Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease

Abstract

Background

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS.

Results

To gain insights into the molecular mechanisms of SOD1^G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca²⁺ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca²⁺ elevations in WT (SH-SY5Y^WT) and G93A (SH-SY5Y^G93A) expressing cells were observed. In contrast, mitochondrial intracellular Ca²⁺ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca²⁺ regulation.

Conclusion

In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca²⁺]i and [Ca²⁺]_mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca²⁺-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.

Background

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a selective loss of motor neurons (MNs) in the brain stem, the spinal cord and the motor cortex, leading to progressive weakness, muscle atrophy with eventual paralysis, and death. Approximately 5–10% of ALS cases are familial [1, 2]. A decade ago, researchers discovered missense mutations in the gene encoding the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial cases; approximately 20% of familial ALS (fALS) is caused by mutations in SOD1 with high inter-subject variation of progression, including the point mutation G93A [3–7]

Multiple cascades have been implicated in the motor neuron death pathway, including mitochondrial dysfunction and deformities [8–15]; complex I, III, and IV abnormalities [16–19]; mitochondrial alteration, increase in Ca²⁺ uptake and increase of cytosolic Ca²⁺ concentration [6, 20], oxidative stress [21]; and glutamate excitotoxicity [22–29]. Alteration and disruption of calcium homeostasis and metabolism [30–40] has also been reported.

The present evidence supports a hypothesis that mitochondrial dysfunction acts with oxidative stress to cause abnormal neurodegeneration via calcium-mediated MN injury. Oxidative stress may lead to increased intracellular calcium, which leads to increased nitric oxide and peroxynitrite formation [21]. Glutamate excitotoxicity may disrupt intracellular calcium homeostasis and reactive oxygen species (ROS) production [27], which may be promoted by oxidative stress as glutamate transporters are particularly susceptible to disruption by oxidants, and oxidative modifications to the transporter have been reported in ALS and the mtSOD1^G93A mouse model [24, 21]. The etiology is likely to be multifactorial because ALS involves the interplay of several mechanisms to initiate disease and propagate the spread of motor neuron cell death [36, 38, 39].

Mitochondrial Ca²⁺ uptake responds dynamically and sensitively to changes in cytosolic Ca²⁺ levels and plays a crucial role in sequestering the large Ca²⁺ loads induced by FCCP-evoked Ca²⁺ influx [41]. The excessive influx of Ca²⁺ into mitochondria may result in mitochondrial dysfunction. Prominent and sustained mitochondrial depolarization follows intense ion channel receptor stimulation and closely parallels the incidence of neuronal death [42]. Substantial Ca²⁺ can be accumulated in mitochondria as a result of overloading the matrix with Ca²⁺; this disrupts the structural and functional integrity of the organelle. Hence, mitochondria may be a critical intracellular target of injury after intense Ca²⁺ channel stimulation and, in this way, may act as a link between massive Ca²⁺ influx and mitochondria-mediated neurotoxicity by mtSOD1^G93A. However, the precise relationship between Ca²⁺ influx, cytosolic Ca²⁺ increase, and mitochondrial Ca²⁺ uptake remains obscure.

Culture systems, such as slice culture or primary cultures of MN, have proven to be valuable tools in the physiological and biochemical characterization of ALS-related pathology [43–45]. Still unknown is whether the presence of mtSOD1^G93A causes morphological mitochondrial abnormalities when expressed at physiological levels, whether the source specificity of mitochondrial Ca²⁺ sequestration and spatiotemporal properties of cytosolic Ca²⁺ ([Ca²⁺]i) signaling in cells transfected with wild-type and G93A-mutant SOD1 varies at physiological levels, and if there are functional consequences of changes in mitochondrial function on Ca²⁺ homeostasis in the presence of mtSOD1^G93A. To elucidate the underlying molecular events and cellular alterations involved in oxidative stress induced by the aberrant Cu-Zn chemistry and the roles of impaired Ca²⁺ handling and oxidative stress induced by ROS in fALS, we used SH-SY5Y neuroblastoma cells transfected with the G93A mutant form of SOD1 typical for fALS (SH-SY5Y^G93A or G93A) and SH-SY5Y cells transfected with wild-type (WT) human SOD1 (SH-SY5Y^WT or WT) and mimic the situation present in heterozygous patients, previously established as an in-vitro cell culture model of ALS, resembling the situation of heterozygous patients [6, 46]. This particular mutation was chosen because it does not affect the activity of SOD1; previously it was shown that SH-SY5Y cells expressing G93A exhibit increased intracellular ROS [47]. The effect of the continuous expression of WT or G93A on mitochondrial morphology and Ca²⁺ signaling were compared. In addition, the consequences and impact of protonophore FCCP and other organelle-specific drugs on mitochondrial vulnerability and Ca²⁺ homeostasis were studied.

Methods

SH-SY5Y^WT and SH-SY5Y^G93A transfected neuroblastoma cell cultures

An in-vitro model to study the cellular alterations associated with mutations of SOD1 was constructed by transfection of the human neuroblastoma cell line SH-SY5Y with G93A-SOD1 [6]. The cell types that we have used in our study are well characterized and have been used as cellular model system for motoneuron disease by several groups [6, 48–50]. The transfected cell lines have relatively low levels of mtSOD/wtSOD and are therefore an attractive model system for the human disease [50]. Upon expression of fALS – SOD1, these cells show several features typical of neurons of both ALS patients and transgenic mice, such as mitochondrial alteration, increased ROS levels and increased cytosolic calcium concentration [see references in [49]].

Transfected human neuroblastoma cell lines constitutively expressing either WT human SOD1 or the G93A mutant form of this enzyme associated with fALS were routinely maintained in Dulbecco's Modified Eagle Medium (DMEM)-F12 (Gibco, Invitrogen, Karlsruhe, Germany) containing 15% fetal calf serum (FCS; Biochrom, Berlin, Germany), 100 U/ml penicillin (Biochrom, Berlin, Germany), and 100 μg/ml streptomycin (Gibco, Invitrogen, Karlsruhe, Germany) at 37°C in a humidified atmosphere with 5% CO₂ [6]. The cell lines were under consistent selection by addition of 200 μg/ml geneticin (G418 sulfate, Gibco, Invitrogen, Karlsruhe, Germany), which was removed two days before performing the experiments.

Intracellular fluorometric Ca²⁺ measurements

The calcium-sensitive fluorescent dye, Fura-2, was introduced into cells by loading the membrane-permeable AM-ester forms (5 μM, 30 min, 37°C, 5% CO₂ and 95% O₂), which is termed bolus loading. Fura-2 AM was purchased from Invitrogen (Carlsbad, CA, USA) and freshly dissolved in dimethyl sulfoxide (DMSO) containing 10% pluronic acid before the experiment. Changes in [Ca²⁺]i were measured in non-transfected SH-SY5Y cells (Parental cell line) and SH-SY5Y cells expressing WT or G93A attached to glass cover slips after 2–5 days in culture. Cell layers were incubated with RPMI-1640 (Biochrom, Berlin, Germany) containing 10% FCS (Gibco, Invitrogen, Karlsruhe, Germany) and 0.846 mM Ca²⁺ (supplier's data). Cells were rinsed with RPMI -1640 and incubated for 20 min at 37°C to allow for complete de-esterification.

Changes in [Ca²⁺]i were monitored by the excitation of Fura-2 AM at 360 nm and 390 nm; emitted light was directed to a dichroic mirror by a beam splitter at 425 nm. In the case of non-transfected SH-SY5Y cells, the excitation of Fura-2 AM was performed at 356 nm and 385 nm and the emitted light was directed to a dichroic mirror with mid-reflection at 425 nm. In some experiments, the light was filtered by a band pass filter of 505–530 nm [See Additional file 1]. Since the background fluorescence is not clearly determinable in AM loaded cells, we regarded the calculated Ca²⁺ concentrations as an approximation of the real concentration [19]. Therefore, for ester-loaded cells, changes in [Ca²⁺]i are given as changes in the Fura-2 ratio (360/390 nm).

Fura-2 (Kd ~ 0.2 μM) was monitored by a computer-controlled monochromator (Polychrome II, TILL Photonics, Germany), which was connected to an Axioscope microscope (Zeiss, Goettingen, Germany) via quartz fiber optics and a minimum number of optical components for maximum fluorescence excitation (objective Achroplan W 63x, 0.9W). The apparatus was equipped with a CCD camera system [51, 52], which displayed 12-bit dynamics (PCO, Germany), and an A/D converter with a 12.5 MHz sampling rate; the binning was set to 4 × 4 and the exposure time was 30–80 ms. Calcium changes in defined regions of interest (ROIs) were monitored online using TILL Vision Software V3.3 (TILL Photonics, Martinsried, Germany). The measured fluorescence ratio at the selected wavelengths was used to calculate the [Ca²⁺]i and was represented as F/F0. Further analysis was performed off-line with the IGOR software (Wavemetrics, Lake Oswego, OR, USA) and Origin software, version 7.5.

Simultaneous recording of changes in [Ca²⁺]i and [Ca²⁺]mito

Simultaneous fluorometric measurements of [Ca²⁺]i and mitochondrial calcium ([Ca²⁺]mito) were performed utilizing calcium-sensitive fluorescent dyes (Fura-2 AM for cytosolic calcium and Rhod-2 AM for mitochondrial calcium). SH-SY5Y cells expressing either WT or G93A were attached to glass cover slips and loaded initially with 5 μM Fura-2 AM at 37°C for 30 min as described above. Subsequently, cells were washed with RPMI-1640 for 20 min and further incubated with 10 μM Rhod-2 AM for 20 min at 37°C (5% CO₂ and 95% O₂). The coverslips were rinsed with RPMI-1640 and further incubated for 20 min at 37°C to allow for complete de-esterification.

To clearly separate and simultaneously monitor the dynamic [Ca²⁺]i and [Ca²⁺]mito profiles with a temporal resolution in the millisecond time domain, we utilized a computer-controlled monochromator that rapidly switched between excitation wavelengths of 390 nm (Fura-2) and 550 nm (Rhod-2) and collected wavelengths above 565 nm for both excitation conditions. Schematic representation of the CCD-imaging setup used for conventional calcium imaging and simultaneous imaging of [Ca²⁺]i and [Ca²⁺]mito signals in the cell line preparation are illustrated in Additional file 1 [See Additional file 1]. For kinetic data analysis, only Fura-2 and Rhod-2 signals from pre-defined regions of cells were used.

The accumulations of mitochondrial and other cell organelles calcium were inhibited by cell organelles specific drug modulators. First, drugs were used, including KCl (K⁺), depolarisation stimulus; FCCP, a protonophore; and oligomycin (oligo), a F₁, F₀-ATPase blocker preventing maintenance of the proton gradient, which disrupt the mitochondrial membrane potential (ΔΨ_m) [53–56]. Secondly, by thapsigargin (thapsi) an extremely tight binding inhibitor of intracellular Ca²⁺ pumps, which raises cytosolic calcium concentration by blocking the ability of the cell to pump calcium into the sarcoplasmic and endoplasmic reticulum which causes these stores to become depleted [57]. Store-depletion can secondarily activate plasma membrane calcium channels, allowing an influx of calcium into the cytosol. Thirdly, by caffeine, a specific activator of Ca²⁺ influx from ryanodine receptor (RyR)-dependent Ca²⁺ stores [58].

Statistical analysis

Each cover slip was used for a single experiment and included more than 4 cells for each imaging experiment. Unless otherwise indicated, values in the text are given as mean ± standard deviation (SD) and the error bars indicate SD too. All values represent at least three separate experiments. Significance was calculated using the two tailed unpaired Student t-test. A p-value < 0.05 was consider statistically significant. Five point smoothing was performed in the case of simultaneous [Ca²⁺]i and [Ca²⁺]mito signals to remove the noise.

Chemical induction of calcium release

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), thapsigargin, and oligomycin were purchased from Sigma-Aldrich Chemie (Deisenhoff, Germany). FCCP was bath applied at a dose of 2 μM. A comparative analysis of the Ca²⁺ influx in WT and G93A transfected cells was done in the presence of FCCP. For these experiments the SH-SY5Y cells were loaded with 5 μM Fura-2 AM through the incubation method and [Ca²⁺]i transients were triggered once Fura-2 AM concentration was equilibrated after washing. Ca²⁺ release from intracellular stores by inhibition of the sarcoplasmic/endoplasmic reticulm Ca²⁺-dependent ATPase pump and inhibition of F₁, F₀-ATP Synthase was done by intervention of thapsi, and oligo, respectively.

For the investigation of endoplasmic reticulum (ER)-dependent Ca²⁺ release, caffeine has been established as a specific activator of Ca²⁺ influx from the RyR-dependent Ca²⁺ stores [58]. To investigate the relative impact of the ER and mitochondria in [Ca²⁺]i regulation together with the association on MN degeneration in G93A transfected SH-SY5Y cells, we used pharmacological agents as a specific activator of the ER-dependent Ca²⁺ release response [59], which not only selectively blocks Ca²⁺ uptake into ER/mitochondrial compartments but also triggers Ca²⁺ release from intracellular storage sites. Caffeine was purchased from Sigma-Aldrich chemie (Deisenhofen, Germany) and dissolved in water (25 mM). It was then diluted to the final concentration immediately before the experiment. We utilized KCl (K⁺) concentrations of 30 mM for depolarisation induced Ca²⁺ transients.

Results

Impaired mitochondrial responses of cytosolic calcium in G93A transfected cells

Representative non-transfected SH-SY5Y neuroblastoma cells (parental cell line) stained with Fura-2 AM are shown in Additional file 2 [See Additional file 2]. Mitochondrial Ca²⁺ release responses were measured to verify the impact of mitochondrial uncoupler FCCP in normal SH-SY5Y cells. The application of FCCP for 2 min fairly inhibited mitochondrial Ca²⁺ and most of the cells had a proper distinguishable and characteristic fluorescence response (See Additional file 2]. In the cells matching set criteria, including a normal morphological appearance of a large soma and intense proper staining, we performed Ca²⁺ imaging 2–5 days after culture from WT-SOD1 and G93A transfected SH-SY5Y cells.

The transfected cells were treated with FCCP (3 min) and the [Ca²⁺]i measured for both WT and G93A-transfected SH-SY5Y cells (Fig. 1). A CCD imaging photomicrograph series of [Ca²⁺]i in 7–8 cells before drug application (0.0s), after peak FCCP challenge (3 min), and after FCCP wash in WT-SOD1 (a-c) and G93A (d-f) transfected cells are shown in Fig. 1A. Figs. 1B and 1C show [Ca²⁺]i as cytoplasmic calcium versus time curves in 6 representative cells during FCCP challenge. The mitochondrial uncoupler induces a fast transient elevation in [Ca²⁺]i and a fast recovery to baseline in WT transfected cells (Fig. 1B, F/F0 = 0.1766 ± 0.0362; N = 3, n = 17). The G93A transfected cells had a comparatively lower fluorescence intensity for transient elevation in [Ca²⁺]i followed by a delayed recovery to baseline (Fig. 1C, F/F0 = 0.0948 ± 0.0223; N = 5, n = 23). The FCCP-induced calcium influx peak fluorescence intensity in G93A transfected cells was diminished by approximately 48% compared to the calcium influx peak fluorescence intensity of WT transfected cells (Fig. 1D; p < 0.001).

Figure 1

FCCP induced mitochondrial depolarization in SH-SY5Y neuroblastoma cells transfected with G93A exhibits reduced peak fluorescence amplitude. A) A CCD imaging photomicrograph series showing [Ca²⁺]i in 7–8 cells before drug application (0.0s), after peak 2 μM FCCP challenge for 3 min, and after FCCP wash in WT (a-c) and G93A (d-f) transfected SH-SY5Y neuroblastoma cells. B) A representative figure of [Ca²⁺]i fluorescence intensity in 6 SH-SY5Y neuroblastoma cells transfected with WT after FCCP application. FCCP (2 μM) induced a fast, transient elevation in [Ca²⁺]i and a fast recovery to baseline. C) In G93A transfected SH-SY5Y neuroblastoma cells, FCCP induced a transient elevation in [Ca²⁺]i fluorescence intensity that was lower in magnitude, followed by a plateau for 1 min, and a delayed recovery to baseline. D) A bar diagram to illustrate the reduction of the sustained Ca²⁺ response in G93A transfected SH-SY5Y neuroblastoma cells (F/F0 = 0.0948 ± 0.0223; N = 5, n = 23) compared to WT transfected SH-SY5Y cells (F/F0 = 0.1766 ± 0.0362; N = 3, n = 17). Values represent means ± SD, **p < 0.001, scale bar = 10 μm. N = Number of experiments; n = Number of cells.

Calcium uptake by the mitochondria is accomplished by the mitochondrial calcium uniporter (MCU) located in the inner mitochondrial membrane [60]. The accumulation of mitochondrial Ca²⁺ may have a perceptible influence on the existence of a [Ca²⁺]i transient. This effect was studied by following the Ca²⁺ signal under healthy and deleterious mitochondrial conditions in the presence of FCPP. We found that, in G93A transfected SH-SY5Y cells, the Ca²⁺ influx (peak fluorescence) was diminished compared to WT transfected SH-SY5Y cells, which, peaked within 2–3 min and was followed by a baseline recovery in approximately 2 min. The G93A transfected cells took almost 5–6 min to achieve a baseline recovery. However, the cells transfected with G93A suffered from reduced [Ca²⁺]mito after depletion of mitochondrial Ca²⁺ stores by FCCP (Fig. 1).

Effect of high K⁺-evoked Ca²⁺ transient and its impact on FCCP-induced Ca²⁺ influx

To analyze the comparative efficiency of mitochondria as a Ca²⁺ sequestering organelle, FCCP was applied to evacuate mitochondrial Ca²⁺after the WT and G93A transfected cells had been exposed to an evoked Ca²⁺ load through a depolarizing stimulus of K⁺. First, the SH-SY5Y cells transfected with WT or G93A were stained with Fura-2 AM and exposed to 30 mM K⁺ for 30 sec, which was followed by a 3 min challenge with FCCP (Fig. 2). As shown in Figs. 2A and 2B, the peak amplitude of FCCP-evoked Ca²⁺ influx after depolarization was 0.2440 ± 0.0696 (N = 3, n = 20) and 0.1497 ± 0.0362 (N = 5, n = 20) for the WT and G93A transfected cells, respectively. Fig. 2C shows the comparative analysis of average calcium release.

Figure 2

The effect of high K⁺ (30 mM) – evoked Ca²⁺ transient and its impact on the FCCP-induced Ca²⁺ influx in WT and G93A transfected SH-SY5Y neuroblastoma cells. A) A representative figure showing effect of perturbing the mitochondrial Ca²⁺ uptake on the calcium transient evoked by 30 mM K⁺ depolarizing stimulus for 30s (red horizontal bar) and by 2 μM FCCP-evoked Ca²⁺ efflux (black horizontal bar). FCCP (2 μM) induced a fast, transient elevation in [Ca²⁺]i and a fast recovery to baseline after depolarization-induced stimulus in 7 SH-SY5Y cells transfected with WT. B) In G93A transfected SH-SY5Y cells, the FCCP-induced [Ca²⁺]i transient elevation after depolarization-induced stimulus was delayed 5–10s and there was a reduction in the magnitude of fluorescence intensity followed by complete recovery to baseline. C) A bar diagram to illustrate the reduction of the sustained Ca²⁺ response in G93A transfected SH-SY5Y neuroblastoma cells (F/F0 = 0.1497 ± 0.0362; N = 5, n = 20) compared to WT transfected SH-SY5Y cells (F/F0 = 0.2440 ± 0.0696; N = 3, n = 20). Values represent means ± SD, **p < 0.001, scale bar = 20 μm. N = Number of experiments; n = Number of cells.

Interaction between ER/mitochondria in differential Ca²⁺ store regulation by inhibition of the sarcoplasmic/endoplasmic reticulm Ca²⁺-dependent ATPase pump after pharamacological intervention

The relationships between mitochondrial calcium pools and those discharged by the Ca²⁺-ATPase inhibitor thapsi, an extremely tight-binding inhibitor of intracellular Ca²⁺ pumps which induces rapid Ca²⁺ release from intracellular stores by inhibition of the sarcoplasmic/endoplasmic reticulm Ca²⁺-dependent ATPase pump without inositol phosphate formation [57, 61] were studied in WT and G93A transfected SH-SY5Y cells to estimate reciprocal functional interplay between the ER and mitochondria. It was previously shown in SOD1^G93A mice that release of ER based Ca²⁺ stores play minimal role and not an essential factors in the death mechanism of ALS vulnerbale MNs [62]. However, contrary to this study it was also shown that Ca²⁺ release from the ER contributes to neuronal cell death because the Ca²⁺release blocker, dantrolene, can protect neurons against bioenergetic failure and cellular damage [63]. While little is known about the exact ER/mitochondrial Ca²⁺ regulation mechanism in WT and G93A transfected cells, the restoration of ER function or attenuation of the secondary dysfunction induced by ER could present a new, highly promising mechansim for pharmacological intervention which could bring new ways to treat or minimize neuronal cell injury in the pathological states of ALS.

Analysis of the Ca²⁺ storing ability of ER and mitochondria in WT and G93A transfected cells was done using thapsi and FCCP. We show here that in fura-2-loaded cells, thapsi stimulated a slow plateau phase increase in cytoplasmic Ca²⁺ concentration in WT and G93A transfected cells. There was a significant quantitative difference between the ER and mitochondrial Ca²⁺ load in WT and G93A transfected cells; the Ca²⁺release response was high in the mitochondria of WT transfected cells. As shown in Fig. 3, the peak amplitude of mitochondrial Ca²⁺ release after application of FCCP plus thapsi was 0.2712 ± 0.0971 and 0.1276 ± 0.0287 where as peak amplitude of ER Ca²⁺ release after application of thapsi was 0.0412 ± 0.0152 and 0.02589 ± 0.0137 in WT and G93A transfected cells, respectively (Figs. 3A–C). The impact of thapsi on the peak amplitude of Ca²⁺ release was nominal compared with FCCP, indicating a lesser role for ER compared with mitochondria in Ca²⁺ regulation. Comparative details of the normalized fura-2 ratio in both WT and G93A transfected cells are summarized in Fig. 3C (N = 3, n = 15).

Figure 3

Analysis of the differential Ca²⁺ storage and regulation of the ER and mitochondria by pharmacological intervention in WT and G93A transfected SH-SY5Y cells. Cells were stimulated using thapsi and FCCP, which interfere with the integrity of the ER and mitochondria, respectively, and were used to release Ca²⁺ from intracellular stores by inhibition of the sarcoplasmic/endoplasmic reticulm Ca²⁺-dependent ATPase pump and mitochondrial stores by protonophore action. A) The quantitative kinetic profile of the thapsi and FCCP-evoked [Ca²⁺]i release in WT transfected SH-SY5Y cells. B) The corresponding quantitative kinetic profile of the thapsi and FCCP-evoked [Ca²⁺]i release in the G93A transfected SH-SY5Y cells. The trace is representative of mean of 4–6 cells in focus stimulated with thapsi (5 μg/ml; 6 min) and 2 μM FCCP (3 min, normalized data). The horizontal black bars indicate the duration of stimulation by thapsi and with FCCP plus thapsi. Fura-2 AM signals are represented as F/F0. C) A bar diagram of thapsi and FCCP plus thapsi-induced Ca²⁺ release in the WT and G93A transfected SH-SY5Y neuroblastoma cells (N = 3, n = 15). Gray bars represent thapsi plus FCCP-induced Ca²⁺ release in WT (F/F0 = 0.2712 ± 0.0971) and G93A (F/F0 = 0.1276 ± 0.0287) transfected SH-SY5Y cells. Striped bars represent thapsi-induced Ca²⁺ release in WT (F/F0 = 0.0412 ± 0.0152) and G93A (F/F0 = 0.0258 ± 0.0137) transfected cells. Values represent means ± SD, *p < 0.01, **p < 0.001. N = Number of experiments; n = Number of cells.

Interaction of ER/Mitochondria calcium stores regulation by activation of Ca²⁺ influx from the RyR-dependent Ca²⁺ stores

As shown in Fig. 4, caffeine led to a relatively slow and weak increase in the [Ca²⁺]i fluorescence signals, which was very similar for both WT and G93A transfected cells (N = 3, n = 14). We found that WT transfected cells (Figs. 4A and 4C) were affected by 5 mM caffeine with a small and slow increase in [Ca²⁺]i (F/F0 = 0.0471 ± 0.0190) and exhibited slightly higher kinetics than the G93A transfected cells ([Ca²⁺]i: F/F0 = 0.0353 ± 0.0120; Figs. 4B and 4C). This suggests that the caffeine-evoked ER-dependent Ca²⁺ release either makes only a minor contribution to mitochondrial mediated toxicity in G93A transfected cells, as reported in other cell types and animal models, or occurs upstream of the mitochondria.

Figure 4

Impact of inhibition of F1, F0-ATP synthase on FCCP-evoked responses of [Ca²⁺]i in WT and G93A transfected SH-SY5Y cells. A) The quantitative kinetic profile of the oligo and FCCP-evoked [Ca²⁺]i release in WT transfected SH-SY5Y cells. B) The corresponding quantitative kinetic profile of the oligo and FCCP-evoked [Ca²⁺]i release in the G93A transfected SH-SY5Y cells. The trace is representative of mean of 4-6 cells in focus stimulated with oligo (5 μg/ml; 6 min) and 2µM FCCP (3 min, normalized data). The horizontal black bars indicate the duration of stimulation by oligo and with FCCP plus oligo. Fura-2 AM signals are represented as F/F0. C) A bar diagram of oligo and FCCP plus oligo-induced Ca²⁺ release in the WT (N=3, n=20) and G93A (N=3, n=19) transfected SH-SY5Y neuroblastoma cells. Gray bars represent oligo plus FCCP-induced Ca²⁺ release in WT (F/F0 = 0.2245 ± 0.0727) and G93A (F/F0 = 0.0827 ± 0.0304) transfected SH-SY5Y cells. Striped bars represent oligo-induced Ca²⁺ release in WT (F/F0 = 0.0454 ± 0.0175) and G93A (F/F0 = 0.0229 ± 0.0161) transfected cells. Values represent means ± SD, *p<0.01, **p<0.001. N= Number of experiments; n= Number of cells.

The cells were treated with FCCP in the presence of caffeine, which resulted in the depolarization of the mitochondria (Fig. 4). We observed a rapid and high amplitude increase of [Ca²⁺]i, which exhibited a very distinct and differential response for WT and G93A transfected cells (N = 3, N = 14). WT transfected cells revealed that caffeine and FCCP together evoked a massive and comparatively fast increase in the Fura-2 fluorescence signals for WT (F/F0 = 0.1883 ± 0.0584, Figs. 4A and 4C) and G93A transfected cells (F/F0 = 0.1154 ± 0.0246, Figs. 4B and 4C).

Manipulation of mitochondria in WT and G93A transfected SH-SY5Y cells by pharmacological Inhibition of F₁, F₀-ATP Synthase

As an uncoupler of mitochondria, FCCP collapses the mitochondrial membrane potential. In turn, this results in a rapid release of calcium from this store and a slower drop in ATP levels [64]. Impairment of the mitochondria with FCCP may block oxidative phosphorylation and has the potential to compromise oxidative phosphorylation; in this state, glycolysis provides the prime means of ATP synthesis. Previously, evidence had suggested that ATP depletion by FCCP in MNs leads to minimal effect on reverse cycle of uncoupling of oxidative phosphorylation ([62], S. Balakrishnan and B.U. Keller, unpublish data). We assume that the sole effect of FCCP on cells is the uncoupling of mitochondria and that this results might lead to a reduction of the proton gradient across the internal mitochondrial membrane. Oligo, an ATP synthase inhibitor, also depletes ATP but does not alter mitochondrial membrane potential and thus does not change calcium transport across the mitochondrial membrane. Oligo was added with FCCP to prevent any accelerated consumption of cellular ATP by the reverse mode of ATP synthase operation [65, 66].

To clarify this issue, we measure the impact of oligo (5 μg/ml) on FCCP-induced Ca²⁺ release in WT and G93A transfected SH-SY5Y cells loaded with fura-2. Very weak effects to those previously observed with the electron transport inhibitors (CN^-) and azide were seen with oligomycin [19, 62]. As shown in Figs. 5A–C, impact of oligo's on the normalized peak fluorescence of fura-2 was 0.0454 ± 0.0175 and 0.0229 ± 0.0161 for WT and G93A transfected SH-SY5Y cells, respectively (Fig. 5C). The impact of oligomycin on the peak amplitude of fura-2 fluorescence was nominal and there was slight difference between WT and G93A transfected SH-SY5Y cells. However, the response to FCCP plus oligomycin in WT transfected cells (Fig. 5C) was almost 2.71-fold higher (F/F0 = 0.2245 ± 0.0727) compared with G93A transfected SH-SY5Y cells (F/F0 = 0.0827 ± 0.0304; Figs. 5A–C).

Figure 5

The simultaneous measurement of cytosolic (Fura-2) and mitochondrial (Rhod-2) calcium concentrations in WT and G93A transfected transfected SH-SY5Y cells during FCCP-evoked mitochondrial Ca²⁺ release. A) The kinetic profile of the FCCP-evoked Ca²⁺ release in the WT transfected SH-SY5Y neuroblastoma cells; the cytosolic (Error bar green, black square trace) and mitochondrial (Error bar red, black circle trace) compartment were measured simultaneously. The trace represents the mean of 5 cells in focus stimulated with 2µM FCCP (5 point smoothing). B) The corresponding kinetic profile of the FCCP-evoked Ca²⁺ release in the G93A transfected SH-SY5Y neuroblastoma cells; the cytosolic (Error bar green, black square trace) and mitochondrial (Error bar red, black circle trace) compartment were measured simultaneously. The trace represents the mean of 5 cells in focus stimulated with 2µM FCCP (5 point smoothing). FCCP-evoked [Ca²⁺]mito signals were smaller in amplitude and exhibited slower kinetics in G93A transfected SH-SY5Y cells compared to WT transfected cells and were altered from [Ca²⁺]i efflux. C) A bar diagram of the cytosolic (green bar) and mitochondrial (red bar) fluorescence signals (F/F0) from WT (F/F0 = 0.1569 ± 0.0235 for [Ca²⁺]i and F/F0 = -0.1069 ± 0.0181 for [Ca²⁺]mito; hollow; N=5, n=17) and G93A (F/F0 = 0.1008 ± 0.0248 for [Ca²⁺]i and F/F0 = -0.0486 ± 0.0043 for[Ca²⁺]mito; striped pattern, N=4; n=17) transfected SH-SY5Y neuroblastoma cells. Values represent means ± SD, **p<0.001. N= Number of experiments; n= Number of cells.

Simultaneous measurement of [Ca²⁺]i and [Ca²⁺]mito

Using FCCP to prevent a [Ca²⁺]mito increase while measuring an increase in [Ca²⁺]i, we were able to efficiently separate cytosolic and mitochondrial Ca²⁺ at a temporal resolution in the millisecond time domain. As shown in Fig. 1, the Ca²⁺ release in G93A transfected cells is smaller in amplitude and exhibits slower kinetics compared to WT transfected cells. To confirm that there are separate Ca²⁺ uptake and release events from the cytosol and mitochondria, the transfected cells were treated with FCCP and the separate Ca²⁺ levels measured simultaneously (Fig. 6). WT transfected cells exhibited a substantial increase in [Ca²⁺]i (F/F0 = 0.1569 ± 0.0235; Fig. 6A, N = 5; n = 17) while [Ca²⁺]mito decreased (F/F0 = -0.1069 ± 0.0181, Fig. 6A, N = 5; n = 17). This suggests that the FCCP-evoked increase in [Ca²⁺]i in Fig. 1 was mainly due to [Ca²⁺]i and that there was very little impact from [Ca²⁺]mito. In G93A transfected cells, FCCP-evoked [Ca²⁺]i was less and exhibited slower kinetics (F/F0 = 0.1008 ± 0.0248; Figs. 6B and 6C, N = 4; n = 17) compared to WT transfected cells, which exhibited a reduction of the [Ca²⁺]mito (F/F0 = -0.0486 ± 0.0043, Figs. 6B and 6C, N = 4; n = 17).

Figure 6

Caffeine stimulates [Ca²⁺]i release with slower kinetics and weaker transient than the FCCP-evoked [Ca²⁺]i signals in SH-SY5Y neuroblastoma cells, particularly in WT transfected cells compared to G93A transfected cells. A) The kinetic profile of caffeine and FCCP-evoked [Ca²⁺]i release in the WT transfected SH-SY5Y neuroblastoma cells. The trace is representative of 5 cells in focus stimulated with 5mM caffeine and 2µM FCCP (normalized data, 5 point smoothing). B) The corresponding kinetic profile of the caffeine and FCCP-evoked [Ca²⁺]i release in the G93A transfected SH-SY5Y cells. The trace is representative of 5 cells in focus stimulated with 5mM caffeine and 2µM FCCP (normalized data, 5 point smoothing). The ER and mitochondrial Ca²⁺ release from these two compartments were measured simultaneously. The horizontal black bars indicate the duration of stimulation by caffeine and with FCCP plus caffeine. Fura-2 AM signals are represented as F/F0. C) A bar diagram of caffeine and FCCP plus caffeine-induced Ca²⁺ release in the WT and G93A transfected SH-SY5Y neuroblastoma cells (N=3, n=14). Gray bars represent caffeine plus FCCP-induced Ca²⁺ release in WT (F/F0 = 0.1883 ± 0.0584) and G93A (F/F0 = 0.1154 ± 0.0246) transfected SH-SY5Y cells. Striped bars represent caffeine-induced Ca²⁺ release in WT (F/F0 = 0.0471 ± 0.0190) and G93A (F/F0 = 0.0353 ± 0.0120) transfected cells. Values represent means ± SD, *p<0.01, **p<0.001. N= Number of experiments; n= Number of cells.

Discussion

Recent evidence suggests that abnormalities in cellular Ca²⁺ signaling are common features in the pathogenesis of a range of neurodegenerative disorders, including ALS [33]. It is well known that Ca²⁺ is one of the most relevant intracellular messengers essential in neuronal development, synaptic transmission and plasticity, and the regulation of various metabolic pathways in the brain. Further evidence for the involvement of a disruption in intracellular Ca²⁺ homeostasis was reported in cellular and experimental animal models; there was an absence of Ca²⁺ binding proteins, such as Calbindin-D_28K and parvalbumin, in MN populations lost early in ALS [67]. Additionally, accumulation of Ca²⁺ into vacuoles in the mtSOD1 mice and low Ca²⁺ buffering in SMNs were also shown [68]. These findings agree with a quantitative comparison of Ca²⁺homeostasis where a low cytosolic Ca²⁺ buffering capacity acts as an important risk factor for degeneration. In contrast, an increase in the cytosolic Ca²⁺ buffering capacity could protect vulnerable MNs from degeneration [69, 35]. MNs store a larger amount of calcium in the mitochondria and disruption of mitochondrial Ca²⁺ uptake has a marked influence on both the peak amplitude of Ca²⁺ response as well as the clearance of [Ca²⁺]i.

Our interest in studying the role of Ca²⁺ regulation and the impact of mitochondrial inhibition in a cellular model of ALS is based on observations that mitochondria act as local calcium buffers, thus shaping the spatiotemporal aspects of [Ca²⁺]i. MN mitochondria have been shown to have a major percentage of Ca²⁺ sequestered intracellularly after influx through the plasma membrane [70, 71]. Our main objective was to characterize the contribution of the mitochondrial buffering of voltage-activated Ca²⁺ loads and how this Ca²⁺ regulatory mechanism is controlled. We observed that Ca²⁺ influx was diminished by approximately 1.86 fold in the presence of G93A compared to WT (Fig. 1). After exposure to FCCP, the capacity of G93A transfected cells to transport [Ca²⁺]i to the extracellular space or to intracellular storage sites was much lower than WT transfected cells and accounted for the increased vulnerability of cells possessing mutant G93A gene.

Interestingly, the depolarization-induced Ca²⁺ transient in the presence of intact mitochondria was smaller in amplitude than with G93A mitochondria; excess Ca²⁺ emerged in the second response and was prominent in WT cells (Figs. 2A–C). This reflects the variability in Ca²⁺ clearing mechanisms of ALS-vulnerable and non-vulnerable MNs and agrees with data obtained from motor nerve terminals [72], cultured MNs [25], and patch-clamped hypoglossal MNs [52] in which significant mitochondrial Ca²⁺ uptake following voltage-activated Ca²⁺ influx was demonstrated. We observed that the depolarization-induced stimulus not only interfered with the post-depolarization recovery of [Ca²⁺]i after FCCP challenge but also increased the peak amplitude of the FCCP-evoked Ca²⁺ influx by approximately 40 and 68% in WT and G93A cells, respectively (compare Figs. 1D and 2C). Therefore, it is evident that in the absence of mitochondrial Ca²⁺ uptake, the [Ca²⁺]i signal is stronger, and mitochondria can actively sequester Ca²⁺ during an on-going Ca²⁺ influx as a result of the opening of voltage-gated calcium channels. A significant difference in the Ca²⁺ accumulation by mitochondria in G93A and WT cells was observed and presumably due to G93A-mediated toxicity. The capacity of mitochondria to restore the Ca²⁺ buffering capacity of cell organelles is heavily compromised in G93A cells. The disturbance of Ca²⁺ trapping in intracellular storage sites and Ca²⁺ extrusion from the cells [41] may account for the reduced oscillations of [Ca²⁺]i we observed in G93A cells.

Molecular interaction underlying mitochondria-endoplasmic reticulum Ca²⁺ strores coupling was evaluated using thapsi and FCCP. We assume that both ER and mitochondrial intracellular pools participate in the generation of Ca²⁺ signals in SH-SY5Y cells shaping their spatiotemporal Ca²⁺ signals patterns. Thapsi- induced Ca²⁺release was significantly less than that evoked by FCCP in WT and G93A transfected cells, and its kinetics were more or less similar in both genotypes (Figs. 3A–C). The application of thapsi on SH-SY5Y cells causing separate Ca²⁺ release response was slightly more in WT transfected cells than in G93A cells, indicating that, in the context of the G93A transfection, the ER may slightly contribute to the motor dysfunction. Furthermore, the application of FCCP after emptying ER stores with thapsi resulted in a separate release event, evident from the bigger [Ca²⁺]i increase. This release was higher than the general Ca²⁺ release caused by FCCP without emptying ER in WT transfected cells but not in G93A altered cells, which suggests an uptake of the released Ca²⁺ from ER by mitochondria in WT transfected cells. This further indicates the explicit action of FCCP in our working model system and the existence of two separate intracellular Ca²⁺ stores, in which the ER seems to play a minimal role in buffering [Ca²⁺]i after Ca²⁺ loads are imposed in G93A transfected cells, and the ER is most likely not impaired during ALS-related motoneuron disease.

We found that ER of SH-SY5Y cells retained a very low amount of calcium (after Ca²⁺ release with thapsi for 6 min application) compared with mitochondria after Ca²⁺ elevation by FCCP (3 min) indicating its low efficiency to sequester Ca²⁺ in the WT and G93A transfected SH-SY5Y cells, which was slightly higher in WT (Figs. 3A–C). This indicates that the conventional mitochondrial Ca²⁺ storing function dominates ER Ca²⁺accumulation in these cells. It is noteworthy that thapsi has a relatively very weak effect on Ca²⁺ release in G93A transfected cells, indicating that G93A alteration might also result in defects in ER Ca²⁺ handling, which may perturb functional domain and contribute to neurodegeneration. These data suggest that the ER of SH-SY5Y cells does not play a significant role in regulating [Ca²⁺]i at the basal level or after imposed Ca²⁺loads in WT transfected cells; though minor ER contribution to the dysfunction of Ca²⁺ loads did not ruled out, suggesting that Ca²⁺dysregulation as a result of the G93A transfection anticipates mitochondrial impairment. Our hypothesis is strengthened by the fact that previously we have shown quite similar results in an animal model of SOD1^G93A mice for fALS; where cyclopiazonic acid (a specific inhibitor of Ca²⁺-ATPase led to a relatively slow and weak increase of [Ca²⁺]i, which occurred with slightly higher kinetics in WT HMNs than the SOD1^G93A mice HMNs [62]. This finding alongwith our results in WT and G93A transfected cells further explains that ER-dependent Ca²⁺release is a minor contributor to mitochondria-mediated toxicity in G93A transfected cells as previously reported in other cell types and animal models [73, 74]. Results indicate close coupling between ER and mitochondria in WT but not in G93A transfected cells which is probably impaired due to G93A transfection. The thapsi-releasable Ca²⁺ pool in WT and G93A transfected cells (Figs 3A–C) was quite similar in quantity and temporal quality release via RyR-sensitive Ca²⁺ pool by caffeine (see Figs 4A–C). This observations suggests that Ca²⁺ uptake by RyR-responsive pool is also sensitive to thapsi. These findings also indicate that thapsi increases [Ca²⁺]i by inhibiting Ca²⁺ uptake into multiple intracellular Ca²⁺ pools and by also promoting entry of extracellular Ca²⁺ dominantly in WT but not in G93A transfected cells. The exact molecular interactions defining the organization of mitochondria, ER and other Ca²⁺ sources varies among different cell types, are questions that remain unanswered but are interesting areas for future investigations.

We expect a decrease in cellular ATP levels during inhibition of mitochondrial electron transport by FCCP and this may affect release of [Ca²⁺]i. To test the impact of ATP depletion on regulation of [Ca²⁺]i, oligo (5 μg/ml) was added before FCCP application. Oligo does not influence [Ca²⁺]i heavily and left basal Ca²⁺ levels, as well as recovery times of Ca²⁺ transients, unaffected for upto several minutes (6 min application; Figs 5A–C) and therefore amplitude of evoke [Ca²⁺]i transients was clearly attributable to FCCP. Since application of oligo alone did not affect the [Ca²⁺]i transients therefore the effect of FCCP is clearly attributable to collapse of mitochondria, and not to inhibition of ATP-synthesis. We assume that the time course of ATP depletion by FCCP is slower but the increase of cytosolic calcium by FCCP is immediate and therefore there is no causal relationships have been demonstrated between fall in ATP level and increase of [Ca²⁺]i. These results are in agreement with those from studies of SOD1^G93A mice in sensitivity of HMNs mitochondria to oligo [62].

Using the CCD camera imaging system, we clearly separated the dynamic profiles of [Ca²⁺]i and [Ca²⁺]mito. Using this method, we demonstrated that in WT cells, the FCCP-evoked increase in [Ca²⁺]i was mainly due to [Ca²⁺]i alone. However, in G93A cells, the FCCP-evoked [Ca²⁺]i was less, exhibited slower kinetics, and reduced [Ca²⁺]mito. This suggests that cells with G93A have a lower affinity for mitochondrial Ca²⁺ upload and likely represents severely disturbed and vulnerable mitochondria. In this study, for the first time, the simultaneous determination of [Ca²⁺]i and [Ca²⁺]mito in WT and G93A transfected SH-SY5Y cells, a culture model of motor neuron disease was achieved (Figs 6A–C). This data is in agreement with previous findings that G93A cells exhibit damaged and stressed mitochondria [6] and increased ROS [47].

Different intracellular pools participate in the generation of neuronal Ca²⁺ signals, shaping their spatio-temporal patterns, and the cell life-death cycle. Mitochondria, in the kinetic and hot spot hypothesis, and the ER, by different classes of channels with distinct properties and highly defined expression patterns, have been implicated in the regulation of [Ca²⁺]_i in many systems [41, 42]. Caffeine was used to investigate ER-dependent Ca²⁺ release and we found that it evoked a small and slow increase in [Ca²⁺]i with slightly faster kinetics in WT cells than G93A cells. This further suggests that ER-dependent Ca²⁺ release has a minor contribution in the mitochondria-mediated toxicity of G93A. The rate of increase in Fura-2 fluorescence following caffeine application was slower and did not resume the baseline, which is likely due to the slow activity of the mitochondrial Na⁺/Ca²⁺ exchanger, the major pathway for mitochondrial Ca²⁺ efflux [75]. Observations of different pharmacological conditions support the concept that the presence of G93A severely disrupts mitochondrial Ca²⁺ regulation. It is also interesting to note that the rate of increase in Fura-2 fluorescence signals following caffeine application was slightly slower compared to FCCP-evoked signals and did not achieve the baseline within a few minutes. This suggests that the dye accumulates in the mitochondria and the mitochondrial uptake and release event is prominently operated by Ca²⁺ efflux rather than by the store-operated Ca²⁺ uptake and release phenomenon. Our fluorescence system may be a valuable tool to determine mitochondrial and Ca²⁺-related defects during G93A-mediated MN degeneration, which closely parallels the incidence of neuronal death in G93A transfected SH-SY5Y cells. Quantitative values of normalized Fura-2 fluorescence signals after different drugs interventions are compared in Table 1.

Table 1 Differential peak amplitudes of [Ca²⁺]i release from different compartment of WT and SOD1 transfected SH-SY5Y cells after multidrugs intervention.

Despite rigorous research since Charcot's description more than 130 years ago, the molecular abnormalities leading to the damage of specific MNs in ALS are still unknown. The selective vulnerability of MNs in ALS-related disease and associated cell culture models is closely linked to exceptional Ca²⁺ signaling mechanisms that are part of the physiological cell function, but seemingly also enhances the risk of Ca²⁺ homeostasis disruption and mitochondrial dysfunction in vulnerable cells. Earlier studies suggested that uncontrolled Ca²⁺ entry and inefficient calcium sequestering cause selective damage leading to the formation of vacuoles derived from the degenerating mitochondria in the MNs of the mouse model of ALS [12, 76, 77]. In contrast to most other neurons, MNs have a low Ca²⁺-buffering capacity due to the low expression of Ca²⁺-buffering proteins and a high number of Ca²⁺-permeable AMPA receptors resulting from low expression of the GluR2 subunit. The combination of these two properties seems to be intrinsic to MNs and is most likely essential for their normal function. However, under pathological conditions, MNs may become over-stimulated by glutamate and overwhelmed by Ca²⁺; though, whether downstream pathways activated by the intracellular Ca²⁺ increase are different in MNs compared to other neurons is not yet known.

Conclusion

Our experiments identified specialized Ca²⁺ homeostasis characterized by low cytosolic Ca²⁺ buffering in which mitochondria play a major role in the regulation of [Ca²⁺]_i transients in vulnerable MNs. Low cytosolic Ca²⁺ buffering enhances the role of low affinity organelle buffers, such as mitochondria, in the cell. For example, large and long-lasting Ca²⁺ domains around influx sites enhance the risk of toxic Ca²⁺ accumulations and subsequent activation of Ca²⁺-dependent neurodegenerative pathways under excitotoxic conditions. Indeed, a strong contribution of mitochondria, as opposed to ER, Ca²⁺ uptake to the buffering of Ca²⁺ profiles was recently demonstrated [19, 52, 62]. There are two reasons that the prominent role of mitochondria in the regulation of moderate Ca²⁺ loads in MNs has important implications for pathological conditions such as ALS. First, the amount of Ca²⁺ taken up by the mitochondria is probably higher in MNs than other cell types [25, 78]. These high Ca²⁺ loads enhance the risk for ROS generation, which may play a major role in initiating the death cycle resulting in MN degeneration [29]. Second, our experiments provide evidence that cytosolic Ca²⁺ depends on intact mitochondrial Ca²⁺ uptake. Thus, when mitochondrial Ca²⁺ uptake is disturbed, as was seen in G93A cells, MNs are directly put at risk of accumulating basal Ca²⁺ levels during repetitive oscillations. Additionally, the decreased ability to limit Ca²⁺ transient amplitudes in the cytosol and, in particular, local domains when mitochondria are depolarized enhances the risk of initiating Ca²⁺ dependent neurodegenerative pathways leading to cell death. A summary of the identified mechanisms is given in Fig. 7; in vulnerable MNs, the calcium buffering machinery is represented by the predominance of mitochondria and calcium binding proteins.

Figure 7

Low Ca²⁺ buffering and excitotoxicity under physiological stress and pathophysiological conditions in motor neuron (MNs). Low Ca²⁺ buffering in amyotrophic lateral sclerosis (ALS) vulnerable hypoglossal MNs exposes mitochondria to higher Ca²⁺ loads compared to highly buffered cells. Under normal physiological conditions, the neurotransmitter opens glutamate, NMDA and AMPA receptor channels, and voltage dependent Ca²⁺ channels (VDCC) with high glutamate release, which is taken up again by EAAT1 and EAAT2. This results in a small rise in intracellular calcium that can be buffered in the cell. In ALS, a disorder in the glutamate receptor channels leads to high calcium conductivity, resulting in high Ca²⁺ loads and increased risk for mitochondrial damage. This triggers the mitochondrial production of reactive oxygen species (ROS), which then inhibit glial EAAT2 function. This leads to further increases in the glutamate concentration at the synapse and further rises in postsynaptic calcium levels, contributing to the selective vulnerability of MNs in ALS.