SH-SY5Y human neuroblastoma cell line transfected with either WT or G93A mutant linked with fALS were routinely cultured in growth medium and were used as a valid and robust in vitro system to investigate the cellular excitotoxicity and mitochondria mediated alterations associated with SOD1-G93A mutations (Carri et al., 1997; Goos et al., 2007; Jaiswal et al., 2009). Detail descriptions about the transfection strategy, cell culture maintenance, cell culture growth medium and procedures are described earlier (Jaiswal et al., 2009).

Toxicity of Na-azide induced mitochondrial inhibition and thereby cell death assay in SH-SY5Y^WT and SH-SY5Y^G93A was done using bright field microscopy, hematoxylin and eosin (H&E) histochemistry and WST-1 assay. Neuroprotection study was done using similar method. SH-SY5Y^WT and SH-SY5Y^G93A transfected cells on glass coverslips were treated for 3 min (acute), 30 min (moderate) and 60 min (chronic) exposure with 3 mM Na-azide (60 min data not shown), cells were fixed with 4% paraformaldehyde (PFA), dehydrated through water/ethanol and histolene and then stained with Meyer’s hemalum reagent (1:1 in H₂O). While choosing the Na-azide concentrations we followed the previous literature and accordingly we selected three different Na-azide concentrations, 1, 3, and 10 mM for cytotoxicity induced cell death assays and neuroprotection study (Hunter et al., 1959; Harvey et al., 1999). Concentration of 1 mM Na-azide not always leads to swelling of mitochondria for acute (3 min) exposure. Also shown by other groups at 0.1 mM or less than 2 mM sodium cyanide (NaCN) was not always adequate to prevent completely spontaneous swelling or swelling induced by low concentrations of phosphate (Hunter et al., 1959; Harvey et al., 1999). Higher concentration, e.g., 10 mM Na-azide might react with components other than cytochrome oxidase, therefore based on our pilot toxicity experiments and previous literature, we have chosen 3 mM Na-azide concentration suitable for our acute toxicity (3 min exposure) and neuroprotection assays. To study the differences in impact of Na-azide–induced cell death and neuroprotection mediated by riluzole and melatonin, SY5Y^WT and SH-SY5Y^G93A cells at a density of 10⁵ cells/cm² were seeded into 24-well plates. SH-SY5Y^WT and SH-SY5Y^G93A cells were exposed to culture medium with 3 mM Na-azide for 3 min (acute), 30 and 60 min (chronic). After 3, 30, and 60 min (60 min data not shown) of exposure, cell photomicrograph was acquired using brightfield microscope. Furthermore, for neuroprotection assay by riluzole and melatonin, both cell lines were exposed to DMEM growth medium with 3 mM Na-azide+100 μM riluzole or 3 mM Na-azide+100 μM melatonin for 3 min (acute). After rinsing the cover slips twice with water, cover slips were dehydrated and Vectashield Medium (Vector Laboratories, Burlingame, CA, USA) was used for mounting the cover slips.

To further investigate the impact of Na-azide exposure on cell viability and neuroprotection by riluzole and melatonin, cells at a density of 10⁵ cells/cm² in 24-well plates were treated with culture medium or medium containing 3 mM Na-azide final concentration. After 3, 30, and 60 min (60 min data not shown) of incubation with 3 mM Na-azide, WST-1 cell proliferation reagent (Roche Applied Science, Mannheim, Germany) was used to determine cell viability. For neuroprotection, similar WST-1 assay were done using 3 mM Na-azide+100 μM riluzole or 3 mM Na-azide+100 μM melatonin. The WST-1 assay is comprises a cleavage of the tetrazolium salt by healthy mitochondria to soluble orange formazan, by the enzyme succinate dehydrogenase present in the ETS of healthy mitochondria. The formazan dye formed after incubation of cells with WST-1 for 2 h was quantified by measuring the absorbance at 490 nm using the Genios multiplate reader (Tecan, Crailsheim, Germany) and correlates with the total number of metabolically viable cells.

Superoxide dismutase 1^G93A Tg 1Gur (Fast Line) mice strain is considered a well-established animal model for human ALS. This mice strain acquired from the Jackson Laboratory (Bar Harbor, ME, USA) and in-house breeding was carried out in our animal facility. The SOD1^G93A Tg mouse develop paralysis in the limb and eventually dies at 4–5 months of age due to the loss of MNs in the brain stem, HMNs, FMNs, and the spinal cord whereas WT littermates were unaffected. The Tg mouse carries a variation of the human mutant G93A-SOD1 gene in which at position 93 glycine is substituted by alanine (Gurney et al., 1994). Breeding and genotyping procedures were adapted from Hoyaux et al. (2000; for further details also see Jaiswal and Keller, 2009). Animal breeding and experimental procedures were approved and carried out in accordance with the guidelines of the ethics committee of the Medical Faculty of Georg-August University, Göttingen, Germany.

Mice were anesthetized until the paw withdrawal reflex disappeared in a chamber containing an ether vapor-enriched atmosphere and immediately sacrificed. Mice brains were quickly removed and then put in 4°C ice-cold aCSF. Transverse 250 μm thick acute brain stem slices were prepared from the 14–15 weeks old WT and symptomatic SOD1^G93A mice in late stage of motor dysfunction according to the procedures described earlier (Ladewig and Keller, 2000; Jaiswal and Keller, 2009) using a vibroslicer (Leika VT 1000S, Göttingen, Germany). Selectively vulnerable brain stem HMNs (Nuc. hypoglossus; XII) region were visually identified by their location nearby to the IV ventricle during the brain stem slice preparations transversely (Figure 3). To achieve maximum oxygen supply to slices, aCSF (in mM: NaCl 118, KCl 3, NaH₂PO₄ 1, CaCl₂ 1.5, MgCl₂ 1, NaHCO₃ 25, glucose 20; pH 7.4; 320 mOsm) was constantly simmered with a mixture of 95% O₂ and 5% CO₂ (carbogen). An essential requirement for the microfluorometric measurements was the preservation of MNs in adult slices near to the slice surface in a physiologically viable and intact condition. This challenging requirement for Ca²⁺ imaging in MNs was accomplished by reducing mechanical tissue disturbances during the thick adult brain slice preparations, slice cutting at 4°C temperature and regular maximum oxygen delivery to keep metabolic conditions optimum for cells viability (For details, see Jaiswal and Keller, 2009). Slices were kept at ∼27°C in continuous carbogen-bubbled aCSF solution at pH 7⋅4 for 30 min and then let it cool down to room temperature (RT, ∼20–21°C) prior to dye loading. Unless indicated otherwise, all experiments were performed at RT.

Intracellular calcium [Ca²⁺]i fluorescence measurement were achieved using an optical recording system including an upright Axioscope microscope (Zeiss, Göttingen, Germany) equipped with a computer-operated monochromator (TILL Photonics, Martinsried, Germany) built on a galvanometric scanner (Polychrome II, TILL Photonics, Martinsried, Germany). Briefly, a modified version of the CCD camera system (TILL Photonics, Planegg, Germany) and Achroplan W 63×, 0.9W objective were used to collect fluorescent signal changes in defined “regions of interest” (ROIs) in cell soma. Fluorescence signals changes were digitized using a 12-bit CCD camera (PCO, Kelheim, Germany), binning of which was put to 4 × 4, with a sampling rate between 3 and 13 Hz using TILL Vision Software (TILL Vision Software V3.3, TILL Photonics, Germany). Dichroic mirror with mid reflection at 425 nm was used for Fura-2 AM calcium dye (Invitrogen, Carlsbad, CA, USA) to direct the emitted light. To achieve the ratiometric calcium imaging, swapping between wavelengths (360 and 390 nm) was achieved in ∼3 ms, thereby allowing fast ratiometric Ca²⁺ measurements.

WT (SH-SY5Y^WT) and SOD1 (SH-SY5Y^G93A) transfected cell layers on cover slips were stained with membrane-permeable Fura-2 AM (K_d ∼ 0.2 μM) dye by incubating with RPMI-1640 medium having 10% FCS (Invitrogen, Carlsbad, CA, USA) and 0.846 mM Ca²⁺ (5 μM Fura-2 AM dissolved in DMSO containing 10% pluronic acid for 30 min at 37°C and continuous bubbled with 95% O₂ and 5% CO₂). After the dye incubation cells were washed with RPMI and incubated in culture medium for ∼20 min at 37°C to permit de-esterification of dye. Baseline [Ca^2+]i were measured in transfected cells 2–5 days in culture. Chemical induction of Ca²⁺ measurements were done by incubating the cells for 3 min with 3 mM Na-azide and effects of 100 μM riluzole and 100 μM melatonin were measured by inhibition of Ca²⁺ with similar measurements (data not shown).

In HMN_S of brain stem slice, calcium signals were measured by defining appropriate ROIs of MN somas as previously described (Lips and Keller, 1998). [Ca²⁺]_i signals were monitored by bath-loading of the brain stem slices with 5 μM final dye concentration of Fura-2 AM for 40 min at 27°C with continuous carbogen bubble (5% CO₂ and 95% O₂). After the dye incubation, prior to onset of imaging slices were washed with aCSF and incubated another 30 min in aCSF for de-esterification of Fura-2 AM. Excitation of Fura-2 was done alternately at 360 and 390 nm by UV light and emitted light was directed to a dichroic mirror having mid-reflection at 425 nm and filtered by a band pass filter of 505–530 nm (Zeiss, Goettingen, Germany) using a computer-controlled monochromator. Changes in [Ca²⁺]_i are denoted as variations in Fura-2 ratio (F/F₀) where F = fluorescence at different time points of experiment and F₀ = baseline fluorescence value before drug/chemical applications (for details see Jaiswal and Keller, 2009). Further calculations and analysis of [Ca²⁺]_i signals were performed offline using IGOR Pro (Wavemetrics, Lake Oswego, OR, USA) and OriginPro 6.0 (OriginLab Corporation, Northampton, MA, USA) Software.

The metabolic state of HMNs from SOD1^G93A mice in the presence and absence of 100 μm riluzole was monitored by measuring and monitoring intrinsic nicotinamide adenine dinucleotide (NADH) fluorescence of HMNs in the slice preparations using a high-speed CCD camera. Briefly, NAD(P)H excitation at 360 nm was carried via a computer-controlled monochromator (TILL Photonics, Martinsried, Germany) reflected onto the surface of the slice using dichroic mirror (400 nm, Zeiss, Goettingen, Germany). The hypoglossal nucleus was easily recognized by a high level of intrinsic fluorescence. Fluorescence emission was collected by using a CCD camera (TILL Photonics, Planegg, Germany). All experiments used a 410 nm LP filter between the dichroic mirror and camera to maximize light capture. Imaging was performed after focusing onto the surface of slices in hypoglossal area using 63× (0.9 NA) Achroplan water objective (Zeiss, Göttingen, Germany) and collected by a 12-bit CCD camera (PCO, Kelheim, Germany) described earlier. Calculations and analysis of NAD(P)H signals were performed offline after the experiment using IGOR Pro (Wavemetrics, Lake Oswego, OR, USA) and OriginPro 6.0 (OriginLab Corporation, Northampton, MA, USA) Software.

Fura-2 AM dye (50mg/vial) was purchased from Invitrogen (Carlsbad, CA, USA) and dissolved in 50 μL DMSO containing 20% Pluronic F-127 to a concentration of 1 mM. Na-azide, riluzole, melatonin and all other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA). To prepare the stock solutions, riluzole and melatonin were dissolved in 100% ethanol to make 10 mM stock solutions and all the time kept at 4°C to avoid ethanol evaporation. Na-azide was dissolved in distilled water. To keep cells viable drug solutions when mixed in the perfusate always simmered with carbogen (95% O₂, 5% CO₂) during the experiments.

SH-SY5Y^WT and SH-SY5Y^G93A transfected cells attached with cover slip was used for single experiments and replicate in five separate experiments (five field of view) for each condition in cell viability assay (cell survival/death) and seven separate experiments (seven field of view for each cover slides for SH-SY5Y^WT and SH-SY5Y^G93A) for neuroprotection assay. All slices were used for a sole experiment and 5–6 HMNs taken from each slice in Fura-2 calcium imaging data experiments. 2–3 slices were used per mouse and 5–6 HMNs selected from each slice for bar graph presentation. In slice experiments, bar diagram represents 12 imaging experiments recorded from 12 separate slices obtained from 5 WT mice and 10 imaging experiments recorded from 10 slices obtained from 5 SOD1^G93A mice in acute Na-azide experiments. In case of riluzole experiments, bar diagram represent 14 imaging experiments recorded from 14 separate slices obtained from 6 WT mice and 13 imaging experiments recorded from 13 slices obtained from 6 SOD1^G93A mice. In case of NADH fluorescence imaging, 5–6 cells from slice for five separate experiments for each condition from five different mice of same genotypes was used for bar diagram representation. Values in the text and error bars in experimental figures are represented as mean ± SD. Significance for all the in vitro assay, slice imaging and pharmacological intervention was calculated using the two-tailed unpaired student t-test and a p-value < 0.05 was considered statistically significant. One-way analysis of variance (ANOVA) with Bonferroni’s method was used as the post hoc test used to recognize statistically significant Na-azide effects, and neuroprotection effects of riluzole and melatonin exposed cells as compared with controls with in groups.

We utilized differentiated SH-SY5Y^WT and SH-SY5Y^G93A cells used previously to generate WT and SOD1^G93A cell culture model of ALS (Jaiswal et al., 2009). Cell death, cell viability and anti-aggregate formation were assessed using bright field microscopy, immunocytochemistry and WST-1 cell proliferation assay (Figures 1 and 2). SH-SY5Y^G93A cells showed slightly higher levels of cell death compared to SH-SY5Y^WT after 3 and 30 min (Figures 1A–Da–c and 2A) exposure to mitochondrial toxin Na-azide (Figures 1A–Da–c and 2A). A similar pattern was observed while investigating the apoptosis and aggregation assessed by bright field microscopy demonstrating typical apoptotic morphology, e.g., condensation/fragmentation of nuclear material and elongation of cell size and cell membranes. Cells displaying fragmentation of nuclear materials (pyknosis)/cell membrane elongation were classified as apoptotic and showed aggregates formation with both cell lines after incubation for 30 and 60 min (Figures 1Aa–c,Ca–c; data not shown for 60 min exposure) but not with acute 3 min (acute) induction (Figures 1Aa–c,Ca–c). We have not quantified the cell numbers after 30 and 60 min exposure to Na-azide due to lump formation after 30 min exposure and inability to separate each cells with in aggregates and almost complete cell death 60 min after the inhibition of mitochondria by Na-azide (Figures 1Ac,Cc). Cell survival/death was assessed by H&E staining and WST-1 test. Cell count for H&E staining for both SH-SY5Y^WT and SH-SY5Y^G93A cells treated with Na-azide for 3 min (77.0 ± 4.7 for WT and 71.5 ± 6.1 for SOD1^G93A) and 30 min (59.2 ± 5.8 for WT and 54.0 ± 5.5 for SOD1^G93A) were significantly lower compared to untreated cells with Na-azide (114.5 ± 2.8 for WT and 107.0 ± 5.1 for SOD1^G93A; Figures 1A–Da–c and 2A; N = 5; ^∗∗∗P < 0.001, ^∗∗P < 0.005, Students two sample t-test, Table 1).

It was previously reported by several groups that riluzole and melatonin ameliorates ALS progression in SOD1^G93A mouse model and extend morbidity of hALS patients. We next attempted to determine whether riluzole and melatonin confers neuroprotection and ameliorate cell death in ALS cell culture model and if yes at what extent. Previously it was shown that riluzole up to 10 μM have no effect on cell survival in DA neurons and SH-SY5Y cells. However, concentrations greater than 50 μM have a remarkable effect on cell survival (Storch et al., 2000); therefore, here we used 100 μM riluzole and melatonin exposure with superfusate and evaluate the protective effects of riluzole on cell survival in SH-SY5Y^WT and SH-SY5Y^G93A cells in vitro (Figures 1A–Dd,e and 2A). Our findings show that the acute inhibition of mitochondrial metabolism and function and thereby cell death by Na-azide is ameliorated by riluzole but there is no significant effects of melatonin in both SH-SY5Y^WT and SH-SY5Y^G93A cells (Figures 1A–Dd,e and 2A). The average number of H&E positive cells in a given field of view (seven field of view) after the treatment of 100 μM riluzole to SH-SY5Y^WT and SH-SY5Y^G93A cells is significantly higher (Figure 2A; 110.0 ± 7.8 for WT and 101.2 ± 6.7 for SOD1^G93A) compared to non-treated cells stimulated with 3 mM Na-azide (Figure 2A; 77.0 ± 4.7 for WT and 71.5 ± 6.1 for SOD1^G93A; for riluzole treated and non-treated cells; ^∗∗P < 0.005, Students two sample t-test; Table 1). In addition to decreasing the cell death, riluzole had significant protective effects on the neuritis, fine cell processes and cell membrane integrity being preserve from mitochondrial inhibition and the effect was most pronounced in SH-SY5Y^WT compared to SH-SY5Y^G93A cells (Figures 1Ad–Dd). However, impact of 100 μM melatonin treatment on Na-azide induced inhibition of mitochondria is not significantly different then non-treated cells in both SH-SY5Y^WT and SH-SY5Y^G93A cells though there are minor increase in cell count of WT and SOD1^G93A cells (Figures 1A–De and 2A; 87.2 ± 7.0 for WT and 82.5 ± 5.3 for SOD1^G93A, Table 1).

Cell viability assessed by WST-1 assay showed significant dose dependent ROS-induced mitochondrial toxicity and cell death. Cell viability of SH-SY5Y^WT and SH-SY5Y^G93A cells in presence of Na-azide for 3 and 30 min leads to a decrease in cell survival (75.0 ± 3.4 and 61.2 ± 6.1% for 3 and 30 min incubation, respectively, for WT; Figure 2B) and were significantly not different with SOD1^G93A cells (73.5 ± 6.2 and 54.0 ± 8.4% for 3 and 30 min incubation, respectively; Figure 2B; Table 2). However, cell viability for both WT and SOD1^G93A treated cells were much lower and significantly different compared to untreated cells without Na-azide toxicity (100 ± 3.4% for WT and 91 ± 8.6% for SOD1^G93A; Figures 1A–Da–c and 2A; N = 5; ^∗∗∗P < 0.001, Students two sample t-test; Table 2). Similarly % of SH-SY5Y^WT and SH-SY5Y^G93A viable cells after the treatment of 100 μM riluzole is significantly higher (Figure 2B; 91.0 ± 4.5 for WT and 88.2 ± 5.2 for SOD1^G93A; Table 2) compared to non-treated cells stimulated with 3mM Na-azide for 3 min (Figure 2A; 75 ± 6.3% for WT and 73.5 ± 6.3% for SOD1^G93A; ^∗∗P < 0.01, Students two sample t-test; Table 2). There is no significant increase in cell survival after melatonin treatment (Figure 2B; 80.2 ± 5.3 for WT and 78.5 ± 4.2 for SOD1^G93A; Table 2).

To test further the efficacy of riluzole on Na⁺-azide induced mitochondrial Ca²⁺ signaling, Fura-2 AM stained HMNs of brain stem slices were exposed to Na-azide in presence and absence of 100 μM riluzole. Sketch diagram of hypoglossal MNs in brainstem slice preparations (Figure 3A) and representative photomicrograph of fura-2 AM stained HMNs in WT and (left) and symptomatic SOD1^G93A mice (right) shown in Figure 3B. We evaluated the protective impact of riluzole on calcium signaling of HMNs of adult WT (Figures 3C,E) and symptomatic SOD1^G93A mice brain stem slices in vitro (Figures 3D,F). We found that riluzole has a moderate effect on inhibition of Na-azide (3 mM) induced [Ca²⁺]_i signaling of MN’s mediated by Na⁺-azide induced excitotoxicity. At higher concentration (>50 μM) riluzole moderately inhibited [Ca²⁺]_i fluorescence in both WT and selectively impaired HMNs of symptomatic SOD1^G93A mice whereas at lower concentrations (<50 μM) there is no significant effect (data not shown). Sodium azide-induced mitochondrial [Ca²⁺]_i release response is significantly different between WT and symptomatic SOD1^G93A mice. The impact of riluzole inhibition is not specific to only SOD1^G93A mice and it also moderately inhibits [Ca²⁺]_i signaling in WT mice as well (Figure 3; Table 3).

As illustrated in Figures 3C,D,G; 3 mM Na-azide caused a rise in the [Ca²⁺]_i transient by mitochondrial complex IV inhibition, with average peak amplitudes (F/F₀) reaching 0.15 ± 0.02 and 0.11 ± 0.01 (Figures 3C,D,G; Table 3) respectively, in HMNs of adult WT (5 mice/N = 12 slice; n = 58 HMNs) and symptomatic SOD1^G93A mice (5 mice/N = 10 slice; n = 46 HMNs) and subsequently comes to baseline over 2–3 min wash out with aCSF. In the presence of 100 μM riluzole, application of 3 mM Na-azide caused a rise in the [Ca²⁺]_i transient, with average of the peak amplitudes (F/F₀) reaching 0.089 ± 0.01 and 0.065 ± 0.01 (Figures 3E–G; Table 3) respectively, in HMNs of adult WT (6 mice/N = 14 slice; n = 63 HMNs) and symptomatic SOD1^G93A mice (6 mice/N = 13 slice; n = 58 HMNs). The inhibition of the peak amplitude of [Ca²⁺]_i in SOD1^G93A mice (F/F₀ = 0.065 ± 0.01) is prominent compared to WT mice (F/F₀ = 0.089 ± 0.01; P < 0.05, Students two sample t-test; quantitative fluorescence value compared in Table 3). The inhibition of [Ca²⁺]_i peak amplitude in SOD1^G93A mice by riluzole might be attributed to a decrease in the entry of Ca²⁺ through a voltage dependent calcium channel (VDCC) and need further investigations. We conclude that riluzole inhibits Na-azide-induced mitochondrial Ca²⁺ signaling and it modestly ameliorate MN degeneration by inhibition of excitotoxicity trigger by [Ca²⁺]_i efflux.

Mitochondrial ETC associated proton efflux causes accumulation of negative charges in the matrix and electrochemical gradient across the mitochondrial membrane. Intrinsic NADH fluorescence in MNs has been shown to serve as a valuable tool to characterize the metabolic signature of intrinsic energy profiles. Accordingly, evaluation of intrinsic NADH fluorescence after inhibition of mitochondrial complex IV with Na-azide indicated that the mitochondrial respiratory chain was significantly disturbed in SOD1 G93A animals which is rescues in acute condition by riluzole by yet unknown mechanism (Figure 4). To confirm that impact of riluzole on mitochondrial NADH fluorescence, experiments were performed, in which respiratory chain activity of HMNs was inhibited by 3 mM Na-azide in absence (aCSF; Figure 4A) and presence of riluzole (100 μM riluzole; Figure 4B). Kinetic profile of HMNs (n = 5) NADH fluorescence in single slice in aCSF (black) and in presence of riluzole (red) shown in Figure 4C for three consecutive application.

To analyze the comparative efficiency of mitochondria of SOD1^G93A mice, HMNs were evoked by a 30 mM K⁺ depolarizing stimulus. When SOD1^G93A mice HMNs were exposed to 30 mM K⁺ for 20 s, there is immediate and slow plateau shape decrease in the NADH fluorescence and subsequently returned to the basal level. To determine whether the mitochondrial depolarization by 30 mM K⁺ has any apparent influence on the lifetime of the NADH fluorescence and riluzole still rescues HMNs metabolism, 100 μM riluzole was added to the aCSF superfusate after K⁺-induced depolarization (Figures 4D,E). Application of 3 mM Na-azide after 20 s depolarization induced by K⁺ resulted in an increase of NADH fluorescence of 0.024 ± 0.003 and 0.049 ± 0.001 in the presence of aCSF (N = 5 slice; n = 38 HMNs) and aCSF+100 μM riluzole (N = 5 slice; n = 41 HMNs), respectively, in SOD1^G93A mice (Figure 4F). Following previous trends there was a slight increase in the NADH fluorescence in presence of 100 μM riluzole but there was no significant difference in NADH fluorescence mean value after 3 mM Na-azide-evoked responses (normalized, F/F₀) post K⁺-induced depolarization with or without 100 μM riluzole.