Review
Extracellular Cu2+ pools and their detection: From current knowledge to next-generation probes

https://doi.org/10.1016/j.ccr.2020.213727Get rights and content

Highlights

  • The notion of exchangeable Cu and the methods to measure it are revised.

  • The exchangeable Cu has arisen as a biomarker for Cu-related diseases.

  • Multiple parameters are crucial to design reversible Cu2+-sensor for bioapplications.

  • Current luminescent and MRI-active Cu2+ probes do not fulfil all these parameters.

  • Combining selectivity and sensitivity is very challenging, especially in the plasma.

Abstract

Copper (Cu) is an essential micronutrient for most organisms and serves mainly as a redox-active catalytic centre in enzymes cycling between Cu+ and Cu2+. Membrane transporters and shuttles are involved to bring and insert Cu into these enzymes and to control tightly the copper metabolism, whose failure can lead to severe diseases. The main oxidation state intracellularly is Cu+, whereas Cu2+ is mainly found in extracellular fluids. A basic approach to investigate Cu metabolism in vivo and in cellulo contemplates the use of luminescent (mostly fluorescent) or magnetic resonance imaging (MRI)-active probes. Here, we focus on sensors of the Cu2+ state. First, Cu metabolism and speciation are revised, focusing on the main extracellular fluids (blood plasma, urine, cerebrospinal fluid, synaptic cleft, milk, saliva, sweat) and cell culture media, and highlighting the notion of exchangeable Cu2+ pool. Indeed, in contrast to bulk Cu measurements, sensors can only detect Cu2+ that is labile and thermodynamically accessible. Thus, the kinetics and thermodynamics of the exchangeable pools determine the quantity of Cu2+ that can be measured and influence the design of the sensor. The study of the best-known exchangeable Cu2+ pool in blood plasma, i.e. serum albumin, shows that a sensor might need a sub-femtomolar affinity for Cu2+ to compete with endogenous Cu2+ ligands. The selectivity of the probe for Cu2+ is also discussed, in particular against Zn2+, which is much more available than Cu2+ in the extracellular fluids (e.g. at least 106 times in the blood). Finally, the analysis of the literature on luminescent and MRI-active Cu2+ sensors applied in extracellular media show indeed how challenging such measurement is, and that none of the sensors reported convincingly and specifically detects Cu2+ in a biological system. Indeed, when considering all the sought parameters, i.e. thermodynamics and kinetics of the Cu2+-sensor, the specificity towards Cu2+, the reversibility, the sensitivity of the luminescent or MRI response and hence the required sensor concentration, it becomes clear that this is a huge challenge and that we stand just at the dawn of this field.

Introduction

Copper (Cu) is an essential redox-active ion for most living organisms. Indeed, the redox cycling between Cu+ and Cu2+ has been exploited by nature to carry out fundamental biochemical processes, such as electron transfer (e.g. in cytochrome c oxidase, COX), oxygen transport (e.g. in Molluscan hemocyanin), and redox catalysis (e.g. in superoxide dismutase, SOD).

However, such redox activity also represents a potential danger, as Cu redox cycling is very competent in catalysing the formation of Reactive Oxygen Species (ROS), which can oxidatively damage biomolecules. Therefore, nature developed a machinery of Cu-binding systems to handle Cu safely and keep the appropriate Cu homeostasis in cells and multicellular organisms (see Fig. 1) [1].

In mammalian cells, the membrane transporters Ctr1/2 and ATP7A/7B regulate respectively the import and the export of Cu (as Cu+) into/from the cytosol and intracellular organelles. Intracellularly, the reduced Cu+ state is predominant due to the high abundance of reducing agents such as ascorbate, NADPH, and glutathione. The latter is a key regulator of cellular redox homeostasis, attaining 1–10 mM concentration. Glutathione exists in both reduced (thiol) GSH and oxidized (disulfide) GSSG form, the GSH:GSSG ratio being a marker of cellular redox status. The cytosolic and mitochondrial glutathione pools are highly reduced, with GSH:GSSG > 10000:1. Conversely, the glutathione pool is much more oxidized in the endoplasmic reticulum, with a GSH:GSSG ratio in the 1–15:1 range. Notwithstanding, reduced GSH is still the major species therein [2]. Such levels of the GSH:GSSG ratio imply a redox potential that contributes to keeping Cu in the reduced Cu+ state in all cell compartments. As a result, Cu2+ exists in the intracellular environment only transiently, in binding sites buried within cuproenzymes, where Cu cycles between Cu+ and Cu2+.

Although an important role of GSH in Cu uptake has been suggested,[3] recent studies showed that intracellular “free” Cu+ is buffered at very low concentration (about 1 aM), below the threshold for Cu+-binding to GSH in mainly tetranuclear Cu-thiolate clusters. Actually, Cu+ is bound to proteins with attomolar affinity, called metallo-chaperones, each of which delivers Cu to a specific target (CCS to SOD1, Atox1 to ATP7A/B, Cox17 to COX). Only in case of Cu overload, GSH and metallothioneins (MTs), a family of thiol-rich proteins, could bind Cu. Indeed, although metallothioneins are the strongest Cu+ ligands in the cell (log Ka ∼19–20), they are normally loaded with Zn2+ [4], [5], [6].

Conversely, in the extracellular space, Cu is carried mainly in the oxidised Cu2+ state. Herein, Cu2+ levels and speciation in different extracellular compartments, are thoroughly revised under both physiological and pathological conditions (see Section 2). Indeed, although a rigorous control of Cu levels and localization exists, failure of Cu homeostasis may occur, resulting in pathological states. For instance, genetic mutations on ATP7A or ATP7B genes result in two severe Cu-related diseases called Menkes’ (MD) and Wilson’s disease (WD), respectively. In MD, Cu accumulates in the intestine causing a systemic Cu deficiency, whereas in WD Cu accumulates mainly in the liver due to impaired biliary excretion. In addition, Cu dyshomeostasis or mislocalization has been also recognized in neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease, as well as in cancer [7]. As a result, altered Cu levels in biological fluids are observed in such Cu-related diseases. Remarkably, the portion of exchangeable (i.e., kinetically-labile) Cu, and not total Cu levels, appears to be increased in serum and urine of WD and AD patients [8].

Consequently, the specific measurement of the exchangeable Cu pool in biological fluids has been gaining interest to study Cu metabolism and also as a potential diagnostic tool. Whilst well-established elemental analysis techniques are routinely used to measure total Cu levels in biological samples, these techniques require preliminary separative steps to selectively detect the exchangeable Cu pool. Unfortunately, such separative processes are prone to altering the native Cu speciation in biological samples and hence separation-free methods minimizing sample treatments are more desirable. Chelators have been used since decades to extract the exchangeable Cu2+ from endogenous ligands, but in most cases the isolation of the Cu-chelator complex from the sample mixture is still performed (see Section 3). In order to simplify the detection of the Cu-chelator complex, a Cu-responsive spectroscopically-active ligand would be clearly useful. In particular, due to their high sensitivity, user-friendliness, and availability of the instrumentation, luminescent (i.e. fluorescent or phosphorescent) Cu2+ probes appear as a potential tool to determine directly the exchangeable Cu (see Section 4). They also show broad applicability, spanning from ex vivo analysis of biological samples to live-cell and in vivo imaging. The latter is more commonly achieved by magnetic resonance imaging (MRI), which is a major tool for diagnostic imaging. Both luminescent and MR imaging can be envisioned as unique tools to investigate temporal and spatial Cu distribution in living organisms. Recently, some Cu2+-responsive MRI contrast agents started to be developed (see Section 5), although very limited in vivo applications have been reported to date. However, the design of Cu2+-responsive probes suitable for the aforementioned applications faces several challenges and pitfalls. Therefore, here we discuss the requisites of a ligand to measure Cu2+ pools in biological samples, we analyse the advantages and the shortcomings of the state-of-the-art luminescent and MRI Cu2+ probes with regards to their application in extracellular Cu2+ sensing, and we provide hints for the improvement of current systems.

Section snippets

Cu pools in extracellular compartments

Cu concentration varies considerably among different biological fluids (see Table 1). Current knowledge on Cu levels, speciation, and metabolism in the main extracellular compartments, under both physiological and pathological states, are revised hereafter.

Total Cu measurement

The determination of trace metals in any kind of sample, including biological systems, is mainly performed by means of elemental analysis techniques, such as atomic spectroscopy and mass spectrometry (MS). In both cases, the sample is first subjected to atomization. Flame (F) and graphite furnace (GF) are the most common atomizers used for Atomic Absorption Spectroscopy (AAS). Besides, Atomic Emission Spectroscopy (AES), also referred to as Optical Emission Spectroscopy (OES), and atomic mass

Types of luminescent sensor

Most luminescent sensors are based on the change of the emission intensity upon interaction with the analyte. Depending on whether the intensity increases or decreases, sensors are referred to as turn-on and turn-off. Turn-on sensors are generally preferred due to easier quantifiability of their response and higher spatial resolution for imaging applications [159]. Moreover, they are considered more specific since turn-off response can be also caused by chemical degradation, photobleaching or

MRI contrast agents

Beside luminescence, MRI can also be used to sense ions such as Cu2+. MRI is one of the most powerful imaging modalities in clinical medicine as it is able to give images that are spatially and dynamically very well resolved. Contrast agents (CA) are used in clinics to enhance the contrast between normal and pathologic tissues. This enhancement reflects their biodistribution, which is mainly non-specific. All commercial CAs are extracellular fluid agents and one is used for the hepatobiliary

Conclusions

The study of Cu2+ speciation in extracellular fluids is paramount to understand Cu metabolism and the pathophysiology of Cu-related diseases. To date, Cu2+ pools in the blood are the best characterized, but several points (e.g. the involvement of α2M, the nature of SCC) still have to be addressed. Conversely, despite their relevance for diagnosis, little is known about Cu2+ pools in urine and CSF, which do need future investigations. For the same purpose, the development of probes for the

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors wish to thank their colleagues and collaborators for helpful discussions, notably Vincent Lebrun (Institut de Chimie, University of Strasbourg-CNRS), Maria Linder (California State University, Fullerton), Olivier Sénèque (CEA, IRIG, LCBM, Univ. Grenoble Alpes-CNRS), Gilles Ulrich (ICPEES, University of Strasbourg-CNRS), Loïc Charbonnière (IPHC, University of Strasbourg-CNRS), Wojciech Bal (Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw). Giuseppe

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