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Annals of Neurodegenerative Disorders

Cite this article: Lautenschläger J, Tadic V, Weidemann L, Prell T, Ruhmer J, et al. (2016) Analysis of ER Ca2+ Homeostasis in Embryonic Spinal Neurons of Wild-Type and G93A hSOD1 Mice. Ann Neurodegener Dis 1(3): 1011.

*Corresponding authorJanin Lautenschläger, Hans Berger Department of Neurology, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany, Tel: 0049 3641 9396630; Email:

Submitted: 28 July 2016

Accepted: 06 September 2016

Published: 10 September 2016

Copyright© 2016 Lautenschläger et al.

OPEN ACCESS

Keywords•Spinal cord neurons•Amyotrophic lateral sclerosis•SOD1•ER•Calcium

Research Article

Analysis of ER Ca2+ Homeostasis in Embryonic Spinal Neurons of Wild-Type and G93A hSOD1 MiceJanin Lautenschläger#*, Vedrana Tadic#, Lisa Weidemann, Tino Prell, Julia Ruhme, Jingyu Liu, Otto W. Witte, and Julian Grosskreutz1Hans Berger Department of Neurology, Jena University Hospital, Germany#These authors contributed equally to this work

Abstract

Calcium dysregulation is strongly implemented in the pathophysiology of neurodegenerative diseases. In amyotrophic lateral sclerosis (ALS), several findings indicate mitochondrial Ca2+ disturbances, however data on Ca2+ handling mechanisms of the endoplasmic reticulum (ER) are sparse. Therefore, we analyzed Ca2+ dynamics of the ER in embryonic neurons of the spinal cord from wild-type and G93A hSOD1 mice. ER depletion with cyclopiazonic acid (CPA) and thapsigargin showed no reduction of kainate-induced cytosolic Ca2+ transients. However, caffeine-induced Ca2+ transients were decreased upon application of CPA. Also, direct measurements of ER Ca2+ dynamics revealed ER Ca2+ release upon application of caffeine, but not upon kainate application. Furthermore, we revealed that caffeine stimulation produces increased ER Ca2+ release in the G93A hSOD1 spinal cord neurons compared to the wild-type neurons. Taken together, our results indicate, that Ca2+ release from the ER contributes only to a minor extent to kainate-induced Ca2+ transients in embryonic neurons of the spinal cord from wild-type mice, as well as G93A hSOD1 mice. Thus, upon excitotoxicity it is probably not an increased ER Ca2+ release which is disastrous, but an increased ER calcium storage and breakdown of ER homeostasis in G93A hSOD1 neurons could have major implications in disease manifestation.

ABBREVIATIONS ALS: Amyotrophic Lateral Sclerosis; AMPA: Alpha-Amino-

5-Methyl-3-Hydroxyisoxazolone-4-Propionat; AUC: Area Under the Curve; CICR: Calcium-Induced Calcium Release; CPA: Cyclopiazonic Acid; CTZ: Cyclothiazide; DRG: Dorsal Root Ganglion; ER: Endoplasmic Reticulum; ERMCC:- ER Mitochondria Calcium Cycle; hSOD1: Human Superoxide Dismutase 1; IP3R: Inositol Triphosphat Receptors; RyR: Ryanodine Receptors; SERCA: Sarco-/Endoplasmic Reticulum ATPase; UPR: Unfolded Protein Response; VGCC: Voltage Gated Calcium Channels

INTRODUCTION In neurons, a strict modulation of cytosolic Ca2+ homeostasis

is crucial for survival and viability, and thus calcium dysregulation is strongly implemented in the pathophysiology of neurodegenerative diseases, like Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic

lateral sclerosis (ALS) [1-4]. In ALS, AMPA-receptor mediated excitotoxicity has been demonstrated as a major key player and kainate-stimulation is a well-established model for selective motor neuron death [5-8]. While several findings indicate that there are disturbances in mitochondrial Ca2+ handling in ALS, data on Ca2+ handling mechanisms of the endoplasmic reticulum (ER) are sparse.

The ER is a large calcium store and can thus function as both a source and a sink of calcium. Ca2+ is released into the cytosol either through IP3R (inositol triphosphat receptors) or RyR (ryanodine receptors) and is taken up from the cytosol into the ER by the sarco-endoplasmic reticulum ATPase (SERCA) [9]. In the pathophysiology of ALS, induction of the unfolded protein response (UPR) clearly indicates an alteration of ER homeostasis [10,11]. Thus, we intended to analyze the role of the ER calcium store in embryonic neurons of the ventral spinal cord from G93A hSOD1 mice and their wild-type littermates. ER Ca2+ dynamics


Lautenschläger et al. (2016)Email:

Ann Neurodegener Dis 1(3): 1011 (2016) 2/9

were manipulated by cyclopiazonic acid (CPA) and thapsigargin, both of which are inhibitors of the sarco-endoplasmic reticulum ATPase. Furthermore, caffeine was used to investigate ER Ca2+ release. Additionally, ER Ca2+ dynamics were directly visualized in primary ventral spinal cord neurons using the ER targeted Ca2+ sensor, D1ER.

METHODS

Animals

Transgenic mice over-expressing the G93A mutant of the human SOD1 [12] (B6.Cg-Tg (SOD1*G93A)1Gur/J, JAX Mice stock number 004435) were obtained by the Jackson Laboratory (Bar Harbor, USA) and mated with C57BL/6J females. Animals were housed under controlled laboratory conditions (temperature 22 °C, relative air humidity 55–60 %, LD 12:12, lights on at 06:00 am) with free access to standard diet and tap water. All animal experiments were conducted in accordance with the requirements of the National Act on the Use of Experimental Animals in Germany.

Motor neuron-enriched cultures of the ventral spinal cord

Motor neuron-enriched cultures were prepared as described previously [5,13]. Ventral spinal cords were dissected from 13-day-old mouse embryos and pooled according to genotype (genotyping protocol recommended by the Jackson Laboratory, Bar Harbor, USA). Centrifugation on a 6.2 % OptiPrep cushion (Axis-shield Poc AS, Norway) was used to separate glial cells and neurons. Glial cells were plated at a density of 50,000 cells per 12 mm coverslip in a 24 well plate. Neurons were seeded on confluent glial feeder layers at a density of 30,000 cells/coverslip. Cultures were used for experiments starting on day 13 in vitro to achieve appropriate maturation. Motor neuron-enriched cultures obtained from wild-type littermates were used as controls.

Monitoring of cytosolic Ca2+ dynamics

For cytosolic Ca2+ measurements, the membrane permeable acetoxymethylester of the high-affinity ratiometric calcium dye fura-2 (Sigma, Germany) was used. Motor neuron-enriched cultures were loaded with 8 µM fura-2 AM for 20 min in a 5 % CO2-humidified incubator at 37°C. After de-esterification at room temperature, cells were placed in the recording chamber and monitored as described in the live cell imaging section.

Ratiometric fluorescent images were obtained (Till Vision Imaging System from TillPhotonics, Gräfelfing, Germany) by alternate excitation of fura-2 at 350 nm and 380 nm (Polychrome V, TillPhotonics, Gräfelfing, Germany) and collection of the emission light passing the dichroic mirror (DCLP410) and the emission filter (LP440, both obtained from TillPhotonics, Gräfelfing, Germany). Exposure time was set to 5 ms and pictures were taken at a sampling frequency of 5 Hz.

Calcium concentration was calculated from the fluorescence ratio according to Grynkiewicz et al., [14]: calcium concentration = KD x β x (R-Rmin)/(Rmax-R) using KD = 245, β = 3.6 and Rmin = 0.08, which was determined using the standard extracellular solution containing no CaCl2, 2 mM EGTA and 2 µM ionomycin (Sigma; Germany), and Rmax = 0.8, which was determined using

the standard extracellular solution containing 30 mM CaCl2 and 10 µM ionomycin as described previously [15].

Monitoring of ER luminal Ca2+ dynamics

To monitor ER luminal Ca2+ dynamics, the ER-targeted calcium indicator D1ER (a gift from R.Y. Tsien; University of California, San Diego, CA) was employed. D1ER is specifically designed to cover the span of Ca2+ concentration in the ER and is targeted to the ER by a calreticulin signalling peptide [16]. Motor neuron-enriched cultures were transfected with the D1ER plasmid DNA using Lipofectamine 2000 (Invitrogene, UK). The standard protocol with a DNA: Lipofectamine 2000 ratio of 1:2 was used. 48 h after transfection, ER Ca2+ dynamics were monitored by live cell imaging, and D1ER distribution was analyzed by confocal microscopy.

D1ER was excited at 436 nm (Polychrome V, TillPhotonics, Gräfelfing, Germany; 440 SP; AF Analysentechnik, Tübingen, Germany) for 100 ms at a sampling frequency of 2 Hz. Emission light was passed by a dichroic mirror (DC 458; AF Analysentechnik, Tübingen, Germany) to the Dual View Beamsplitter equipped with a dichroic mirror (DC 510) and the emission filters 465/30 and 535/30 (Till Potonics, Gräfelfing, Germany). One picture with the projection of the CFP fluorescence at one site and the citrine fluorescence at the other site was obtained (Till Vision Imaging System from TillPhotonics, Gräfelfing, Germany) and the citrine/CFP ratio was calculated.

Confocal analysis of D1ER distribution

After transfection with the D1ER plasmid (see section Monitoring of ER luminal calcium dynamics), motor neuron-enriched cultures were fixed with 4 % paraformaldehyde diluted in phosphate buffered saline (pH 7.4) for 20 min. After washing with PBS and blocking, dishes were incubated with the primary antibody SMI32 (mouse monoclonal antibody; 1:1000; Covance, CA, USA), diluted in PBS with 0.3 % Triton X-100 and 2 % normal donkey serum for 2 h at room temperature. The cultures were washed in PBS and incubated for 1 h at room temperature with the secondary antibody Alexa 594 (goat anti mouse; 1:200; Invitrogen, UK) in 10 % donkey serum. For nuclear staining, 4’,6-Diamidino-2-phenylindole (DAPI, Sigma, Germany) was used. The immunocytochemical staining was examined by confocal laser scanning microscopy (LSM 710, Zeiss, Germany). All images were captured using a 60X oil immersion objective.

Live cell imaging

Motor neuron-enriched cultures were imaged using an upright microscope (Nikon Microscope Eclipse FN1, Tokyo, Japan) with a 40X/0.8W water immersion objective (Nikon, Tokyo, Japan) equipped with a cooled CCD camera (iXONEM+, ANDORTM, Belfast, UK).

The recording chamber was continuously perfused with a standard extracellular solution containing (in mM) HEPES 11.6, NaCl 129.1, KCl 5.9, glucose 11.5, MgCl2 1.2, and CaCl 3.2 adjusted to pH 7.4 with NaOH. During measurements with kainate application, cells were superfused with the standard extracellular solution containing 200 µM verapamil and 0.5 µM tetrodotoxin. A custom-made application system, which has previously been




described in detail by Grosskreutz et al., was used to apply precise caffeine or kainate pulses inducing cytosolic Ca2+ transients [17].

Chemicals

Stock solutions of the following chemicals were prepared as indicated: 1 mM tetrodotoxin-citrate (Biotrend, Switzerland), 10 mM kainate (Ascent Scientific, UK), 1 M caffeine (Sigma, Germany) in purified water, 20 mM verapamil (Sigma, Germany) in a 1:4 mixture of DMSO and purified water, 30 mM cyclopiazonic acid (Sigma, Germany) and 4 mM thapsigargin (AppliChem; Germany) in DMSO. Chemicals were diluted to their final concentration in standard extracellular solution before measurements were taken.

Statistics

Values are given as mean ± sem. Student’s t-test was used for normally distributed data. For multiple comparisons, one-way ANOVA with Bonferroni correction was applied. For comparisons of not normally distributed data, the Mann-Whitney U-test was used. The Kruskal-Wallis test was applied for multiple comparisons, following Mann-Whitney U-test and Bonferroni correction to test for discrete differences (IBM SPSS Statistics 20). Significance was considered at p < 0.05.

RESULTS AND DISCUSSION

Role of the ER calcium store upon AMPA receptor stimulation

The impact of SERCA inhibition upon kainate-induced Ca2+ transients: Short kainate pulses of 2 s duration, which have previously been shown to produce cytosolic Ca2+ transients in the range of spontaneous Ca2+ activity [5], were applied. In order to test whether kainate-induced cytosolic Ca2+ transients are amplified by Ca2+ release from the ER, CPA, a reversible inhibitor of the SERCA, was used. Since Ca2+ is no longer able to enter the ER upon SERCA inhibition, CPA leads to a depletion of the ER calcium store [18]. In the test protocol, all neurons were checked for appropriate calcium buffering by applying an initial 2 s kainate (100 µM) pulse, followed by further 2 s kainate applications at an interval of 100 s. The application of CPA (30 µM) was started 50 s before the second kainate pulse, lasting in total 100 s. Compared to neurons exposed solely to kainate pulses, no reduction of kainate-induced cytosolic Ca2+ transients was verifiable upon treatment with CPA. The relative peak amplitude of the Ca2+ transients was not significantly decreased (Figure 1A).

Furthermore, also a higher concentration of CPA (100 µM) had no significant influence on the relative peak amplitudes (Figure 1B). To ensure a sufficient time of incubation for ER depletion, 2 s kainate pulses were applied at an interval of 15 minutes and CPA (30 µM) was washed in immediately after the first kainate pulse, lasting until the second kainate pulse. Even under these conditions, no decrease in the peak amplitudes was seen and the relative peak amplitude was not statistically decreased. Furthermore, a protocol with 30s kainate pulses was run, but similarly no decrease of the cytosolic Ca2+ transients was seen upon application of CPA (30 µM) (Figure 1C). In addition, thapsigargin, an irreversible inhibitor of the SERCA [19] was tested. Kainate pulses of 2 s were applied with an interpulse interval of 100 s, thapsigargin was washed in starting 50 s before

the second kainate pulse, lasting in total for 100 s. No decrease of the kainate-induced Ca2+ transients was seen and the relative peak amplitude was not statistically decreased (Figure 1D). Two different concentrations of thapsigargin (0.5 µM and 3 µM) were tested. No difference was seen between wild-type and G93A hSOD1 spinal neurons.

The impact of ER Ca2+ depletion with caffeine upon kainate-induced Ca2+ transients: A depletion of the ER calcium store can also be achieved by application of caffeine, leading to the release of Ca2+ through RyR [20-22]. Caffeine (10 mM) application began 50 s before the second 2 s kainate pulse, lasting until the end of the experiment. The application of caffeine triggered a transient increase in the cytosolic Ca2+ concentration; however no change in the relative peak amplitude of the kainate-induced Ca2+ transients was seen upon ER depletion with caffeine (Figure 2A).

The impact of CPA upon cyclothiazide-induced Ca2+ activity: In addition, cyclothiazide (CTZ), an inhibitor of AMPA receptor desensitization, which thereby amplifies spontaneous Ca2+ activity, was used to test a protocol independent from kainate. After recording stable cytosolic Ca2+ activity for 60 s in the presence of CTZ, a solution containing CTZ plus CPA was applied for additional 60 s. Nevertheless, cytosolic Ca2+ transients were steadily present upon ER depletion by SERCA inhibition with CPA (Figure 2B).

These results obtained by different approaches indicate that Ca2+ release from the ER calcium store contributes only to a minor extent to kainate-induced Ca2+ transients in mouse embryonic neurons of the spinal cord. However, studies investigating glutamate-, AMPA- or NMDA-induced Ca2+ transients in rat cerebellar granule neurons, Purkinje neurons, cochlear neurons or hippocampal neurons generally report a proportion of ER Ca2+ release of 40 % or more [23-28]. In particular, kainate-induced Ca2+ transients in rat retinal neurons were found markedly reduced upon store depletion with caffeine [29]. In rat spinal motor neurons, spontaneous calcium activity mediated by AMPA/kainate receptors was almost completely blocked upon application of CPA and dantrolene [30]. On the contrary, Metzger et al., did not found any reduction of glutamate-induced Ca2+ transients upon application of CPA in rat spinal neurons [31]. Summarizing the data on depolarization-induced Ca2+ transients draws a more tessellated picture. Several reports revealed that ER Ca2+ release contributes to approximately 50 % or more in rat dorsal root ganglia (DRG) neurons [32-34]. However, only a small Ca2+ release or none at all was observed from the ER in rat sympathetic neurons, Purkinje neurons, septal neurons, DRG neurons and also motor neurons [21,22,35-39]. In neurons from frog, a Ca2+-induced Ca2+ release (CICR) is repeatedly shown [40-46]. However, studies with depolarization-induced Ca2+ transients in mice were not able to show any Ca2+ release from the ER [47,48]. Likewise, Guatteo et al., mentioned no change in the amplitude of kainate-induced Ca2+ transients upon application of CPA in mouse spinal neurons [49]. However, one study in mouse hippocampal neurons reported that ryanodine (100 µM) was able to decrease action potential-induced Ca2+ transients to about 15 %, indicating that there is possibly only a small contribution of ER Ca2+ release in these neurons [50].




Figure 1 Inhibition of the SERCA pump does not change kainate-induced Ca2+ transients(A) Kainate (100 µM) was applied as repetitive 2 s pulses in an interval of 100 s. CPA 30 µM application was started 50 s after the first kainate application, lasting for 100 s (CPA, n=55). No changes were verifiable in the peak amplitude of the following kainate-induced Ca2+ transients when compared to neurons solely treated with kainate (CTRL, n=31). Baseline Ca2+ increased with the application of CPA. (B) CPA 100 µM application was started 50 s after the first kainate application, lasting for 100 s (CPA, n=37). No changes were verifiable in the peak amplitude of the following kainate-induced Ca2+ transients when compared to neurons solely treated with kainate (CTRL). (C) Kainate (100 µM) was applied as repetitive 30 s pulses in an interval of 100 s. CPA 30 µM application was started 50 s after the first kainate pulse, lasting for 100 s (CPA, n=11). No changes were verifiable in the peak amplitude of the following kainate-induced Ca2+ transients when compared to neurons solely treated with kainate (CTRL, n=8). (D) Thapsigargin 3 µM application was started 50 s after the first kainate application, lasting for 100 s (Thap, n=12). No changes were verifiable in the peak amplitude of the following kainate-induced Ca2+ transients when compared to neurons solely treated with kainate (CTRL). Every trace illustrates the mean course of Ca2+ concentration for all cells analyzed.

ER Ca2+ release induced by caffeine application

Caffeine (10 mM and 20 mM) was able to induce cytosolic Ca2+ transients in cultured mouse embryonic neurons of the ventral spinal cord, demonstrating a functional intracellular calcium store in these neurons. When caffeine was applied for periods lasting 50 s or more, Ca2+ concentration returned to basal levels, even in the presence of caffeine. Similar effects of caffeine have been shown before in DRG neurons, hippocampal neurons, granule neurons and Purkinje neurons from rat and mice [20,21,33,48,51].

Furthermore, we were able to show a pronounced effect of ER Ca2+ depletion on the caffeine-induced Ca2+ release. We used a test protocol applying 2 s caffeine (10 mM) instead of kainate at an interval of 100 s, and CPA (30 µM) application was started 50 s before the second caffeine pulse, lasting for 100 s. The second caffeine pulse still produced a large cytosolic Ca2+ transient, possibly because Ca2+ had first to be released from the ER to obtain Ca2+ depletion. The third and the fourth caffeine pulse either triggered minor cytosolic Ca2+ transients or none at all. After this, further caffeine pulses induced increasing cytosolic

Ca2+ transients, indicative of a slow refilling of the ER calcium store. The relative peak amplitude of the caffeine-induced Ca2+ transient after application of CPA was significantly reduced compared to neurons solely exposed to caffeine pulses (for each p<0.001; Figure 3). Thus, we clearly demonstrated Ca2+ release from an intracellular compartment with SERCA-dependent refilling in mouse embryonic spinal neurons. And we revealed the dynamics of ER Ca2+ depletion and refilling upon reversible SERCA inhibition. However, no difference was seen between wild-type and G93A hSOD1 spinal neurons.

Direct measurements of calcium dynamics in the ER

However, to have a direct look on Ca2+ changes in the ER, the genetically targeted ER calcium indicator D1ER was used for single cell measurements. The intracellular distribution of the D1ER protein is shown in SMI32 stained neurons of the ventral spinal cord by confocal images (Figure 4A). Application of caffeine (20 mM) was able to induce a decrease of the citrine/CFP fluorescence ratio of the D1ER protein, indicating Ca2+ release from the ER. No decrease was seen upon application of kainate (100 µM). To ensure that there were no disturbances




Figure 2 ER Ca2+ depletion does not change kainate-induced Ca2+ transients or cyclothiazide-induced Ca2+ activity(A) ER Ca2+ depletion with caffeine does not change kainate-induced Ca2+ transients. Kainate (100 µM) was applied as repetitive 2 s pulses in an interval of 100 s. Caffeine 10 mM application was started 50 s after the first kainate application, lasting until the end (Caff, n=11). The Ca2+ concentration steeply increased upon application of caffeine, returning to basal levels even in the presence of caffeine. No changes were verifiable in the peak amplitude of the following kainate-induced Ca2+ transients when compared to neurons solely treated with kainate (CTRL, n=11). Every trace illustrates the mean course of Ca2+ concentration for all cells analyzed. (B) Inhibition of the SERCA pump does not change cyclothiazide-induced Ca2+ activity. The application of cyclothiazide (CTZ) 100 µM immediately induced cytosolic Ca2+ transients. 60 s after initiation, a solution containing cyclothiazide and CPA 30 µM was applied for 60 s, then it was switched back to the solution only containing cyclothiazide. No change in cytosolic Ca2+ transients was verifiable upon addition of CPA. Every trace illustrates one single cell.

Figure 3 Inhibition of the SERCA pump by CPA decreases caffeine-induced Ca2+ transients(A). Caffeine (10 mM) was applied as repetitive 2 s pulses in an interval of 100 s. CPA 30 µM application was started 50 s after the first caffeine application, lasting for 100 s (CPA, n=40). Baseline Ca2+ increased with the application of CPA. The second caffeine pulse still produced a large cytosolic Ca2+ transient, the third and the fourth caffeine pulse triggered no or only minor cytosolic Ca2+ transients, even when CPA was already removed. The following caffeine pulses induced increasing cytosolic Ca2+ transients. (B). The relative peak amplitudes of caffeine-induced Ca2+ transient were significantly reduced upon application of CPA (CPA, n=40) when compared to spinal neurons solely exposed to caffeine pulses (CTRL, n=29). Every trace illustrates the mean course of Ca2+ concentration for all cells analyzed. *** p<0.001.




during consecutive measurements, a protocol was used applying 9 s pulses at an interval of 100 s, firstly with caffeine followed by kainate or vice versa. Three individual neurons were tested using caffeine first and then applying kainate. A clear decrease in the citrine/CFP fluorescence ratio was apparent upon the first application with caffeine. The second application produced an initial decrease of the citrine/CFP ratio, which is due to residual caffeine in the perfusion system used. The third application with kainate produced no detectable decrease of the citrine/CFP ratio (Figure 4B). Even if kainate was used during the first application, no decrease of the citrine/CFP fluorescence ratio was detectable. A switch to caffeine for the second and the third application demonstrated the responsiveness of both cells (Figure 4C).

These results clearly indicate an emptying of the ER Ca2+ store by caffeine, whereas no release was seen upon kainate exposure. The detection of small Ca2+ changes upon kainate stimulation

could be limited, however, in rat DRG neurons a decrease of ER Ca2+ concentration upon depolarization has been revealed using mag-fura-2 (Kd = 221 µM) [52]. D1ER, which has a half maximal dissociation constant of 200 µM [53], should be able to report similar ER Ca2+ changes. Therefore, our results support the hypothesis of a minor contribution of ER Ca2+ release upon kainate stimulation in mouse ventral spinal cord neurons.

Increased ER calcium storage in G93A hSOD1 ventral spinal cord neurons

So far, like the wild-type neurons, the ventral spinal cord neurons of G93A hSOD1 mice showed no reduction of kainate-induced Ca2+ transients, but a decrease of caffeine-induced Ca2+ transients upon ER Ca2+ depletion with CPA. However, G93A hSOD1 spinal cord neurons demonstrated an increased caffeine-induced Ca2+ release from the ER.

Figure 4 ER Ca2+ store depletion by caffeine but not upon kainate stimulation(A). ER Ca2+ changes were monitored with the genetically targeted ER calcium indicator D1ER. Neuronal cultures transfected with D1ER (green), counterstained for the motor neuron marker SMI32 (red) and DAPI (blue). (B). Application of caffeine (20 mM) for 9 s was able to induce a decrease of the citrine/CFP fluorescence ratio, while application of kainate (100 µM) for 9 s induced no change in the citrine/CFP fluorescence ratio. (C) Vice versa, when kainate was applied first no change was verifiable in the citrine/CFP fluorescence ratio. While subsequent application of caffeine induced a decrease of the citrine/CFP fluorescence ratio. Every trace illustrates one single cell.




Upon repetitive 2 s caffeine application we observed increased relative peak amplitudes in the G93A hSOD1 compared to the wild-type spinal neurons. This was confirmed in a protocol applying 9 s caffeine pulses to trigger a higher ER Ca2+ release. The relative peak amplitude of the second caffeine-induced Ca2+ transient was significantly elevated in the G93A hSOD1 spinal neurons. Furthermore, the area under the curve (AUC) demonstrated an increased Ca2+ release in the G93A hSOD1 compared to the wild-type spinal neurons (Figure 5).

While several studies were able to show mitochondrial Ca2+ handling disturbances in ALS, we show for the first time a disturbance of the ER Ca2+ homeostasis in G93A hSOD1 ventral spinal cord neurons. A breakdown of the delicate balance of ER Ca2+ homeostasis will have major implications, as protein folding and processing are strictly Ca2+ dependent processes [54-56]. Therefore, ER stress and activation of the unfolded protein response would be possible consequences [10]. Studies using Alzheimer disease models with mutant presenilin 1 have also indicated increased caffeine-induced Ca2+ transients in cortical neurons [57-59], thus demonstrating the significance of our results in the understanding of neurodegenerative diseases.

CONCLUSIONIn conclusion, our study revealed that Ca2+ release from the

ER contributes at most to a minor extent to kainate-induced Ca2+ transients in mouse embryonic neurons of the spinal cord. Thus, we demonstrate that upon excitotoxicity, it is probably not an increased ER Ca2+ release, but rather a breakdown of the

delicate balance of intra-ER Ca2+ homeostasis that plays a critical role in the pathophysiology of ALS. To maintain cytosolic Ca2+ homeostasis, the ER and mitochondria are highly coupled in the ER mitochondria calcium cycle (ERMCC) [10]. Upon failure of the initial Ca2+ buffering capacity of mitochondria [5], the ER is possibly able to take up more Ca2+, serving as a compensatory mechanism.

Beside the mitochondrial dysfunction reported so far, we now show for the first time that ER calcium storage is increased in G93A hSOD1 spinal neurons and thereby demonstrate that changes in ER calcium homeostasis could represent a pathological hallmark of neurodegenerative diseases.

AUTHORS’ CONTRIBUTIONS JL, LW, VT, JR and CL were responsible for the cytosolic

calcium measurements with pharmacological intervention and the culture of embryonic ventral spinal cord neurons. JL carried out the transfection of spinal neurons and measurements of ER calcium dynamics. JL and VT performed the statistical analysis. JL, OW and JG were responsible for the design of the study. JL, VT and TP drafted the manuscript. All authors read and approved the final manuscript.

ACKNOWLEDGEMENTSThis research is supported by a BMBF (the Bundesministerium

für Bildung und Forschung) grant to JG (ERMCC-NDEG) in the framework of the ERANET-NEURON program and to JG in the E-RARE program (PYRAMID) of the European Union.

Figure 5 Increased caffeine-induced Ca2+ release in G93A hSOD1 spinal neurons(A). Caffeine (10 mM) was applied as repetitive 9 s pulses in an interval of 100 s. In G93A hSOD1 spinal neurons (G93A, n=11) caffeine application induced an increased Ca2+ release compared to wild-type spinal neurons (wt, n=15). (B). The relative peak amplitude of the second caffeine-induced Ca2+ transient was significantly increased. (C). The area under the curve calculated from the beginning until the end of the caffeine-induced Ca2+ transient was significantly increased for all four caffeine-induced Ca2+ transients in G93A hSOD1 spinal neurons compared to the wild-type neurons. Every trace illustrates the mean course of Ca2+ concentration for all cells analyzed. * p<0.05




We thank the Laboratory of Roger Y. Tsien for kindly providing us with the plasmid of D1ER. We thank N. Kroegel and G. Simpson for language editing.

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Lautenschläger J, Tadic V, Weidemann L, Prell T, Ruhmer J, et al. (2016) Analysis of ER Ca2+ Homeostasis in Embryonic Spinal Neurons of Wild-Type and G93A hSOD1 Mice. Ann Neurodegener Dis 1(3): 1011.

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Analysis of ER Ca2+ Homeostasis in Embryonic Spinal Neurons of … · 2018. 1. 22. · Calcium...

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