A rapid method for the evaluation of compounds with mitochondria-protective properties

A rapid method for the evaluation of compounds with mitochondria-protective properties

Journal of Neuroscience Methods 92 (1999) 153 – 159 www.elsevier.com/locate/jneumeth A rapid method for the evaluation of compounds with mitochondria...

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Journal of Neuroscience Methods 92 (1999) 153 – 159 www.elsevier.com/locate/jneumeth

A rapid method for the evaluation of compounds with mitochondria-protective properties Rony Nuydens a,b, Jesu´s Novalbos d, Gwenda Dispersyn c, Claudia Weber e, Marcel Borgers a,b, Hugo Geerts a,* a

Department of Cell Physiology, Janssen Research Foundation, Turnhoutseweg 30, Beerse 2340, Belgium Department of Molecular Cell Biology and Genetics, Maastricht Uni6ersity, Maastricht, The Netherlands c Department of Biochemistry, Uni6ersity of Antwerp, Antwerp, Belgium d Department of Clinical Pharmacology, Princesa Hospital, Department of Pharmacology and Therapeutics, Faculty of Medicine, Uni6ersidad Autonoma de Madrid, Madrid, Spain e Preclinical De6elopment, Janssen Research Foundation, Neuss, Germany b

Received 19 January 1999; received in revised form 25 June 1999; accepted 18 July 1999

Abstract Mitochondrial dysfunction has been implicated in a number of neurodegenerative diseases, such as ischemia and Parkinson’s disease. We present here a method that allows the rapid quantification of interventions, aimed at inhibiting the effect of mitochondrial membrane potential uncouplers, based on the ratioing properties of the fluorescent probe 5,5%,6,6%-tetrachloro1,1%,3,3%-tetraethylbenzimidazolcarbocyanine iodide (JC-1), by using currently available 96-well fluorescent plate readers. A method is presented for evaluation of cross-talk between the two excitation/emission channels. Further characterization of the probe shows that the effect of plasma membrane potential changes on JC-1 fluorescence ratio are negligible, but that the signal is very sensitive to pH. One of the most exciting applications is the possibility to perform end-point measurements, thanks to the ratioing properties of the probe. The system is tested in different culture types with different mitochondrial uncouplers. As an example of a quantitative evaluation, we show that flunarizine is able to inhibit, dose-dependently, FCCP mediated JC-1 signal increase. The procedure is simple and allows for the fast screening of mitochondria-protective compounds © 1999 Elsevier Science B.V. All rights reserved. Keywords: Fluorescence; Microplate; Mitochondrial membrane potential; Neurons; Toxicity

1. Introduction Mitochondrial dysfunction has been implicated in a number of neurodegenerative diseases, including ischemia, Parkinson’s and Alzheimer’s disease (for a review see: Tatton and Chalmers-Redman, 1998). Preservation of the mitochondrial membrane potential is considered to be an essential process in rescuing neurons from energy-depletion in anoxic situations and inhibition of release of pro-apoptotic components from the mitochondrial intermembrane space (Susin et al., 1998). * Corresponding author. Tel.: +32-14-60-3614; fax: + 32-14-605788. E-mail address: [email protected] (H. Geerts)

The mitochondrial membrane potential (MMP) is hyperpolarized (estimated between − 120 and −180 mV). This hyperpolarization is a consequence of H+ distribution and is associated with well-respiring mitochondria. In situations of pathological energy depletion, where ATP production falls rapidly, the mitochondrial membrane potential is depolarized. This can be monitored by a multitude of means. Radioactive TPP accumulation has been shown to be related to mitochondrial membrane potential (Sanchez et al., 1988), however, this method, being an equilibrium method, precludes any fast transient measurement of the MMP. Furthermore, the radioactive waste removal excludes any large scale use of this technique.

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A number of fluorescent probes have been described to insert preferentially into hyperpolarized mitochondria (Macho et al., 1996). Probes, such as Rh 123, a cell-permeant, cationic dye that is readily sequestered by active mitochondria and chloro-methylX-rosamine (CMXRos), are however singlewavelength probes. Although they are excellent for the study of transient and short term phenomena, they suffer from a number of drawbacks (Salvioli et al., 1997). For instance, uniform and identical loading of the probe in all conditions is not guaranteed and photobleaching may affect the dynamic readout of the fluorescence signal. For some it has been documented that they either show a lower sensitivity (Rh123) or that they also respond to changes in plasma membrane potential (3,3%-dihexyloxocarbocyanine iodide, DiOC6(3)). JC-1 is a new ratiometric probe, which selectively inserts into the mitochondrial membrane (Reers et al., 1991; Cossarizza et al., 1993). The spectral characteristics of this fluorescent probe depend on the mitochondrial membrane potential. The monomeric form is excited at 488 nm and fluoresces into the green wavelength. The aggregated form is excited at 560 nm and fluoresces into the red wavelength. The former is associated with a depolarized MMP, whereas during hyperpolarization the molecules become aggregated and displays a large shift in emission (590 nm). We took advantage of the dual excitation-emission properties of this probe to develop a fast screening method, based on conventional fluorescent plate readers. We optimized the experimental procedures of probe loading, cell density, filter cross-talk and further characterized some aspects of the cell biology of this fluorescent probe.

2. Materials and methods

and 50 ng/ml Nerve Growth Factor for the PC12 cells and to complete medium supplemented with 25 nM staurosporine for the SHSY-5Y cells. The cells were used 4–7 days after plating.

2.2. Measurements of mitochondrial membrane potential JC-1 (Molecular Probes, Leiden, The Netherlands) fluorescence is determined by the mitochondrial membrane potential. At depolarized (− 100 mV) membrane potentials JC-1 exists as green monomers with emission peak around 527 nm. As the membrane is hyperpolarized (− 140 mV) JC-1 forms J-aggregates and the emission shifts towards 590 nm. Cells were loaded by changing the culture medium to Phosphate Buffered Saline (PBS, GIBCO, BRL) containing 1g/l glucose and 10 mM JC-1 for 10 min at 37°C. Thereafter the cells were washed once and the actual experiment was started. Fluorescence was measured in a Cytofluor (Perkin-Elmer) plate reader which allows for the sequential measurement of each well at the respective wavelengths as indicated above. In each cycle a scan at 480/530 and at 530/590 is taken. The ratio of measured fluorescence intensities at both wavelengths is an indication of the mitochondrial membrane potential. At the indicated timepoints PBS was changed to PBS containing the different test compounds. For pretreatment with flunarizine, the compound was added with the first wash solution and remained present throughout the entire experiment. After seven consecutive cycles the medium was removed and replaced with PBS containing FCCP and the test compound. All compounds were dissolved in DMSO as 100 times concentrated stock solutions so that the final DMSO levels never exceeded 1%.

2.1. Cell culture

2.3. Data analysis

PC12 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, BRL) supplemented with 7.5% horse serum (HS, GIBCO BRL), 7.5% fetal calf serum (FCS, HyClone), 2 mM L-glutamine (GIBCO, BRL) and 0.5 mg/ml gentamycine. SHSY-5Y cells were grown in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, non-essential amino acids (GIBCO, BRL) and 0.5 mg/ml gentamycine. The cells are maintained in a humidified incubator aerated with 95% air and 5% CO2 at 37°C. For the actual experiments the cells (20 000/well) were plated in poly-L-lysine pre-coated 96-well plates (Biocoat, Falcon) in complete medium, 4 h later the medium was changed for DMEM containing 1% HS

The average of the first row (containing no cells) is used as a background and this value is subtracted from all the other intensities. Usually a 10 min pretrigger period is taken in order to measure basal values of the two intensities allowing us to determine a basal mitochondrial membrane potential. All subsequent ratios are normalised against this base-line ratio. The effect of the uncoupling can be calculated as the area under the curve during a fixed period after the trigger. This way, the effect of small organic compounds on the trigger-induced JC-1 change can be quantified. Appropriate statistical analysis was carried out with the JMP package.

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3. Results

3.1. E6aluation of the possible cross-talk between the two spectral measurements The main advantage of JC-1 is its dual-spectral property. The fluorescence can be detected either from the monomeric form or the aggregated form. Therefore we need to spectrally separate the two observations. This can be done by measuring a dilution series of two proteins, a goat anti-rabbit IgG tagged with fluoresceine and another goat anti-rabbit IgG tagged with rhodamine (both commercially available). These fluorochromes have essentially the same spectral characteristics as both wavelengths used for measuring the JC-1 ratio. This standardization has to be performed with the same settings of gain and offset of the photomultiplier tubes. Table 1 shows the intensities measured in the two channels of both single probe solution and a mixture of various proportions, suggesting that over a large ratio of protein mixtures the cross-talk is limited to 1% in the FITC channel and 5% in the TRITC channel.

3.2. Optimalization of JC-1 loading and stability o6er time Since previous reports had illustrated the accelerated extrusion of JC-1 (Smiley et al., 1991) another parameter that required validation was the stability of the probe once incorporated into the living cells. Cells were loaded with different concentrations of JC-1, washed,

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and the signal followed over an extended period of time. The intensities of both wavelengths did not change dramatically (Fig. 1) even over a period of 1 h, essentially suggesting the absence of photobleaching during the subsequent scans. Concentrations of 10 mM were needed for reaching sufficient fluorescence readout at 590 nm to detect accurately the J-aggregates.

3.3. Application in different cell types We then studied the kinetics of various mitochondrial uncouplers in different cell types. Fig. 2 illustrates the response of the JC-1 probe in two different neuronal cells upon FCCP application. In PC12 cells the peak response immediately after FCCP addition is lower when compared to SHSY-5Y cells and the effect seems to decline in time. For instance, a 92% increase in the ratio is the result of a 35% increase in the 485/530 channel and a 40% decrease in the 530/590 channel, suggesting that both wavelengths react in the appropriate way. Fig. 2 gives the dose-dependent and quantitative evalution of the FCCP responses in the two cell types. The ED50 value for PC12 cells is 550 nM and for SHSY-5Y cells this value is 200 nM. Notice also that the response clearly decreases at the highest FCCP concentrations, especially in the SHSY-5Y cells. Thapsigargin has also been documented as a mediator of mitochondrial changes in a number of cellular paradigms. In PC12 cells concentrations up to 50 mM were needed to acutely modify the mitochondrial potential (Fig. 3), this is about 500 times higher then the concentration necessary to induce intracellular Ca2 +

Fig. 1. Illustration of the JC-1 signal stability and intensity after loading PC12 cells with different concentrations JC-1 (0.1, 1 and 10 mM). Only at the concentration of 10 mM enough J-aggregates are formed to produce a reasonable signal at 530 nm excitation.

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Exc 485/Em 530 Protein mixture FITC-GAR TRITC-GAR FITC-GAR/TRITC-GAR a

Exc 530/Em 590 1/50 3124.83 5.83 2422.83

1/100 1481.33 2.33 1197.83

1/200 761.83 1.33 609.83

1/400 381.83 0.83 312.33

1/800 186.83 0.83 156.83

1/1600 98.33 0 0

1/3200 0 0 0

1/50 79.34 1150.84 1201.84

1/100 36.84 574.84 591.84

1/200 19.34 283.34 298.34

1/400 9.34 138.84 151.84

1/800 4.84 68.34 77.84

The measurements were performed with the same settings of the photomultiplier-gain as necessary for determining the JC-1 ratio in all subsequent experiments.

1/1600 2.34 35.84 38.84

1/3200 0 0 0

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Table 1 Quantification of individual (FITC-GAR, TRITC-GAR) or mixed fluoresceine and rhodamine tagged proteins (FITC-GAR/TRITC-GAR) at the two different excitation/emission combinations necessary for use with JC-1a

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Fig. 3. Thapsigargin up to 10 mM does not acutely change JC-1 ratio readout. At 50 mM there is only a slow increase in MMP upon addition (arrow) of thapsigargin.

Addition of 3-NP to PBS, the medium used in the acute experiments, lowers the pH to:3, but when compensated for these changes the acute effects completely disappear. This result clearly suggests that the JC-1 fluorescence is clearly pH dependent.

Fig. 2. Illustrates the acute effect of FCCP addition (arrow) on MMP in PC12(a) and SHSY-5Y cells; and (b). In SHSY-5Y cells 500 nM FCCP still induces an abrupt increase in JC-1 signal while in PC12 cells 1000 nM is necessary to induce immediate changes although even at this concentration the response is slower when compared to SHSY-5Y cells. This illustrates the differential sensitivity of neuronal cells to mitochondrial uncouplers.

release. We also evaluated 3-NP (3-nitro-propionic acid), an inhibitor of succinate dehydrogenase, as possible mediator of mitochondrial uncoupling. Unexpectedly, we observed (Fig. 4) that the acute changes in MMP induced by high concentrations of 3-NP were largely carried by the differences in extracellular pH. Table 2 End-point measurements of FCCP induced mitochondrial membrane potential changes in 48 h treated SHSY5Y cellsa FCCP (nM)

JC-1 ratio (%)

Solvent control 5 10 50 100 500 1000 5000 10000

100.0 9 7.7 99.49 4.1 100.79 7.1 111.59 9.9 108.39 14.9 100.49 3.6 106.99 8.9 161.09 3.9 261.09 11.4

a

The JC-1 ratio is normalized to its value in the untreated sample.

Fig. 4. Fifty micromolar 3-NP induced acute effects observed in NGF treated PC12 cells in unbuffered solution (b), and, are abolished when the extracellular pH is adjusted to physiological values (a).

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decrease has been observed in the time-dependent changes in JC-1 ratio in PC12 cells during the acute experiments.

3.6. Pharmacological modulation of MMP changes

Fig. 5. Membrane depolarization was induced in JC-1 loaded PC12 cells by increasing extracellular K+ concentrations from 10 to 50 mM (arrow). There is only a small ( B 10%) transient change in JC-1 ratio after addition of 50 mM extracellular K+.

3.4. JC-1 is not influenced by plasma membrane depolarization JC-1 rearranges itself either in monomeric or aggregated forms depending upon the membrane potential. In our system the selectivity of JC-1 for the mitochondrial over the plasma membrane potential was unknown. Therefore we evaluated the contribution of plasma membrane depolarization on the JC-1 signal by inducing membrane depolarization through addition of high K+ ext to PC12 cells. According to the Nernst equation, a 50 mM K+ concentration yields a membrane potential of −26 mV (a decrease of 57 mV) and at 100 mM K+ even − 8 mV (a depolarization of 75 mV). We have documented before that these cells indeed respond to a K+ depolarization by an increased Ca2i + (Dispersyn et al., 1997), suggesting the presence of voltage-sensitive Ca2 + channels. Fig. 5 shows the effect, in PC12 cells of membrane depolarization, by a K+ ext pulse, on the JC-1 signal. The change in JC-1 ratio turns out to be in the range of 5 – 10%, much lower than the effect elicited by FCCP, suggesting that the JC-1 response to reasonable plasma-membrane potential changes does not interfere with the readout of the mitochondrial membrane potential.

As an example of a mitochondria protective compound we evaluated the effect of flunarizine on FCCP induced acute changes in PC12 cells. FCCP applied at 100 nM induces a steap rise in JC-1 ratio which decreases towards a steady state level after about 15 min. This initial increase is completely inhibited by pretreating the cells with flunarizine 10 mM for 7 min. Even at 1 mM there is still a partial inhibition of the initial peak increase. After prolonged incubations however, the JC1 ratio slightly increases and reaches control values (Fig. 6). If one calculates the normalised area under the curve as a quantitative parameter (see Section 2), we find a response of 479 10% (range 35–56 over six experiments) for 10 mM flunarizine.

4. Discussion This report documents the development of a highthroughput system for detecting changes in the mitochondrial membrane potential triggered by mitochondrial uncouplers. The system is based on currently available fluorescent plate readers and allows a large numbers of conditions to be tested within a reasonable time. The time resolution of the instrument is adequate to retrieve a reliable estimate of the changes induced by various interventions. In our instrument, a complete 96-well plate is read in about 32 s, giving a time resolution of 64 s for a dual scan. In principle one can increase the time resolution by limiting the number of wells to be read, or, ultimately reduce this number to just one well. In this case, the attainable time resolution

3.5. The use of JC-1 in long-term mitochondrial membrane potential changes Because of its dual spectroscopic character, the probe lends itself extremely well to ratiometric measurements of end-point changes. To illustrate this, we monitored changes in JC-1 readout after 48 h exposure of SHSY5Y cells to FCCP, this is shown in Table 2. The effect was prominent only at the higher concentrations (5–10 mM). This could be a reflection of the reversibility of the FCCP effects upon prolonged exposure. A similar

Fig. 6. Pretreatment for 7 min with flunarizine at 10 mM but not 1 mM inhibits the FCCP induced acute changes in MMP. In this particular experiment, the inhibition by 10 mM flunarizine is about 65%, whereas at 1 mM essentially no inhibition is observed.

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is limited by the change of the filter wheels. A disadvantage of using plate reader based systems is the loss of information at the single cell level. Cellular responses may vary and even within the same cell mitochondrial responses may be different (Wadia et al., 1998). We have shown that the system is able to accurately monitor shifts in JC-1 fluorescence induced by FCCP, a known mitochondrial uncoupler. The effects of thapsigargin on the mitochondrial membrane potential are quite small and are present only at high concentrations. This is about 500 times the concentration needed for triggering an endoplasmic reticulum mediated Ca2 + response so that an aspecific effect on the mitochondrial membrane potential cannot be excluded. The lack of specific effect of NP-3 could be attributed to its low penetration into the living cells, given its highly polar character. Modulation of mitochondrial functioning can be followed in a number of cell types, such as rat PC12 cells and human neuroblastoma cells. A density of 20 000 cells/well is sufficient to detect reasonable changes in the ratio of JC-1. JC-1 reports changes originating predominantly from the mitochondrial membrane potential. The induction of plasma membrane depolarization by application of high K+ ext did not induce changes in the JC-1 ratio. It turns out that plasma membrane depolarization gives only an increase of JC-1 ratio in the order of 5 –10%, suggesting that the major contribution of JC-1 signal originates from the mitochondrial membrane. This probably also represents the lowest sensitivity of the probe to detect physiological changes in the mitochondrial membrane potential. Because of its dual spectroscopic properties JC-1 has several advantages compared to its single-wavelength predecessors such as DiCO6(3), Rh 123 and CMXRos. The use of ratioing implies that the concentration effects associated with probe loading are minimalised. Therefore JC-1 can uniquely be used in end-point measurements as is shown by the chronic exposure of SHSY-5Y cells to FCCP. In addition, the ratiometric approach increases the dynamic range to monitor effects of interventions on changes in mitochondrial membrane potential. An unexpected finding is the fact that the JC-1 ratio is pH sensitive. This means that experiments need to be performed in buffered conditions. In addition, the system presented here allows for the evaluation of compounds aimed at protecting mitochondria against depolarization. The complete inhibi-

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tion by flunarizine of the FCCP induced depolarization is a nice demonstration of the applicability of this system. These results are in agreement with other studies (Elimadi et al., 1998) illustrating the inhibition of the mitochondrial megapore formation by flunarizine. In conclusion, the data illustrate that the combination of JC-1 and microplate based fluorescence systems allows for the rapid evaluation of compounds with regard to their effects at the mitochondrial level.

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