Oral Presentation 24th Australian Conference on Microscopy and Microanalysis 2016

Using FLIM to shed light on the neuroprotective mechanism of action of the bis(thiosemicarbazonato) CuII(atsm) (#46)

Janine L. James 1 , James Hickey 1 , Clare A. Henderson 1 , Kate A. Price 1 , Alexandra I. Mot 1 , Gojko Bucnic 1 , Peter Crouch 1 , Anthony White 1 , Trevor A. Smith 1 , Paul Donnelly 1
  1. The University of Melbourne, Melbourne, VIC, Australia

Introduction & Background

The metal complex CuII(atsm) is neuroprotective in multiple animal models of Parkinson’s disease, and Amyotrophic Lateral Sclerosis. There is strong evidence to suggest the mechanism of neuroprotection in ALS is due to copper release from the bis(thiosemicarbazonato) backbone. This study aims to shed light on the mechanism of neuroprotection by visually studying the intracellular localisation of CuII(atsm) via the addition of a bodipy fluorophore, determining if copper is released from the bis(thiosemicarbazonato) backbone using fluorescence lifetime imaging (FLIM)

Methods

A bodipy fluorophore (ligand, ‘L’) was synthesised and conjugated to CuII(atsm), (denoted CuII(atsm)-L), enabling the sub-cellular localisation of CuII(atsm)-L to be tracked in live cells. The sub-cellular localisation of CuII(atsm)-L was investigated using confocal live cell microscopy. Experiments to show the fluorescence life time of CuII(atsm)-L were performed on a FLIM capable, live cell, confocal microscope.

Major Findings

CuII(atsm)-L was found to accumulate in lipid droplets as well as in lysosomes, providing potential insight into the neuroprotective mechanism of action of CuII(atsm). The different fluorescent lifetimes of the metal free ligand and its Zn and Cu complexes could be used to identify the presence of each species inside the cell. Treatment of cells with Cu(atsm)-L led to the expected spectroscopic signature of the Cu complex but also evidence for the presence of both the free ligand and the zinc complex presumably due to reduction of the Cu(II) to Cu(I) and loss of the Cu from the ligand. The ligand is then able to bind zinc.  

Concluding statement

CuII(atsm)-L is readily taken up by the cell and accumulates in both lysosomes and lipid droplets, and is capable of releasing copper via reduction and subsequently picking up zinc

Hickey, James, et al. (2015). "Intracellular Distribution of Fluorescent Copper and Zinc Bis(thiosemicarbazonato) Complexes Measured with Fluorescence Lifetime Spectroscopy." Inorganic Chemistry