Ubiquitinated neuronal aggregates containing TDP-43 are pathological hallmarks in the spectrum of frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS). In affected neurons, TDP-43 undergoes C-terminal fragmentation, phosphorylation, and ubiquitination and forms aggregates in the cytoplasm or nucleus. Although in vitro studies have been able to recapitulate these features using transfected cell culture models, little is known about the biochemical mechanisms that underlie pathological changes to endogenous TDP-43. As altered metal ion homeostasis and increased oxidative stress are central features of neurodegeneration, including FTLD and ALS, we sought to determine the affects of these factors on endogenous TDP-43 metabolism in mammalian cells. Treatment of SY5Y neuronal-like cells expressing endogenous TDP-43 with zinc (Zn) induced depletion of TDP-43 expression and formation of inclusions that were TDP-43 positive. TDP-43 was also detected in the cytosol of Zn-affected cells but this was not aggregated. No evidence of C-terminal fragmentation, phosphorylation, or ubiquitination was observed. The depletion and aggregation of TDP-43 were associated with the specific action of Zn but were not seen with copper, iron, or H(2)O(2). These studies describe for the first time specific induction of endogenous TDP-43 aggregation in neuronal-like cells and suggest that specific Zn-associated processes could affect TDP-43 metabolism in neurodegenerative diseases.

Sustained activation of glial cell epidermal growth factor receptor by bis(thiosemicarbazonato) metal complexes is associated with inhibition of protein tyrosine phosphatase activity.

Bis(thiosemicarbazonato) metal complexes (M(II)(btsc)) have demonstrated potential neuroprotective activity in cell and animal models of Alzheimer's disease (AD). Metal complexes can activate the epidermal growth factor receptor (EGFR), leading to inhibition of amyloid peptide accumulation in neuronal cells. As glial cells also have an important role in modulating neuronal health and survival in AD, we examined the effect of M(II)(btsc) on activity of EGFR in an astroglial cell line. Our findings reveal potent activation of glial EGFR by glyoxalbis(N(4)-methylthiosemicarbazonato)Cu(II)] (Cu(II)(gtsm)). Activation of EGFR by Cu(II)(gtsm) involved phosphorylation of multiple tyrosine residues and was mediated by a cognate ligand-independent process involving M(II)(btsc) inhibition of protein tyrosine phosphatase (PTP) activity. EGFR activation resulted in release of growth factors and cytokines with potential modulatory effects on neuronal function. These studies provide an important insight into the mechanism of action of a neuroprotective M(II)(btsc) and provide a basis for future studies into this novel approach to AD therapy.