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Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells


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The Journal of Neuroscience, October 1, 2000, 20(19): Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells Manus W. Ward, A. Cristina Rego, Bruno
The Journal of Neuroscience, October 1, 2000, 20(19): Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells Manus W. Ward, A. Cristina Rego, Bruno G. Frenguelli, and David G. Nicholls Neurosciences Institute, Department of Pharmacology and Neuroscience, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom The relationship between changes in mitochondrial membrane potential ( m ) and the failure of cytoplasmic Ca 2 homeostasis, delayed Ca 2 deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM ) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of m and plasma membrane potential ( P ). Glutamate usually caused an immediate decrease in m of 10 mv, attributable to Ca 2 accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca 2 that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca 2 ([Ca 2 ] c ) has risen to the set point at which mitochondria retain Ca 2. In the presence of oligomycin no increase in [Ca 2 ] c occurs during this depolarization. We conclude that transient Ca 2 loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca 2 extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD. Key words: glutamate excitotoxicity; mitochondrial membrane potential; delayed calcium deregulation; glutamate receptors; TMRM; rhodamine-123 Pathological activation of NMDA receptors, with consequent disturbance in Na and Ca 2 gradients across the plasma membrane, is a primary cause of delayed neuronal death after brain anoxia or ischemia (Rothman and Olney, 1986; Choi and Rothman, 1990; Yoshimura et al., 1998). Prolonged NMDA receptor activation in cultures of primary neurons from spine (Tymianski et al., 1993a), cerebellum (Kiedrowski et al., 1994; Kiedrowski and Costa, 1995; Budd and Nicholls, 1996a; Castilho et al., 1998), forebrain (Hoyt et al., 1992; Rajdev and Reynolds, 1994), striatum (Greene et al., 1998), or hippocampus (Dubinsky and Rothman, 1991; Randall and Thayer, 1992; Dubinsky, 1993; Keelan et al., 1999; Vergun et al., 1999) can result in a failure of the cell to maintain low, stable cytoplasmic free calcium ([Ca 2 ] c ). This delayed Ca 2 deregulation (DCD) precedes and reliably predicts the subsequent necrotic death of the cell (Tymianski et al., 1993b) and has been used extensively to model aspects of in vivo neuronal necrosis. The potential across the inner mitochondrial membrane ( m )is the central parameter that controls mitochondrial respiration, ATP synthesis, and Ca 2 accumulation (for review, see Nicholls and Ferguson, 1992; Nicholls and Budd, 2000) as well as the generation of reactive oxygen species (Boveris et al., 1972; Van Belzen et al., 1997). Because each of these factors can influence the survival of the cell directly or indirectly, the monitoring of m in glutamateexposed neurons provides important information on the mechanism by which mitochondria influence the survival of glutamateexposed neurons. The matrices of the in situ mitochondria load with Ca 2 during glutamate exposure (White and Reynolds, 1995; Budd and Nicholls, 1996a; Wang and Thayer, 1996; White and Received May 28, 2000; revised June 21, 2000; accepted July 19, This research was supported by grants from the Wellcome Trust (054633/Z/98) and the Biomed program of the European Union (FMRX-CT ). M.W.W. is supported by a Medical Research Council studentship. Correspondence should be addressed to Dr. David Nicholls, Buck Center for Research in Aging, Novato, CA (courier mail) or P.O. Box 638, Novato, CA (mail). Copyright 2000 Society for Neuroscience /00/ $15.00/0 Reynolds, 1997), and there is a general consensus that exposure of the neurons to glutamate results in a qualitative mitochondrial depolarization (Ankarcrona et al., 1995; Budd and Nicholls, 1996a; Isaev et al., 1996; Khodorov et al., 1996; Schinder et al., 1996; White and Reynolds, 1996; Kiedrowski, 1998; Prehn, 1998; Scanlon and Reynolds, 1998; Stout et al., 1998; Almeida et al., 1999; Keelan et al., 1999; Vergun et al., 1999). However, the cause, extent, and bioenergetic consequences of such depolarization remain unclear and need further clarification. Conversely, previous depolarization of mitochondria under conditions that prevent ATP depletion protects cultured neurons against DCD (Budd and Nicholls, 1996a; Castilho et al., 1998; Stout et al., 1998). Fluorescent membrane-permeant cations are used widely to monitor m in these circumstances (for review, see Nicholls and Ward, 2000), but interpretation of the complex signals obtained at single-cell resolution is far from trivial and has led to confusing and contradictory conclusions. To help resolve these issues, we have compared experimental traces with those obtained from a simple simulation of the whole-cell fluorescence in response to imposed changes in m and plasma membrane potential ( p ). Results are consistent with both mitochondrial depolarization and failed Ca 2 extrusion from the cell, the factor precipitating DCD depending on which first becomes rate-limiting under the experimental conditions. MATERIALS AND METHODS Materials. Fura-2 acetoxymethyl ester (fura-2 AM), rhodamine-123, and tetramethylrhodamine methyl ester (TMRM ) were obtained from Molecular Probes (Leiden, The Netherlands). Fetal calf serum and MEM were from Life Technologies (Paisley, Strathclyde, UK). Oligomycin, rotenone, and all other reagents were from Sigma (Poole, Dorset, UK). Preparation of cerebellar granule cells. Granule cells were prepared as previously described (Courtney et al., 1990) from 6 7 d postnatal Wistar rats. Cells were plated on poly-d-lysine-coated glass coverslips (13 mm circular for nonperfusion experiments and 22 mm square for confocal microscopy) at a density of 280,000 cells per coverslip. Cells were cultured in MEM containing Earle s salts (Life Technologies) plus 10% (v/v) fetal calf serum, 25 mm KCl, 30 mm glucose, 2 mm glutamine, 100 g/ml Ward et al. Mitochondrial Membrane Potential and Glutamate Excitotoxicity J. Neurosci., October 1, 2000, 20(19): streptomycin, and 100 U/ml penicillin. After 24 hr 10 M cytosine arabinoside was added to inhibit non-neuronal cell proliferation. Cells were maintained at 37 C in a humidified atmosphere of 5% CO 2 /95% air and were used after 6 7 d in vitro (DIV). Incubation conditions. Unless otherwise stated, incubations were performed at 37 C in medium containing (in mm) 120 NaCl, 3.1 KCl, 0.4 KH 2 PO 4, 5 NaHCO 3, 1.2 Na 2 SO 4, 1.3 CaCl 2, and 20 TES [N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid] ph-adjusted to 7.4 at 37 C with NaOH. Unless otherwise stated, incubation media are Mg 2 -free. Epifluorescent imaging. Single-cell imaging was performed in a MiraCal Imaging facility (Life Science Resources, Cambridge, UK) with a Nikon DIAPHOT-TMD inverted epifluorescence microscope equipped with a 40 oil immersion objective and Sutter filter wheel. The imager was equipped with a Lambert intensifier 1187 (Life Science Resources) providing a 30 enhancement of the fluorescent signal, thus limiting photo toxicity. For imaging TMRM, we equilibrated the cells with 50 nm TMRM (unless otherwise stated) for 30 min at 37 C before the experiment. In most experiments the dye, which was also present during the experiment, was excited via an Omega 485DF22 filter with peak transmission at 485 nm. The absorbance of TMRM is only 10% of maximum at this wavelength, but this has the advantage of limiting photodynamic damage to the cells at the concentrations of TMRM that are required to observe matrix quenching. Additionally, the use of this filter allows a dichroic/barrier filter transmitting at 520 nm to be used for both single dye and combined TMRM plus fura-2 imaging. However, experiments performed with 340/380/535 nm excitation and a Chroma fura-2/rhodamine dichroic gave essentially the same results (data not shown). For dual loading the cells were loaded with 50 nm TMRM and 3 M fura-2 AM for 25 min at 37 C in incubation medium containing additionally 30 g/ml bovine serum albumin, 15 mm glucose, and 1.2 mm MgCl 2. After washing, the cells were incubated in the presence of 50 nm TMRM and were excited at 340/380/485 nm with emission 20 nm. Background subtraction and autofluorescence were corrected for, and controls were performed with cells loaded singly with either TMRM or fura-2, which established the absence of any significant crosstalk between the dyes at the wavelengths that were used. Fura-2 fluorescence is reported in terms of 340/380 nm excitation ratios to avoid potential errors because of any changes in the quenching of the 520 nm emission by cytoplasmic TMRM. For rhodamine-123 fluorescence a commonly used empirical loading protocol was applied (Khodorov et al., 1996; Hagen et al., 1997; Buckler and Vaughan-Jones, 1998; Schuchmann et al., 1998; Vergun et al., 1999). Cells were equilibrated with the probe (1 g/ml, i.e., 2.6 M) for 15 min at 22 C before the experiment; the probe was not added to the experimental medium. Then the cells were washed before the experiment (excitation 485 nm; emission 520 nm). It was found that an alternative loading paradigm [10 g of rhodamine-123/ml (26 M) for 15 min] sensitized the cells to photo-induced damage (data not shown). TMRM fluorescence within the matrix of isolated mitochondria. TMRM (100 nm or 2 M) was added to a medium containing (in mm) 100 NaCl, 25 TES (Na salt), and 2 NaH 2 PO 4 plus 16 M albumin, ph 7.0, 37 C, in a thermostatted cuvette inserted in a Perkin-Elmer LS50B fluorometer. Fluorescence was recorded (excitation 543 nm, emission 580 nm) during the sequential additions of rat liver mitochondria (0.25 mg of protein/ml incubation), prepared as previously described (Nicholls, 1978), and2mmsuccinate. Fluorescent quenching after uptake of the probe was determined. The cuvette contents were centrifuged immediately for 30 sec in an Eppendorf microcentrifuge to pellet the mitochondria. The fluorescence of the supernatant was measured to determine the contribution of extramitochondrial TMRM to the total signal, and the mitochondrial pellet was resuspended in water to determine the fluorescence of the mitochondrial TMRM after release from the matrix. The assumption that the matrix fluorescence became invariant above the stacking concentration was tested by incubating isolated mitochondria in the presence of two widely differing concentrations of TMRM, each sufficient to exceed the stacking concentration in the matrix, as confirmed by the decrease in cuvette fluorescence when the mitochondria were energized (Fig. 1). The contribution of the quenched matrix TMRM to the total cuvette fluorescence was quantified by redetermining the fluorescence of the incubation after removing the mitochondria by centrifugation as described above. Figure 1 shows that the fluorescence of the matrix-located probe was similar for mitochondria equilibrated with both 100 nm and 2 M TMRM, although the actual concentration of TMRM in the matrix, confirmed by lysis of the mitochondria to release TMRM, differed 12-fold. Simulating single-cell fluorescence of neurons loaded with membranepotential probes. The simulation (see Appendix) is based on the following premises. (1) Cationic lipophilic probes are nonselectively permeant across both plasma and mitochondrial membranes. (2) Probe distribution tends to a Nernst equilibrium across both membranes. (3) The rate at which the probe equilibrates across the small, highly invaginated inner mitochondrial membrane is much faster than across the plasma membrane because of the differing surface volume relationships of the mitochondrial matrix and the cell soma. (4) The quantum yield of the probe is similar in both cytoplasm and matrix until a threshold concentration is reached in the latter, above which nonfluorescent H-aggregates form (Bunting, 1992) (Fig. 1). The initial p was taken to be 60 mv (Becherer et al., 1997). The Figure 1. The fluorescence of TMRM in the mitochondrial matrix is concentration-independent above the quench threshold. A suspension of rat liver mitochondria was equilibrated with the indicated concentrations of TMRM in the absence of substrate, and the fluorescence was monitored. The addition of succinate as a substrate caused a decrease in fluorescence as the indicator was accumulated into the matrix. The contribution of the extramitochondrial probe to the total fluorescence ( C) was determined after the removal of mitochondria by centrifugation. The difference ( A) attributable to the fluorescence of matrix TMRM was similar at both concentrations although the total matrix TMRM (B), determined by resuspending the mitochondria in water, differed by 12-fold. initial value of 150 mv for m is in agreement with values determined by the distribution of the lipophilic cation TPP in synaptosomes after correction for the plasma membrane potential (Scott and Nicholls, 1980). The mitochondrial matrix within isolated nerve terminals accounts for 3% of terminal volume (Scott and Nicholls, 1980), whereas the lower value of 1% taken here for the soma reflects the relatively thin annulus of cytoplasm surrounding the nucleus. The model is used to reproduce the behavior of two commonly used fluorescent probes, rhodamine-123 and TMRM. The tetramethylrhodamine ester equilibrates more rapidly across membranes than the slowly permeant rhodamine-123 (Bunting, 1992), and empirical fits with experimental traces were obtained with a value for the permeability constant k for equilibration across the somatic plasma membrane in the presence of glutamate of 0.02/sec for TMRM and 0.001/sec for rhodamine-123. It should be emphasized that these values are for the somata of cultured cerebellar granule cells and would be lower for larger cells or conversely increased for thin neurites. In the absence of glutamate, probe equilibration was limited by constraints of charge neutralization across the plasma membrane (see Fig. 10), and lower values of k were determined empirically. The simulated loading conditions for the two probes followed the empirically determined optimal conditions that were used in this study. Because TMRM is rapidly lost from cells in the absence of external probe, the granule cells were equilibrated with probe, usually 50 nm, and this concentration was also present continuously in the incubation to allow continuous reequilibration across the plasma membrane. The less permeant rhodamine-123 is normally loaded by a brief exposure (insuffi- 7210 J. Neurosci., October 1, 2000, 20(19): Ward et al. Mitochondrial Membrane Potential and Glutamate Excitotoxicity cient for Nernstian equilibration across the plasma membrane) to a relatively high concentration of probe, typically 2 20 M (Vergun et al., 1999). After this the cells are washed, and the subsequent experiments usually are performed in the absence of external probe. Where indicated, the simulation reflects this. In view of the lower permeability of this probe, loss across the plasma membrane is sufficiently slow to permit most (but not all; see Fig. 2) short-term experiments to be performed without excessive loss of probe from the cells. Statistics. Each set of single-cell responses shown is representative of at least 60 individual cell somata that were monitored in at least three independent experiments from different cell preparations. Significance was assessed by unpaired variance Student s t test. RESULTS Validation of the simulation Figure 2 displays a gallery of fluorescence traces obtained by the addition of elevated KCl and FCCP/oligomycin to granule cells exposed to 2.6 M rhodamine-123 for 15 min at 22 C before the experiment (Fig. 2A) or equilibrated with either 50 nm (Fig. 2B,C,E) or 10 nm TMRM (Fig. 2D). Also shown are curves generated by the cell simulation for step changes in p and m that would be induced by these agents. The simulated curves were fit to the experimental traces by varying the plasma membrane permeability coefficient, k, appropriate for TMRM and rhodamine-123 (see Appendix Eq. 9) until a satisfactory fit was obtained. Values of 0.003/sec (TMRM ) and /sec (rhodamine-123) were adopted for these traces. A further empirically determined constant is the threshold concentration at which aggregation and quenching of the probe occur in the matrix. This was established by determining the loading concentration for TMRM at which protonophore-induced dequenching disappeared. Thus equilibration with 50 nm TMRM allows dequenching to be observed (Fig. 2C), but this vanishes when the loading concentration is reduced to 10 nm (Fig. 2D). With the initial conditions of a p of 60 mv and a m of 150 mv, this corresponds to a quench threshold (c [stacking]) (see Appendix Eq. 4) of 50 M within the matrix, and this value was adopted for subsequent simulations. Several features are revealed by these calibrating traces. When the cells are equilibrated with sufficient probe to exceed the matrix quench threshold, acute mitochondrial depolarization produces a spike followed by a decay in signal (Fig. 2A,B); when loading is subthreshold (Fig. 2D), only the decay phase is seen. This may reconcile some of the confusion in the literature as to the signal expected from a mitochondrial depolarization. Because the total fluorescence of a single cell is the sum of that originating from the cytoplasm and matrix whereas the matrix fluorescence is invariant above the quench threshold, the changes in fluorescence mainly reflect changes in cytoplasmic probe concentration. The short-term insensitivity of rhodamine-123 to changes in p (Fig. 2A) can be ascribed to the low permeability coefficient, although as will be seen later (see Fig. 8) there are important conditions for which this is not valid. In contrast, TMRM redistribution across the plasma membrane must be considered under all conditions, particularly with small somata such as those of granule cells. Equilibration of TMRM across the plasma membrane after KCl depolarization is slower than after protonophore (Fig. 2B), consistent with the increased buffering in the presence of the mitochondrial pool (see Appendix). Finally, the decreased cell fluorescence after the decay of the FCCP/oligomycin spike (e.g., Fig. 2B) is attributable to the loss of the unquenched component of the mitochondrial TMRM fluorescence. The cell simulation was devised to interpret the single-cell fluorescence responses during the complex, slow changes in m associated with glutamate exposure and the delayed Ca 2 deregulation that occurs as a consequence of glutamate excitotoxicity (Tymianski et al., 1993b; Budd and Nicholls, 1996a). Figure 3 investigates the effects of inhibitors of the respiratory chain and ATP synthase. Because the membrane potential of in situ mitochondria in the presence of respiratory chain inhibitors is supported by ATP synthase reversal and hydrolysis of glycolytic ATP, respiratory chain inhibitors cause only a slight depolarization in cells with acti
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