A new mitochondrial fluorescent zinc sensor

Cell Calcium 34 (2003) 281–284

A new mitochondrial fluorescent zinc sensor Stefano L. Sensi a,b , Dien Ton-That a , John H. Weiss a , Anca Rothe c , Kyle R. Gee c,∗ b

a Department of Neurology, University of California, Irvine, CA 92697-4292, USA Department of Neurology, CESI-Center for Excellence on Aging, University ‘G. d’Annunzio’, Chieti, 66013, Italy c Molecular Probes, Inc., 4849, Pitchford Avenue, 29851 Willow Creek Rd., Eugene, OR 97402, USA

Received 11 March 2003; received in revised form 7 May 2003; accepted 11 May 2003

Abstract A novel cationic fluorescent zinc (Zn2+ ) indicator (RhodZin-3) with nanomolar affinity for Zn2+ has been synthesized. RhodZin-3 exhibits large pH-independent fluorescence increases in the orange region of the visible wavelength spectrum with increasing zinc concentrations, and no sensitivity to physiologically relevant Ca2+ concentrations. Experiments in neuronal cell cultures show that RhodZin-3 effectively localizes into mitochondria and detects changes of intramitochondrial free Zn2+ ([Zn2+ ]m ). © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Fluorescence; Intracellular; Neuron; Zn2+ ; RhodZin-3

1. Introduction The zinc(II) ion (Zn2+ ) has been long known to play critical roles in protein structure and function [1]. Recently Zn2+ has emerged as an important player in neurotransmission [2] and neuronal injury [3,4]. Much of the total biological zinc is tightly bound to proteins and enzymes [5]. Rapid rises in intracellular free Zn2+ ([Zn2+ ]i ) have been linked to neuronal injury in transient global ischemia and epilepsy [2,4]. The mechanisms by which Zn2+ exerts potent neurotoxic effects are still largely unknown. We and others have suggested that among the intracellular targets of Zn2+ dependent neurotoxicity, Zn2+ sequestration into mitochondria may play a critical role [6–8]. As with Ca2+ , upon excessive cytosolic Zn2+ loading, mitochondria take up Zn2+ and help to restore intracellular Zn2+ homeostasis [8]. However, once in the mitochondria, Zn2+ can trigger a prolonged disruption of the functioning of these organelles. Indeed, Zn2+ has been shown to have potent effects on mitochondria [7–14]. For instance, in neuronal mitochondria, rises in [Zn2+ ]m promote loss of mitochondrial membrane potential (Ψ m ) and generation of reactive oxygen species (ROS) as well as release of pro-apoptotic factors [7,8,14,15]. Direct measurements of [Zn2+ ]m have previously exploited the Zn2+ sensitivity of rhod-2, a mitochondrial probe usually employed to detect mitochondrial Ca2+ uptake [15]. ∗ Corresponding

author. Tel.: +1-541-465-8366; fax: +1-541-984-5652. E-mail address: [email protected] (K.R. Gee).

However, the high Ca2+ sensitivity of rhod-2 potentially confounds such zinc measurements. Thus, better tools are critically needed to effectively explore changes in [Zn2+ ]m . Herein we described a novel fluorogenic zinc-specific sensor that localizes into mitochondria of cultured neurons and can effectively detect rises in [Zn2+ ]m .

2. Materials and methods 2.1. Materials Reagents were obtained from Sigma Chemical Company (St. Louis, MO, USA) or Aldrich Chemical Company (Milwaukee, WI, USA) and used as received. Probe synthesis was performed at Molecular Probes, Inc. 2.2. In vitro characterization of RhodZin-3 Absorbance and emission spectra, dissociation constants, and fluorescence enhancements were measured in standard fashion [16]. Spectra were measured at 22 ◦ C in 100 mM KCl, 50 mM MOPS, pH 7.0. Free [Zn2+ ] in buffered solutions was determined using WEBMAXC v2.10. Free Zn2+ solutions of 0.7, 2.75, and 11 nM were prepared using 0.2, 0.5, and 0.8 mM, respectively, zinc chloride in 1 mM EGTA. Free Zn2+ solutions of 27 nM, 109 nM, 436 nM, 974 nM, and 1.9 ␮M were prepared using 0.2, 0.5, 0.8, 0.9, and 0.95 mM, respectively, zinc chloride in 1 mM of the weaker chelator ADA (N-(2-acetamido)iminodiacetic acid).

0143-4160/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0143-4160(03)00122-2

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2.3. Confocal fluorescence microscopy For neuronal imaging, murine forebrain cultures, derived from E-15 embryos, were plated on previously established astrocytic monolayers and used between 13 and 16 days in vitro [17]. Cortical cultures were loaded with RhodZin-3 AM (5) (10 ␮M+0.1% pluronic acid) at 4 ◦ C for 30 min and then left at 37 ◦ C for 4 h for de-esterification. In some experiments, cultures were co-loaded with MitoTracker Green FM (200 nM, 37 ◦ C, 30 min). Experiments were carried out using a simplified Ca2+ -free HEPES-buffered medium (HSS) whose composition was (in mM): 120 NaCl, 5.4 KCl, 0.8 MgCl2 , 20 HEPES, 15 glucose, 10 NaOH, pH 7.4. Series of confocal images (seven planes each 2 ␮m deep) were obtained using either an Olympus fluoview (Olympus USA, Melville, NY), or a Bio-Rad MRC 600 (Bio-Rad Laboratories; Hercules, CA) confocal system equipped with 60× and 40× objectives, respectively, and argon (Ex: 488 nm; Em: >510 nm, for MitoTracker Green) and krypton (Ex: 568 nm; Em: >648 nm, for RhodZin-3) lasers.

3. Results and discussion Condensation of the aldehyde 1 [18] with two equivalents of 3-dimethylaminophenol afforded the unstable dihydroxanthene 2, which was quickly oxidized with p-chloranil to give the xanthene 3 (Scheme 1). The methyl esters were

Fig. 1. Spectroscopic response of RhodZin-3 (4) to increasing concentrations of buffered zinc chloride at pH 7.0 (50 mM MOPS, 100 mM KCl). Zero Zn2+ measurements were made in the presence of the zinc chelator TPEN. Excitation was at 545 nm.

removed by saponification, and the resulting salt form of RhodZin-3 (4) converted into its cell permeable AM ester [19] form 5 by acidification and reaction with bromomethyl acetate. RhodZin-3 utilizes the cationic rhod fluorophore [20], coupled to the N,N,N -triacetic acid chelator contained in the zinc fluoroionophore FluoZin-3 [18,21]. Titration of 4 with buffered Zn2+ solutions in a cuvette revealed that 4 is essentially non-fluorescent but becomes

Scheme 1. (a) 3-dimethylaminophenol, propionic acid, (b) p-chloranil, (c) KOH, and (d) (i) HCl, (ii) BrCH2 OC(O)CH3 , DIEA.

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Fig. 2. Co-localization of RhodZin-3 AM (5) and the mitochondrial selective probe MitoTracker Green. Cultures were co-loaded with RhodZin-3 (red fluorescence) and the mitochondrial marker, MitoTracker Green (green fluorescence), and imaged with confocal microscopy. Note the substantial overlap between the probes (yellow), indicating that they largely target the same intracellular organelles. Bar = 10 ␮m.

Fig. 3. RhodZin-3 detects changes in [Zn2+ ]m . Neurons loaded with RhodZin-3 AM (5) were exposed for 5 min to 50 ␮M Zn2+ in presence of a depolarizing buffer (high K+ ). Upper left, bright field; upper right, basal RhodZin-3 fluorescence; lower left, RhodZin-3 fluorescence changes after exposure to 50 ␮M Zn2+ and high K+ ; lower right, after exposure to the cell permeable Zn2+ chelator TPEN (20 ␮M). Note the substantial increase in RhodZin-3 fluorescence upon cytosolic Zn2+ loading and its quenching by the addition of TPEN.

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brightly fluorescent orange as the Zn2+ concentration is increased. A 75-fold fluorescence increase is observed as the solution goes from TPEN (no Zn2+ ) to saturating Zn2+ , and a dissociation constant (KD ) of 65 ± 10 nM is observed (Fig. 1). No Ca2+ sensitivity is observed at ≤40 ␮M. Screening of 4 against other metal ion solutions revealed modest sensitivity to Fe3+ (KD ∼ 5 ␮M), very weak sensitivity to 500 ␮M Hg2+ and 500 ␮M Cd2+ , and no response to 500 ␮M Pb2+ , Ni2+ , Mn2+ , Mg2+ , Ga3+ , or 200 mM Na+ . Fe2+ and Cu2+ were completely quenching, as is normal with paramagnetic metals. To verify that RhodZin-3 effectively localizes into mitochondria, cortical neurons were loaded with the AM ester of RhodZin-3 (5) and the mitochondrial marker, MitoTracker Green [22,23]. When co-loaded with MitoTracker Green, neurons showed a strong co-localization of these probes, with distinct speckled pattern of fluorescence, most prominent in the perinuclear region, characteristic of mitochondria staining (Fig. 2). We then tested if RhodZin-3 could effectively detect changes in [Zn2+ ]m . Cortical neurons loaded with RhodZin-3 AM (5) were exposed to 50 ␮M Zn2+ in the presence of a depolarizing buffer (60 mM K+ ) in order to increase cytosolic Zn2+ by allowing entry through the opening of voltage sensitive Ca2+ channels. Consistent with our prior observations of mitochondrial uptake of cytosolic Zn2+ loads [8,15], Zn2+ entry into the neurons caused an increase in the mitochondrial signal (44.8 ± 4.3% increase; 177 neurons from 8 experiments). This indicated that the observed changes in RhodZin-3 fluorescence were indeed due to Zn2+ uptake, punctate regions of strong fluorescence were attenuated by addition of the Zn2+ selective chelator, TPEN (Fig. 3). Recent years have seen a rapid increase in our understanding of the complex neurobiological effects of Zn2+ . A better understanding of the intracellular systems controlling [Zn2+ ]i homeostasis, and in particular on the role played by mitochondria in such processes is needed. Present results indicate that RhodZin-3 might be a valuable tool for detecting physiologically and pathophysiologically relevant changes in [Zn2+ ]m . Acknowledgements K.R.G. and A.R. thank Dr. Iain Johnson for helpful discussions, and Hans Engel for expert technical assistance. We also thank Simin Amindari for expert assistance with the cell cultures. This work was supported by NIH grants NS30884 and AG00836 (JHW), AG00919 (SLS), and a grant from the Alzheimer’s Association (JHW). References [1] J.M. Berg, Y. Shi, The galvanization of biology: a growing appreciation for the roles of Zn2+ , Science 271 (1996) 1081–1085.

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